Poster abstracts
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1. Structural basis of tRNA recognition by arginyl-tRNA-protein transferase 1
Thilini Abeywansha (Case Western Reserve University, School of Medicine, Biochemistry Department, Cleveland, OH), Wei Huang (Case Western Reserve University, School of Medicine, Department of Pharmacology, Cleveland, OH), Xuan Ye (Case Western Reserve University, School of Medicine, Center for RNA Science and Therapeutics, Cleveland, OH ), Xin Lan (Case Western Reserve University, School of Medicine, Biochemistry Department, Cleveland, OH), Allison Nawrocki (Case Western Reserve University, School of Medicine, Biochemistry Department, Cleveland, OH), Yi Zhang, Derek Taylor, Eckhard Jankowsky (Case Western Reserve University, School of MedicineCleveland, OH )
Abstract:
Arginyl-tRNA protein transferase (ATE1) catalyzes the conjugation of arginine to the N-terminal or midchain Glu/Asp residues (also known as arginylation) of its substrates. Protein arginylation is solely mediated by ATE1 in an arginyl-tRNAArg-dependent manner and is a vital post-translational modification in many biological processes. ATE1-mediated arginylation is dysregulated in breast, prostate, skin, and many other cancers. This indicates that ATE1 could be a potential target for cancer therapy, and developing drugs that target ATE1 could be a promising approach to treating cancer. However, the mechanisms by which ATE1 regulates tumorigenesis have yet to be understood due to a lack of understanding of the catalytic mechanism of ATE1. Here, we present our findings on ATE1 structure and the catalytic mechanism as a continuation of our effort to elucidate the role of ATE1 as a tumor suppressor. We solved structures of ATE1 from Saccharomyces cerevisiae (scATE1) in apo form and in complex with tRNAArg using CryoEM. The structure of scATE1 displays overall new folds with a compact conformation consisting of three domains, including a highly conserved GNAT fold, a putative polypeptide substrate binding domain, and a variable domain unique to yeast ATE1. ATE1 interacts with the tRNAArg via helix αf in the assembled complex and mainly contacts the major groove of the tRNAArg acceptor arm, resulting in structural rearrangements of the ATE1 protein. These rearrangements facilitate arginine transfer from the tRNA cofactor to the protein substrate. Based on these data, we propose a detailed catalytic mechanism of ATE1-mediated protein arginylation. Intracellular ATE1 displays a high affinity for arginyl-tRNAArg and leads to the formation of ATE1.arginyl-tRNAArg complex inducing a conformational change in ATE1 and increasing its affinity to protein substrates, which bind weakly to free ATE1. The arginine is then transferred from the tRNA to the protein substrate, generating an arginylated protein and an uncharged tRNA. The reduced affinity of tRNA following the arginine transfer reaction facilitates its release and subsequent recharging by argRS to complete the catalytic cycle.
References:
Abeywansha, T., Huang, W., Ye, X. et al. The structural basis of tRNA recognition by arginyl-tRNA-protein transferase. Nat Commun 14, 2232 (2023). https://doi.org/10.1038/s41467-023-38004-8
Keywords: Arginylation, ATE1, tRNA
2. Myotonic Dystrophy type I (DM1) Impairs Cardiac Bioenergetics and Disrupts Cardiac Mitochondrial Fusion-Fission Dynamics
Oluwafolajimi Adesanya (Department of Biochemistry, University of Illinois Urbana-Champaign), Pouya Nabie (Department of Biochemistry, University of Illinois Urbana-Champaign), Subhashis Natua (Department of Biochemistry, University of Illinois Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, University of Illinois Urbana-Champaign)
Abstract:
Background: DM1 is the commonest cause of adult-onset muscular dystrophy, affecting 1-in-2100 people in the U.S. DM1 is a (CTG)n trinucleotide repeat expansion disease in the 3’UTR of the DMPK gene. Once expressed, the repeat RNA form toxic hairpins, which sequester the muscle blind-like (MBNL) family of splicing factors. This inducing tissue-wide disruption of alternative splicing events, resulting in DM1 symptoms, including muscle weakness, neurological defects, and cardiomyopathy. Here, we explored the effect of DM1 on cardiac mitochondria function.
Methods: Using an inducible, cardiomyocyte-specific DM1 mouse model, we performed extracellular flux analysis on mitochondrial fractions obtained from heart tissue of control and DM1 mice. This was accompanied by quantification of ATP production, to determine OXPHOS efficiency. Further, we performed TOM20 immunofluorescence (IF) microscopy to assess the integrity of mitochondrial morphology, and gene expression studies to identify mis-splicing events involving mitochondria-related genes in heart tissues obtained from the DM1 mice and deceased human DM1 subjects.
Results: DM1 induced a multi-state increase in cardiac oxygen consumption rate (OCR) which was however accompanied by decreased ATP levels (2.83µM v. 2.45µM, p=0.0079) indicating decreased efficiency of cardiac OXPHOS. DM1 also induced a disruption in mitochondria network, with increased mitochondria fragmentation on IF microscopy. These findings correlated with an increased exclusion of Mitochondria Fission Factor (Mff) exon 6 in DM1 mice (ΔΨ: -16.8%, p=0.0069) and human heart samples (ΔΨ: -19%, p<0.01). Interestingly, this resulted in an increased expression of a shortened 29kDa MFF protein isoform in DM1 mice (Δ%29kDa: 47%, p=0.0003) and human (Δ29kDa: 21.1%, p<0.0001) heart samples.
Conclusion: DM1 impairs cardiac bioenergetic function potentially through the mis-splicing of critical genes regulating mitochondrial fusion-fission dynamics.
References:
1. Johnson, N. E. et al. Population-Based Prevalence of Myotonic Dystrophy Type 1 Using Genetic Analysis of Statewide Blood Screening Program. Neurology 96, e1045–e1053 (2021).
2. Wheeler, T. M. & Thornton, C. A. Myotonic dystrophy: RNA-mediated muscle disease. Curr. Opin. Neurol. 20, 572–576 (2007).
3. Jiang, H., Mankodi, A., Swanson, M. S., Moxley, R. T. & Thornton, C. A. Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum. Mol. Genet. 13, 3079–3088 (2004).
4. Miller, J. W. et al. Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J. 19, 4439–4448 (2000).
Keywords: Myotonic Dystrophy, Mitochondria, Bioenergetics
3. Reusable Microfluidic Chambers for Single-Molecule Microscopy
Janan Alfehaid (Department of Physics, Kent State University), Sineth G. Kodikara (Department of Physics, Kent State University), Tuqa Alhajri (Department of Physics, Kent State University), Mohammad Lutful Kabir (Department of Chemistry and Biochemistry, Kent State University), Hamza Balci (Department of Physics, Kent State University)
Abstract:
Maintaining a stable environment in single-molecule microfluidic chambers with surface-bound molecules involves time-consuming cleaning and surface passivation. Even with such measures, variations in non-specific binding and background signals are often seen across different chambers. Reusing these chambers without surface degradation offers both practical and fundamental benefits, but it requires removing surface-bound molecules like DNA, proteins, lipids, or nanoparticles. Biotin-streptavidin is a common method for attaching these molecules since biotin can easily be incorporated. In this study, we demonstrate through single-molecule fluorescence experiments that chambers can be effectively reset and reused at least 10 times by using photocleavable biotin (PC-biotin) and UV light. Unlike other methods, this approach avoids harsh chemical treatments. We show that a 5-minute exposure to UV light at a specific wavelength successfully removes all bound molecules using various PC-biotin attachment chemistries. Non-optimal wavelengths and light sources showed varying degrees of success. Importantly, our method causes no detectable surface degradation, as confirmed by the low non-specific binding of fluorescently labeled DNA and proteins, as well as the recovery of DNA secondary structure and protein activity. This resetting process is fast, efficient, cost-effective, and broadly applicable to a wide range of single-molecule experiments due to the common use of biotin-streptavidin attachments.
Keywords: single molecule, chamber recycling, UV-exposure
4. Protein bL38 facilitates incorporation of uL6 during assembly of the 50S subunit in Flavobacterium johnsoniae
Md. Siddik Alom (1Ohio State Biochemistry Program, 2Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Dominic Arpin, Haojun Zhu (3Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada; 4Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada), Brenna N. Hay (5Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, V3T1Z4, Canada.), Leonard J. Foster (5Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, V3T1Z4, Canada.), Joaquin Ortega (3Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada; 4Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada), Kurt L. Fredrick (1Ohio State Biochemistry Program, 2Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA, 6Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.)
Abstract:
Previous studies of the 70S ribosome from Flavobacterium johnsoniae revealed a novel ribosomal protein, bL38, which interacts with uL6 on the 50S subunit. This 5.6 kDa protein is conserved across the Bacteroidia and encoded downstream of bL28 and bL33 in a three-gene operon. Here, we show that bL38 is critical for the growth of F. johnsoniae, and depletion of bL38 leads to accumulation of immature 50S particles which lack uL6 and retain precursor rRNA sequences. Cryo-EM analysis of these particles reveals several putative assembly intermediates, all which show an absence of electron density for uL6 and the entire uL12 stalk base, and additional density corresponding to unprocessed ends of the pre-23S rRNA. One structural class reveals a Der-bound pre-50S intermediate in which the GTPase is tucked behind and appears to reposition helix H68. Extra copies of the uL6 gene can rescue the phenotypes caused by bL38 depletion, strongly suggesting that bL38 facilitates uL6 incorporation during 50S subunit biogenesis. Cryo-EM analysis of 50S particles from this rescued strain reveals nearly twice as many intermediates, suggesting a broader and more robust assembly landscape. Collectively, these data show the importance of bL38 and its role in the assembly of the 50S subunit.
Keywords: 50S Assembly, bL38, Der
5. Substrate specificity and 3'-5'-polymerization capabilities of two tRNA His guanylyltransferase-like proteins
Thaanya Amarasekara (Department of Chemistry and Biochemistry, The Ohio State University), Malithi Ishara Jayasinghe (Department of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Department of Chemistry and Biochemistry,The Ohio State University)
Abstract:
The discovery of tRNAHis guanylyltransferase-like proteins (TLPs), which catalyze nucleotide addition in the opposite direction (3'-5') to all standard DNA/RNA polymerases, has raised the possibility of developing a versatile 3'-5'-polymerase for multiple biotechnology applications, such as RNA 5'-end labeling. Although the physiological substrates identified so far for TLPs have all been highly structured RNAs, such as tRNA, two TLPs isolated from Acanthamoeba castellanii (Aca) and Dictyostelium discoideum (Ddi), known as AcaTLP2 and DdiTLP4, catalyze efficient 3'-5'-template-dependent nucleotide addition to short RNA duplexes in vitro. As the first members of the TLP family to act on non-tRNA substrates, understanding the biochemical properties is essential for developing these enzymes as broadly active RNA labeling tools. Based on the flexibility in substrate specificity exhibited by these enzymes compared to the other TLPs studied so far, we hypothesize that AcaTLP2 and DdiTLP4 will exhibit preferences for specific sequences and structures rather than acting uniformly on all small duplex RNA substrates. To comprehensively characterize these biochemical preferences, we designed an RNA substrate pool with nine randomized nucleotides, generating more than 200,000 sequences that can form various RNA structures to assess 3'-5'-polymerization using a high-throughput in vitro 3'-5'-extension assay. AcaTLP2 and DdiTLP4 successfully catalyzed the time-dependent addition of variable numbers of nucleotides to RNAs in this substrate pool. Characterization of the extension products by isolation and RNA-seq is ongoing to identify the most efficient substrates. Further characterization of selected RNAs by filter binding and activity assays will reveal the biochemical preferences for 3'-5'-polymerization by AcaTLP2 and DdiTLP4.
References:
Jayasinghe, M. I.; Patel, K. J.; Jackman, J. E. Thg1 family 3′–5′ RNA polymerases as tools for targeted RNA synthesis. RNA 2024, 30 (10), 1315–1327.
Chen, A. W.; Jayasinghe, M. I.; Chung, C. Z.; Rao, B. S.; Kenana, R.; Heinemann, I. U.; Jackman, J. E. The role of 3′ to 5′ reverse RNA polymerization in tRNA fidelity and repair. Genes 2019, 10 (3), 250.
Keywords: RNA substrates, 3-5-polymerization, TLPs
6. RNA-Targeted Drug Design: Impact of pH on Fragment-Based Ligand Discovery
Deborah K. Amesaki (Department of Chemistry & Biochemistry, Ohio University), Md Ismail Hossain, Emily A. Fairchild (Department of Chemistry & Biochemistry, Ohio University), Jennifer V. Hines (Department of Chemistry & Biochemistry, Ohio University)
Abstract:
Regulatory noncoding RNA plays an essential role in bacteria, highlighting the significance of RNA as a target for antibacterial development amidst growing concerns over antibiotic resistance. Effective targeting of the RNA requires a knowledge of all the factors that can affect RNA structure, including nucleobase protonation. Nucleobase protonation of A or C can occur when the local environment results in a pKa shift towards neutrality. This study investigates possible nucleobase protonation effects on the bacterial T box riboswitch by combining structure analysis, computational docking and experimental techniques. Based on analysis of crystallographic structure data and in silico protonation, we have identified nucleobases in the T box riboswitch that are consistent with protonation. To explore possible ligand-binding interactions and the effect of nucleobase protonation, we computationally docked fragment and small molecule libraries to the antiterminator and discriminator regions of the T box riboswitch. Analysis of docking data using Principal Component Analysis (PCA) and clustering methods results in 4 clusters. A set of fragments and compounds clustered with known inhibitors of the riboswitch and will be prioritized for ligand binding assays to test as potential hits.
Keywords: Regulatory noncoding RNA, in silico protonation, Fragment-Based Ligand Discovery
7. Identification and Characterization of Small Molecules Targeting Oncogenic Pre-miR-31
Grace Arhin (Biophysics Program, University of Michigan), Lily Haghpassand (Department of Chemistry, University of Michigan), Sarah C. Keane (Biophysics Program, Department of Chemistry, University of Michigan)
Abstract:
Abnormal levels of microRNA 31 (miR-31) is well documented in several pathological states including cancers, neurodegenerative and cardiovascular disorders. Therefore, the ability to modulate miR-31 levels in cells can have immense therapeutic potential. One promising strategy to achieve this goal involves the use of small molecule ligands to control the biogenesis of miR-31. Following transcription, primary (pri-) miR-31 is processed to form precursor (pre-) miR-31 and ultimately mature miR-31 by the enzymes Drosha/DGCR8 and Dicer/TRBP respectively. Mature miR-31 is a 22 nucleotide single stranded RNA which lacks structural complexity needed to provide binding pockets for small molecule targeting. In contrast, pre-miR-31, folds into a highly structured RNA with cavities capable of accommodating small molecule ligands. Targeting pre-miR-31 could inhibit the production of mature miR-31 by modulating its processing by the Dicer/TRBP enzyme complex.
Herein, we employed a structure-guided approach to screening for small molecule ligands to pre-miR-31. Our approach involves initial virtual screening of 1,493 drug-like small molecules from the National Cancer Institute’s DTP library against pre-miR-31’s ligandable cavities. Our virtual screening campaign identified 40 initial hits with binding energies at least two standard deviations more negative than the average binding energy of compounds in the library. We next experimentally validated ligand binding for these hits using saturation transfer difference (STD) NMR spectroscopy and identified nine compounds that bound pre-miR-31 in vitro. Heteronuclear single quantum coherence (HSQC) chemical shift mapping experiments revealed that two of these compounds bound pre-miR-31 at the Dicer/TRBP cleavage site, which may be linked to the inhibition of its maturation. We are currently evaluating if these compounds modulate the Dicer/TRBP cleavage of pre-miR-31 in vitro
Keywords: MicroRNAs, Virtual Screening, NMR
8. Investigating the interactome and functions of the UPF3 paralogs in Nonsense Mediated mRNA Decay
Rene Arvola (Department of Molecular Genetics, The Ohio State University), Kristen Nedza (Department of Molecular Genetics, The Ohio State University), Sharon Amacher (Department of Molecular Genetics, Department of Biological Chemistry and Pharmacology, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, The Ohio State University)
Abstract:
Nonsense Mediated mRNA Decay (NMD) degrades both aberrant transcripts containing Premature Termination Codons (PTCs) and “normal” transcripts with specific features that mimic PTCs. Regulation by NMD is pervasive and estimated to impact around 10% of the transcriptome. UPF3 is a key NMD factor that engages with the mRNA bound Exon Junction Complex (EJC) and the rest of the NMD machinery and aids in PTC recognition. In mammals, there are two paralogs of UPF3, UPF3A and UPF3B. In mice, Upf3A knockout is embryonic lethal [1], whereas Upf3B knockouts are viable, but exhibit neurodevelopmental defects [2]. Moreover, mutations in UPF3B are linked to various forms of intellectual disability in humans (summarized in [3]). We and others have found that both human paralogs function in NMD, to varying extents [4]. To better understand the functions of the human UPF3 paralogs in NMD, we performed immunoprecipitation followed by mass spectrometry from a human colorectal carcinoma cell line (HCT116) to identify proteins associated with UPF3A and UPF3B. In addition to EJC and NMD components, we identified transcriptional regulators to be among some of the most enriched factors in UPF3A and UPF3B immunoprecipitation. Ongoing work includes investigating the role of these factors in UPF3-mediated NMD. Furthermore, we have generated zebrafish mutants of upf3a and upf3b to investigate their roles in development. Taken together, this work will deepen our understanding of how mRNA decay processes such as NMD can become dysregulated in development and disease.
References:
[1] Shum EY, et al. The Antagonistic Gene Paralogs Upf3a and Upf3b Govern Nonsense-Mediated RNA Decay. Cell. 2016 Apr 7;165(2):382-95
[2] Huang L, et al. A Upf3b-mutant mouse model with behavioral and neurogenesis defects. Mol Psychiatry. 2018 Aug;23(8):1773-1786
[3] Deka B, Chandra P, Singh KK. Functional roles of human Up-frameshift suppressor 3 (UPF3) proteins: From nonsense-mediated mRNA decay to neurodevelopmental disorders. Biochimie. 2021 Jan;180:10-22
[4] Yi Z, et al. Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding. EMBO J. 2022 May 16;41(10):e109202
Keywords: Nonsense Mediated mRNA Decay (NMD), Exon Junction Complex, mRNA decay
9. Identification of segmentation clock oscillatory transcripts in the zebrafish presomitic mesoderm
Clare C. Austin (Molecular Genetics, The Ohio State University ), Thomas L. Gallagher, Kathryn G. Thompson, Sharon L. Amacher (Molecular Genetics, The Ohio State University )
Abstract:
The segmentation clock is an oscillatory gene network that regulates somitogenesis, the formation of embryonic somites (segments) that later develop into vertebrae and musculature. Proline-rich nuclear receptor cofactor 2 (Pnrc2), a mRNA decay adaptor, is necessary for rapid decay of oscillatory gene transcripts in the presomitic mesoderm (PSM) in zebrafish. To identify additional oscillatory genes, I am characterizing transcripts identified via RNA-seq that are upregulated in pnrc2 mutant embryos compared to wild-type siblings. Using qPCR, I have validated 15 genes that are both oscillatory in human and mouse PSM and upregulated in pnrc2 mutant embryos and observed that at least 11 show enhanced and expanded expression in pnrc2 mutant embryos at mid-segmentation by in situ hybridization. I have also identified putative novel oscillating transcripts. To discover if any function as segmentation clock regulators, I will use CRISPR-mediated knockdown to observe whether loss of function affects somite formation.
Keywords: oscillatory expression, segmentation
10. Generating ProXp-x Active Site Mutations to Determine the Mechanism of D-aa-tRNA Selection
Aouss Azzouz (Department of Chemistry, Earlham College), Gary Coker (Department of Chemistry, Earlham College), Jalyn Kelsey (Department of Chemistry, Earlham College), Amanda Vasconcellos (Department of Chemistry, Earlham College), Alana Weaver (Department of Chemistry, Earlham College), Alexandra Kuzmishin Nagy (Department of Chemistry, Earlham College)
Abstract:
Aminoacyl-tRNAs that carry D-amino acids, rather than the L-enantiomers used in protein synthesis, can deplete the pool of charged tRNA available to the cell, resulting in impaired bacterial growth and cytotoxicity.[1, 2] Bacteria that are sensitive to the presence of D-amino acids show reduced fitness and growth when cultured in media rich in D-enantiomers.[1, 3, 4] ProXp-x is a trans-editing enzyme found in soil bacteria that can hydrolyze D-amino acids from aminoacyl-tRNAs,[5] however the specific mechanism for D-amino acid editing has not been elucidated. It is also not understood if this mechanism protects the organism from D-amino acids. This research aims to understand how ProXp-x selects and binds mischarged D-aminoacyl-tRNA (D-aa-tRNA) species within the active site. Conserved active site residues of ProXp-x were selected for mutation; previous work implicated candidate residues in catalysis or substrate orientation.[5] Site-directed mutagenesis was used to mutate active site residues of wild type (WT) and K45A ProXp-x encoded on pET15b plasmids. The K45A mutation renders ProXp-x and other INS-like Superfamily members catalytically inactive, allowing for studies of substrate binding without catalysis to degrade the D-aa-tRNA. These amino acid residues are hypothesized to play a direct role in orienting the substrate within the active site, however their specific interactions are unknown. The use of active site point mutations provides a gateway to understanding how these residues are involved in the activity of ProXp-x. Future studies include using these ProXp-x variants in kinetic and binding assays to assess how the targeted residues contribute to substrate selection.
References:
1. Soutourina, O., et al., Formation of D-tyrosyl-tRNATyr accounts for the toxicity of D-tyrosine toward Escherichia coli. J Biol Chem, 2004. 279(41): p. 42560-5.
2. Englander, M.T., et al., The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center. PNAS, 2015. 112(19): p. 6038-43.
3. Soutourina, J., P. Plateau, and S. Blanquet, Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells. J Biol Chem, 2000. 275(42): p. 32535-42.
4. Soutourina, J., S. Blanquet, and P. Plateau, D-tyrosyl-tRNA(Tyr) metabolism in Saccharomyces cerevisiae. J Biol Chem, 2000. 275(16): p. 11626-30.
5. Bacusmo, J.M., et al., Quality control by trans-editing factor prevents global mistranslation of non-protein amino acid alpha-aminobutyrate. RNA Biol, 2017: p. 1-10.
Keywords: aminoacyl-tRNA, trans-editing, D-amino acids
11. Meiotic viability of telomere-free circular chromosomes in the eukaryote Saccharomyces cerevisiae
Rebecca L. Bailey (Department of biology and chemistry, Morehead State University), Devan W. Herald (Department of biology and chemistry, Morehead State University), Mark T. Wilson (Department of biology and chemistry, Morehead State University), Melissa A. Mefford (Department of biology and chemistry, Morehead State University)
Abstract:
Prokaryotes generally have a single circular chromosome, while eukaryotes have multiple linear chromosomes capped by specialized repetitive DNA sequences called telomeres. Telomeres shorten as we age because they cannot be completely replicated before the cell divides. To overcome this shortening, telomeres can be lengthened by the ribonucleoprotein enzyme telomerase. However, more than 85% of cancer cells upregulate telomerase, allowing the cancerous cells to divide uncontrollably. Since telomeres and telomerase contribute to aging and cancer, we set out to understand why eukaryotes have linear chromosomes with telomeres instead of circular chromosomes. To this end, we have used a genetic engineering strategy to circularize two individual chromosomes in the single-celled eukaryote Saccharomyces cerevisiae. In asexually dividing haploid cells, the circular chromosomes exhibit no obvious phenotypes. A current hypothesis posits that eukaryotes evolved linear chromosomes to permit meiosis and sexual reproduction. To test this hypothesis, we mated haploid yeast to form diploid cells with a single circular chromosome, as well as diploid cells with two copies of a circular chromosome. Upon starvation, the diploid cells will undergo meiosis to form four haploid cells, which can be dissected and tested for viability. We find that circular chromosomes decrease the number of viable spores by >30%, consistent with the hypothesis that linear chromosomes evolved to allow sexual reproduction. In the future, we plan to characterize the meiotic defects caused by the presence of a circular chromosome to shed further light on why linear chromosomes evolved in eukaryotes.
Keywords: telomere, chromosome, meiosis
12. Temporally controlled expression of a splicing factor in single cells coordinates the metabolic and proliferative activities of regenerating livers
Nick Baker (Department of Biochemistry, University of Illinois, Urbana-Champaign), Sushant Bangru (Department of Biochemistry, University of Illinois, Urbana-Champaign), Ullas V. Chembazhi (Department of Biochemistry, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, University of Illinois, Urbana-Champaign)
Abstract not available online - please check the booklet.
13. Protection of Long Telomeric Overhangs by Shelterin
Hamza Balci (Physics Department, Kent State University), Sajad Shiekh (Physics Department, Kent State University), Amanda Jack (University of California, Berkeley), Sineth Kodikara (Physics Department, Kent State University), Janan Alfehaid (Physics Department, Kent State University), Ahmet Yildiz (University of California, Berkeley)
Abstract:
We present single molecule fluorescence (including FRET, FRET-PAINT, and PIFE) studies that probe the folding patterns and accessibilities of telomeric overhangs of physiologically relevant lengths (up to 28 GGGTTA repeats). In particular, we investigate the impact of shelterin complex and POT1 on the accessibility and protection of these otherwise vulnerable genomic regions. In addition to length-dependent periodic accessibility patterns, we present the impact of POT1 and Shelterin on these accessibility patterns. On average POT1 reduces the telomeric accessibility by ~2.5-fold and Shelterin by ~5-fold. Our studies also illustrate the junction between single and double stranded telomeres to be highly accessible in the absence of shelterin. We present recent work in which we focused on this junction region to understand the minimum requirements to attain effective protection by Shelterin or POT1, and the impact of 5-phosphorylation and sequence variations on protection of this region.
References:
1- Sajad Shiekh, …, Ahmet Yildiz, Hamza Balci, "Protection of the Telomeric Junction by the Shelterin Complex", JACS (2024), DOI:10.1021/jacs.4c08649
2- Sineth G. Kodikara, …, Hamza Balci, "Detecting Secondary Structure Formation with FRET-PAINT", RSC-Analyst (2023) DOI: 10.1039/d3an01118f
3- Sajad Shiekh, Sineth G. Kodikara, Hamza Balci, "Structure, Topology, and Stability of Multiple G-quadruplexes in Long Telomeric Overhangs" Journal of Molecular Biology (2023)
DOI: 10.1016/j.jmb.2023.168205
4- Sajad Shiekh, …, Ahmet Yildiz, Hamza Balci , "Shelterin reduces the accessibility of telomeric overhangs", Nuc. Acids Res. (2022). DOI: 10.1093/nar/gkac1176
5- Sajad Shiekh, …, Hamza Balci, "Emerging Accessibility Patterns in Long Telomeric Overhangs", PNAS (2022), DOI: 10.1073/pnas.2202317119
Keywords: Telomere, G-quadruplex, Shelterin
14. Enzymatic Characterization of Full-length Glutamyl-Prolyl-tRNA Synthetase (EPRS) and Disease-associated Mutants
Morgan Bauer (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, USA. *Bauer.715buckeyemail.osu.edu ), Brianna Young (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, USA.), Sabat Gonzalez-Serrano (William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, USA ), David Wood (William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, USA ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, USA. )
Abstract:
Glutamyl-prolyl-tRNA synthetase (EPRS) catalyzes the attachment of proline and glutamic acid onto their respective tRNAs. Out of all the aminoacyl-tRNA synthetases, EPRS is the only bifunctional synthetase, with two catalytic cores connected by a linker domain. EPRS resulted from a fusion event between stand-alone glutamyl- and prolyl-tRNA synthetases (ERS and PRS, respectively) during evolution around the creation of metazoans, and remained encoded in all animals. Fox and co-workers proposed that this fused protein is metabolically advantageous for organisms by keeping the proline and glutamic acid pool at cellular homeostasis. Whether EPRS catalytic activity differs relative to the stand-alone synthetases is unknown. Previous work on EPRS has shown that it performs many non-canonical functions, such as translational repression of inflammatory mRNAs, increasing the intake of long-chain fatty acids and repressing viral replication. However, the full-length enzyme has never been characterized in vitro. We successfully purified full-length EPRS from mammalian cells for the first time and characterized its enzymatic activity. Compared to ERS and PRS domains alone, the fused catalytic domains of EPRS are more efficient at aminoacylating tRNA. These preliminary findings reveal a new advantage of ERS and PRS fusion. Mutations in EPRS are linked to patients with hypomyelinating leukodystrophy and other neurological disorders. Novel bi-allelic point mutations from five affected individuals have recently been identified. Ongoing studies are aimed at characterizing disease-associated mutations in both full-length EPRS and separate ERS and PRS domains. Our long-term goal is to identify the disease mechanisms underlying EPRS-related disorders and uncover future therapeutic strategies.
Keywords: tRNA
15. tRNA synthetase activity is required for stress-induced RNP granule assembly
Max Baymiller (Department of Human Genetics, University of Michigan), Stephanie L. Moon (Department of Human Genetics, University of Michigan)
Abstract:
During the cellular response to stress, translation initiation is suppressed via phosphorylation of eIF2α and non-translating mRNAs condense into RNP granules including stress granules and P-bodies. The stress response is active in many diseases including neuropathies caused by mutations in tRNA synthetases, where preventing the stress response is therapeutic. We hypothesized that tRNA synthetase activity is required for stress-induced RNP granule formation by facilitating ribosome runoff. We found that despite P-eIF2α induction by tRNA synthetase inhibitors, stress granule and P-body formation was inhibited. Formation of these RNP granules was rescued upon mRNA release from ribosomes by puromycin. tRNA synthetase activity was also required for arsenite-induced stress granules, further indicating loss of tRNA charging traps mRNAs within polysomes during stress. We observed ribosome-associated quality control factor ZNF598 was activated upon tRNA synthetase inhibition, an indication of ribosome collisions. Yet, ZNF598 depletion did not increase P-eIF2α levels or further decrease stress granules, suggesting ribosome collisions are a minor contributor to tRNA charging stress. Together, these results show that tRNA synthetases are critical for mRNA to cycle from the translating pool into stress-induced RNP granules. Defects in translation elongation that occur in other stresses such as UV and amino acid starvation may also impair RNP granule assembly in this manner. Identifying molecular mechanisms of ribosome rescue that enable RNP granule formation could inform therapeutics for diseases associated with tRNA synthetases or tRNA metabolism.
Keywords: stress granule, tRNA
16. Investigating functional transcription start site switching in breast cancer
Dana Beseiso (Biological Chemistry, University of Michigan ), Dr. Rachel Niederer (Biological Chemistry, University of Michigan )
Abstract:
Alternative transcription start sites (TSS) direct the majority of transcript diversity across tissues. Alternative TSSs can give rise to transcripts with 5′ Untranslated Region (5′ UTR) isoforms. Since 5′ UTRs often harbor regulatory elements that alter ribosome recruitment to mRNAs, these isoforms may drive differential translation facilitating translational reprogramming, a hallmark of cancer. Using luciferase reporters, we show that annotated 5′ UTR isoforms of the metastasis-associated proteins, NODAL, NANOG, and SNAIL, are differentially translated in human cancer extracts and identify novel translational control motifs. To map global 5′ UTR isoforms in a disease context, we utilize a series of breast cancer cell lines representing increasingly aggressive cancer states and perform ReCappable-seq. We anticipate that widespread TSS switching throughout metastatic progression will produce distinct 5′ UTR isoforms that alter translational output via enhancer or repressor motifs. Identifying novel translational control elements in 5′ UTRs will elucidate the 5′ UTR regulatory code and inform novel anticancer therapeutic strategies.
Keywords: 5 UTRs, Translational Control, TSS Switching
17. Epigenetic And LncRNA Regulation In Vascular Cells Under Cancer Therapy-Induced Stress
Chayan Bhattacharya (Center for Molecular Medicine and Genetics Wayne State University, Detroit, MI), Miguel Nieto-Hernandez (Center for Molecular Medicine and Genetics,Wayne State University, Detroit, MI), Sudarshan Anand (Knight Cancer Institute, Oregon Health and Science University, Portland, OR.), Cristina Espinosa-Diez (Center for Molecular Medicine and Genetics,Wayne State University, Detroit, MI)
Abstract:
Genotoxic stress from cancer treatments disrupts vascular homeostasis, and can lead to lasting changes in epigenetic modifications, such as DNA methylation and long-non-coding RNAs (lncRNAs), which influence vascular cell fate. We hypothesize that cancer therapy leads to chronic vascular dysfunction due to this epigenetic reprogramming, which may affect the expression of critical vascular health regulators like lncRNAs. To assess the differential DNA methylation under genotoxic stress, we subjected human vascular endothelial cells (HUVECs) to ionizing radiation, followed by reduced representation bisulfite sequencing (RRBS). Our data revealed differentially hypomethylated and hypermethylated gene promoters, including 5% from lncRNA genes. To assess the role of these lncRNAs in vascular function, we employed an in-silico pipeline to identify novel lncRNA candidates, considering factors such as the number of isoforms, their coding potential, conservation across mammals, neighboring genomic location, and basal expression in blood vessels and vascularized tissues. From an initial pool of 52 lncRNAs potentially altered by DNA methylation in response to ionizing radiation, we identified 5 lncRNA candidates that met our selection criteria. To validate the potential changes in expression driven by DNA methylation alterations, we performed RT-qPCR for these lncRNA candidates in endothelial cells treated with doxorubicin, a genotoxic stressor. We identified three lncRNA candidates— DIO3OS, LINC00354, and RP5-998H6—in response to genotoxic stress that changed expression in response to doxorubicin, suggesting that they can be integral components of a broader regulatory network that helps endothelial cells adapt to and survive DNA damage. Our findings suggest that cancer therapies induce significant epigenetic reprogramming in vascular endothelial cells, particularly affecting lncRNAs. Future experiments will further validate the functional roles of these lncRNA candidates through gain and loss of function studies.
Keywords: LncRNAs, Vascular Biology, genotoxic stress
18. Engineered Extracellular Vesicles for Combinatorial TNBC Therapy: SR-SIM-Guided Design Achieves Substantial Drug Dosage Reduction
Abhjeet Bhullar (b.Interdisciplinary Biophysics Graduate Program, The Ohio State University, Columbus, Ohio 43210, United States.), Kai Jin (a.Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy and Comprehensive Cancer Center. The Ohio State University, Columbus, Ohio 43210, USA;), Peixuan Guo (a.Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy and Comprehensive Cancer Center. The Ohio State University, Columbus, Ohio 43210, USA;), Daniel W. Binzel (a.Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy and Comprehensive Cancer Center. The Ohio State University, Columbus, Ohio 43210, USA;), Dan Shu (a.Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy and Comprehensive Cancer Center. The Ohio State University, Columbus, Ohio 43210, USA;)
Abstract:
Triple negative breast cancer is an aggressive subtype of breast cancer that has no therapeutic targets, relies on chemotherapeutics for treatment, and is in dire need of novel therapeutic approaches for improved patient outcomes. Extracellular vesicles serve as intercellular communicators and have been proposed as ideal drug delivery vehicles. Here, extracellular vesicles were engineered with RNA nanotechnology to develop highly effective tumor inhibitors. Utilizing Super Resolved-Structured Illumination Microscopy, extracellular vesicles were optimized for precise Survivin siRNA conjugated to chemotherapeutics loading and CD44 aptamer ligand decoration, thereby enhancing specificity towards triple negative breast cancer cells. Conventional treatments typically employ chemotherapy drugs Gemcitabine and Paclitaxel at dosages on the order of mg/kg respectively, per injection (IV) in mice. In contrast, engineered extracellular vesicles encapsulating these drugs saw functional tumor growth inhibition at significantly reduced concentrations: 2.2 µg/kg for Gemcitabine or 5.6 µg/kg for Paclitaxel, in combination with 21.5 µg/kg Survivin-siRNA in mice. This approach resulted in a substantial decrease of chemotherapeutic dose required, by orders of magnitude, compared to standard regimens. In vivo and in vitro evaluations in a triple negative breast cancer orthotopic xenograft mouse model demonstrated the efficacy of this reduced dosage strategy, indicating potential for decreased chemotherapy-associated toxicity.
Keywords: Extracellular Vesicles, RNA nanotechnology, Cancer therapy
19. Sterical configuration beyond conformation to RNA-protein interaction for executing functions in biological systems
Aliza Klein (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmacology; College of Medicine and James Comprehensive Cancer Center. The Ohio State University), Daniel W Binzel (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmacology; College of Medicine and James Comprehensive Cancer Center. The Ohio State University), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmacology; College of Medicine and James Comprehensive Cancer Center. The Ohio State University)
Abstract:
RNA has been known to regulate cellular functions in part by interacting with protein through mechanisms of electrostatic interaction, conformation capture, and induced fit. Most of the human genome that does not code for proteins codes for non-coding RNA. One non-coding RNA from bacterial virus phi29 has been shown to form a hexameric ring that gears the phi29 DNA packaging motor by interacting with the motor connector protein. Here we report that configuration instead of conformation plays a key role in governing the RNA/protein interaction to make the packing motor work. Configuration is the spatial arrangement of molecules that cannot be easily interconverted, while conformation is the arrangement of molecules that can easily interconvert. The connector is promiscuous toward any form of RNA and DNA, whether single or double stranded, sequence-specific or nonspecific. It was found that the interaction of the connector with these nucleotides resulted in the formation of a heptameric rosette rather than a hexameric ring of pRNA. Since there is no conformational change in the connector before and after rosette assembly, the rosette formation depends on spatial configuration. However, pRNA interaction with the connector while the procapsid protein shell is present results in the formation of a functional hexameric RNA ring that gears DNA into the procapsid. This is because the capsid’s configuration enables the 5'-3' paired domain of the pRNA to extend away from the capsid protein, enabling intermolecular hand-in-hand interaction between the pRNA molecules. These findings highlight the importance of spatial configuration rather than conformation in the RNA protein interaction process. It is expected that configuration might play an essential role in the assembly of RNA protein machinery in human or animal cells.
Keywords: Steric Configuration, RNA-protein interactions
20. Engineering Exosomes Through Versatile Oligonucleotide Tethers
Christopher G. Blum (Chemistry Department Carnegie Mellon University), Phil G. Campbell (Biomedical Engineering Department Carnegie Mellon University), Subha R. Das (Chemistry Department Carnegie Mellon University)
Abstract:
Exosomes’ function as biological couriers between cells makes them an ideal vehicle for drug delivery. Engineering exosomes requires minimizing disruptions to their desirable innate characteristics while introducing desired functions. Recently, we have demonstrated the use of oligonucleotide tethers for exosome engineering. The oligonucleotide tethers consist of an anchor strand and display strand. The double stranded design reduces the length and cost of each individual strand, while also allowing for a modular approach to functionalization. Here, we expand this oligonucleotide tether strategy to incorporate aptamers. We have designed anchor and display strands with aptamers to engineer exosomes for a variety of applications. Display strands can use DNA or RNA aptamers to target specific surface proteins on cells for enhanced targeting and uptake. Additionally, anchor strand designs allow for different exosome populations to be selectively functionalized. The development of these oligonucleotide tether systems allows for a versatile method of engineering exosomes for both diagnostic and therapeutic delivery applications.
Keywords: Exosome, Aptamer
21. Magnesium Ions Increase, but Potassium Ions Decrease, the Thermal Stability of the MALAT1 RNA Triple Helix
Koppany T. Bodor (Department of Chemistry and Biochemistry, University of Notre Dame), Mika J. Schievelbein (Department of Chemistry and Biochemistry, University of Notre Dame), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)
Abstract:
Accumulation of human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a nuclear-localized long noncoding RNA, is associated with cancer. A unique feature of MALAT1 is a 3′-terminal triple helix, which prevents degradation. The triple helix has one confirmed binding-partner: methyltransferase-like protein 16 (METTL16), an mRNA methyltransferase that binds but does not modify the triple helix. Previous studies have suggested that METTL16 preferentially binds to a less thermostable form of the MALAT1 triple helix and that the thermal stability of the MALAT1 triple helix depends on salt concentrations. Salt concentrations are generally lower in cancer, which may favor a less stable triple helix that stabilizes the MALAT1 triple helix•METTL16 complex. This study tests how salt conditions modulate triple helix thermal stability and the MALAT1 triple helix•METTL16 binding interaction. Here, low (0.1 mM MgCl2 and 0-150 mM KCl), middle-low (0.5 mM MgCl2 and 0-150 mM KCl), middle-high (1 mM MgCl2 and 0-150 mM KCl), and high (5 mM MgCl2 and 0-150 mM KCl) ionic conditions were tested at pH 7 and pH 5. UV thermal denaturation assays showed up to three melting transitions, which represent a structured region of unknown identity (50-60°C), Hoogsteen interactions specific to the triple helix (60-70°C), and Watson-Crick interactions (70-80 °C). For the Hoogsteen melting transition, an increase in MgCl2 concentration correlates with increased stability, while KCl reduces stability. A lack of KCl hyper stabilizes the triple helical Hoogsteen interactions, as Hoogsteen and Watson-Crick denaturation peaks are indistinguishable. This result contrasts with the Watson-Crick melting transition, where the thermal stability positively correlates to increasing salt, whether it be monovalent or divalent. Salt-dependent trends at pH 5 are similar. However, at pH 5, the Hoogsteen melting transition is more stable (60-80 °C) due to increased cytosine protonation at the N3 position (pKa = 4.3). These findings will be correlated with electrophoretic mobility shift assays of MALAT1 triple helix•METTL16 to determine the relationship between triple helix stability and binding affinity. These results will show how cancer cell conditions may affect the MALAT1 triple helix•METTL16 complex.
Keywords: RNA Triple Helix
22. Interaction of a hexameric RNA with a dodecameric protein complex to produce a stable structure without induced-fit but with conformational capture
Margaret Bohmer (College of Pharmacy, Division of Pharmaceutics and Pharmacology; Center for RNA Biology. The Ohio State University), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmacology; College of Medicine and James CCC; Center for RNA Biology. The Ohio State University)
Abstract:
DsDNA translocation is ubiquitous in living systems for diverse biological functions such as cell mitosis, bacterial binary fission, DNA replication, genome trafficking, DNA repair, homologous recombination, viral genome packaging, and nuclear transport. A common sequential revolving mechanism without rotation using the asymmetrical ATPases has been used by nature to avoid coiling and tangling while translocating the lengthy dsDNA genome, as reported in bacteriophages, human viruses, and bacteria. The DNA packaging motor of the phi29 is a prototype model in dsDNA translocation. The motor uses a six-subunit pRNA ring to gear the motion for dsDNA translocation. How can six copies of pRNA associate with the 12-submit motor protein? If such mismatch is due to the quasi-equilibrium to enforce the RNA-protein interaction, then what is the novel mechanism for such interaction? In this study, we constructed chimeric pRNA with doubled size and revealed that the stoichiometry of the biologically active pRNA on the motor is a common multiple of 2 and 3, which is 6. We evaluate the data from combinational approaches including crosslinking, complementary modification, AFM, and computation modeling to assess the interaction of the pRNA structure. Our new structural analysis reveals that the phi29 pRNA hexameric ring contains 12 protruding regions, each pRNA contributing two regions, to bind to the three positively charged amino acids (Arg-Lys-Arg) of each connector subunit extending from the N-terminal of the motor connector.
Keywords: DNA packaging, RNA structure, ATPase
23. Synonymous codon substitutions enhance transcription and translation of an upstream gene in E. coli
Christopher D. Bonar (University of Notre Dame), Jacob D. Diehl (University of Notre Dame), Anabel Rodriguez (University of Notre Dame), Patricia L. Clark (University of Notre Dame)
Abstract:
Synonymous codon substitutions are genetic mutations that do not change the encoded protein sequence. Although often assumed to be phenotypically silent, synonymous codon substitutions can cause diverse effects on mRNA transcription and protein translation. Yet, the full range of effects of synonymous codon substitutions on the production and maintenance of a functional proteome remains poorly understood. We are using the well characterized Tet ON/Tet OFF divergent expression system to broadly test the effects of synonymous codon substitutions on gene expression. In Tet ON/Tet OFF, the expression level of TetR, encoded upstream of a gene of interest (GOI), regulates expression of both the GOI and tetR, by binding to and repressing a divergent promoter between the GOI and tetR. We found that synonymous mutations within a GOI can enhance transcription from a transcriptional start site (TSS) identified within the GOI. The effects of specific codon substitutions on transcription from this intragenic TSS are poorly predicted by existing prediction algorithms. Surprisingly, we found that transcription from this TSS bypasses canonical repression of tetR transcription. As a result, TetR protein accumulation increases, repressing GOI expression. This mechanism of transcription enhancement was corroborated by inserting an antisense terminator downstream of the intragenic TSS, which led to the expected reduction in tetR mRNA level. Even more surprisingly, although the mRNA produced from the intragenic TSS constitutes a minor fraction of tetR-encoding RNA, its level is predictive of TetR abundance, whereas total tetR mRNA level is not. We are currently testing whether this unexpected correlation between TetR and the minor fraction of tetR mRNA originating from the intragenic TSS is due to enhanced translational efficiency of the longer mRNA, or whether the longer RNA serves as a regulator of translational efficiency of canonical tetR mRNA. Collectively, these results demonstrate that synonymous codon substitutions can enhance intragenic RNA transcription to regulate upstream gene expression.
References:
Rodriguez, A., Diehl, J.D., Wright, G.S., Bonar, C.D., Lundgren, T.J., Moss, M.J., Li, J., Milenkovic, T., Huber, P.W., Champion, M.M., Emrich, S.J., and Clark, P.L. (2024). Synonymous codon substitutions modulate transcription and translation of a divergent upstream gene by modulating antisense RNA production. Proc. Natl. Acad. Sci. U. S. A. 121, e2405510121, 1–10.
Keywords: Synonymous codon substitutions, Intragenic transcriptional start site, Gene expression
24. Understanding how ncRNA Impact the NRF2 Antioxidant Response in Lung Cancer
Jae A. Bucknor (Cellular and Molecular Biology, University of Michigan), Brittany M. Bowman (Biological Chemistry, University of Michigan), Chase A. Weidmann (Biological Chemistry, University of Michigan)
Abstract:
Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. More than 25% of cases of NSCLC display elevated levels of NRF2 protein, a transcription factor and master regulator of the cellular antioxidant response pathway. The accumulation of NRF2 results in increased activation of cytoprotective genes, that are responsible for increasing detoxification, efflux, and resistance to oxidative stressors. Thus, elevated NRF2 activity contributes to increased NSCLC therapy resistance and enables cancer progression. Many noncoding RNAs of unknown function are also expressed when NRF2 is activated, and these noncoding RNAs (ncRNAs) could represent novel ways to target the NRF2 pathway therapeutically. Depletion of ncRNAs that support NRF2 activity could sensitize NSCLC to existing therapies. I confirmed the expression of dozens of ncRNAs in multiple NSCLC cell lines with activated NRF2 by RNA sequencing. I hypothesize that NRF2 activated ncRNA may contribute to NSCLC by supporting NRF2 stability, NRF2 transcriptional activity, and or antioxidant activity. I identified several high confidence ncRNA candidates (activated in all or almost all cell lines tested), including previously identified ncRNA LUCAT1 and NMRAL2P and uncharacterized ncRNAs LOC102723825 and LOC105374811. I aim to understand the mechanism of NRF2 pathway activity by these ncRNA. To do this, I will characterize RNA secondary structures and protein-interaction networks through live cell chemical probing of NSCLC lines (SHAPE-MaP and RNP-MaP). After identifying these motifs, I will identify the critical RNA motifs that interact with 1) KEAP1 to support NRF2 stabilization, 2) DNA to support NRF2 transcriptional activity, or 3) NRF2 effector proteins to support antioxidant activity. Then, I will evaluate these ncRNA’s functional contribution to NRF2 signaling and on NSCLC proliferation and migration. The ultimate goal of this work is to evaluate NRF2 activated ncRNA’s potential as NSCLC therapeutic target.
References:
1. Siegel et al., CA Cancer J Clin. 2021.
2. GBD 2019 Cancer Risk Factors Collaborators, Lancet. 2022.
3. Jason Hoffman, Healthline. 2022.
4. Campbell et al., Nat Genet. 2016.
5. He et al., Carcinogenesis. 2020.
6. Abdalkader et al., Front Neurosci. 2018.
7. Wu et al., Toxicol Sci. 2011.
8. Venugopal et al., Proc Natl Acad Sci U S A. 1996.
9. Kobayashi et al., Nat Commun. 2016.
10. Singh et al., PLoS Med. 2006.
11. Siegfried et al., Nat Methods. 2014.
12. Weidmann et al., Nat Biotechnol. 2021.
13. Dodel et al., Nat Methods. 2024.
Keywords: ncRNA, lung cancer, antioxidant
25. Determination of the effect on translation of 2’-O-methylation at positions 32 and 34 in eukaryotic tRNA
Anh Bui (Chemistry and Biochemistry, Northern Kentucky University), Dani Maki (Chemistry and Biochemistry, Northern Kentucky University), Holly Funk (Chemistry and Biochemistry, Northern Kentucky University), Linh T Le (Chemistry and Biochemistry, Northern Kentucky University), Michael P. Guy (Chemistry and Biochemistry, Northern Kentucky University )
Abstract:
Post-transcriptional modification of tRNA is vital for efficient protein translation, with many human diseases associated with modification defects. Mutations in the widely conserved eukaryotic methyltransferase TRM7 causes non-syndromic X-linked intellectual disability in humans and slow growth in yeast. Yeast Trm7 interacts with Trm732 and Trm734 to perform 2’-O-methylation at C32 and G34, respectively, on tRNAPhe, and corresponding proteins are responsible for these modifications on human tRNA. In budding yeast, lack of 2’-O-methylation at both C32 and G34 causes growth defects, but if only one modification is missing, cells are still healthy. Consequently, the individual role of Cm32 and Gm34 is not clear. Furthermore, because both UUU and UUC code for Phe, it is not clear how each modification affects translation of each codon. Bidirectional expression of red and green fluorescent proteins (RFP and GFP) via the RNA-ID reporter system in yeast allows for the quantitative analysis of the effect of tRNA modifications on the translation of codons in cells. We have placed additional UUU and UUC codons at the start of the GFP, allowing us to test the effect of Cm32 and Gm34 on translation of these codons by determining the GFP/RFP ratio of reporters in trm734∆ and trm732∆ mutants, and then comparing those values to the reporters in wildtype strains. We are also determining the effect of UGG and UUA codons on GFP expression in these mutants, because these codons are decoded by other tRNAs that receive Nm32 and Nm34 modifications. Understanding how Trm7-dependent tRNA modifications affect translation in yeast will provide insights into how these modifications may affect translation in other eukaryotes, including humans.
Keywords: tRNA, yeast, modification
26. Live Cell Imaging of Cell Cycle Stages And Its Effect on Stress Response
Erika Calle Urgiles (University of Michigan Program in Biomedical Sciences), Ben Dodd (University of Michigan Department of Human Genetics and Center for RNA Biomedicine), Stephanie Moon (University of Michigan Department of Human Genetics and Center for RNA Biomedicine)
Abstract:
The integrated stress response (ISR) promotes cell survival of stress in
eukaryotic cells through transcriptional and translational adaptation. Activation of the
ISR by stress-sensing kinases allows for the selective translation of stress-induced
genes and repression of global translation(2). Translationally repressed mRNAs
condense into stress granules, which may sequester RNAs, small ribosomal subunits,
and pre-initiation complexes from degradation and translation machinery. Further,
mutations in stress granule RNA binding proteins are associated with amyotrophic
lateral sclerosis and frontotemporal dementia (4). Stress granules and the ISR may be
especially important in post-mitotic cells like neurons which are no longer able to go
through mitosis or regenerate. Also, dysregulation of the ISR has been associated with
cancer cells, which have a perturbed cell cycle and exhibit aberrant cell survival (1).
Therefore, investigating whether and how the cell cycle plays a role in stress response
will be important in gaining insight into neurodegenerative disease and cancer. Our
hypothesis is that the cell cycle stage affects a cell’s stress response. To test this, we
will track stress granule formation, induced upon arsenite stress, throughout each cell
cycle stage over a 24 hour live cell imaging period. We have expressed human bone
osteosarcoma epithelial cells with mScarlet-G3BP1, which marks the time at which
stress granules form and DHB-mVenus marks cell cycle change. The DHB-mVenus
reporter alters between a cytoplasmic localization during dephosphorylation by
cyclin-dependent kinase 2 (CDK2) to a nuclear localization during phosphorylation in
newly divided cells (3). CDK inhibitors, Palbociclib and RO-3306, offer another avenue
to track cell cycle change since they trap and synchronize cells in the G1 and G2/M
border cycle stage respectively. Through this research, we can learn if the cell cycle is
interrelated to a cell’s integrated stress response when forced into stressful conditions.
Our findings can be applied to the role that the cell cycle and cell differentiation plays in
the development of cancer and neurodegenerative disease.
References:
1. Costa-Mattioli, M., & Walter, P. (2020). The integrated stress response: From
mechanism to disease. Science (New York, N.Y.), 368(6489).
2. English, A. M., Green, K. M., & Moon, S. L. (2022). A (dis)integrated stress response:
Genetic diseases of eIF2α regulators. Wiley interdisciplinary reviews. RNA,
13(3), e1689.
3. Spencer, S. L., Cappell, S. D., Tsai, F. C., Overton, K. W., Wang, C. L., & Meyer, T.
(2013). The proliferation-quiescence decision is controlled by a bifurcation in
CDK2 activity at mitotic exit. Cell, 155(2), 369–383.
4. Vance, C., Rogelj, B., Hortobágyi, T., De Vos, K. J., Nishimura, A. L., Sreedharan, J.,
… Shaw, C. E. (2009). Mutations in FUS, an RNA processing protein, cause
familial amyotrophic lateral sclerosis type 6. Science (New York, N.Y.),
323(5918), 1208–1211.
Keywords: integrated stress response, translation, neurodegenerative
27. Nucleotide-resolution Mapping of RNA N6-Methyladenosine (m6A) in Malaria Pathogen Plasmodium falciparum and Determining m6A’s Role in Host–Pathogen Interactions
Carli Camporeale (Department of Biological Sciences, University of North Carolina, Charlotte, NC, USA, United States of America), Cassie Catacalos, Kaitlin Klotz (Department of Biological Sciences, University of North Carolina, Charlotte, NC, USA, United States of America), Doaa Hasan, Sahiti Shomalraju, Sarath Chandra Janga (Department of BioHealth Informatics, Luddy School of Informatics, Computing and Engineering, Indiana University Indianapolis IUI, Indianapolis, Indiana, United States of America), Matthew Tegowski, Kate Meyer (Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina, United States of America), Arthur Hunt (Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA, United States of America), Kausik Chakrabarti (Department of Biological Sciences, University of North Carolina, Charlotte, NC, USA, United States of America)
Abstract:
The expression of a precise mRNA transcriptome is crucial for establishing cell function. To this end, the N6-methyladenosine (m6A) modification is emerging as the most prevalent and abundant chemical modification in eukaryotic mRNAs, which plays important roles in various biological processes and can regulate all aspects of mRNA metabolism, ranging from 5′ end-capping, polyadenylation, pre-mRNA splicing and translation. Thus, this chemical modification is of profound importance to eukaryotic gene expression regulation. Malaria, caused by apicomplexan parasite Plasmodium falciparum, is the deadliest vector-borne disease in the world, claiming more than 600,000 lives each year. m6A modifications have been implicated in malaria gene regulation. However, m6A’s mechanistic role in mRNA metabolism in this clinically important protist remains unknown. The survival of P. falciparum in the human host is often threatened by two important stressors – febrile temperature resulting from human innate immune response and antimalarial drugs, which induce oxidative stress. These stress conditions significantly decrease the sensitivity of the parasite to the drug as a survival response, but the underlying molecular mechanisms remain poorly understood. Since m6A’s role as a newly emerging layer of genetic control in stress responses is becoming more and more evident, here we seek to determine the extent of m6A modifications in the malaria epitranscriptome and investigate the heat and drug-induced stress response on m6A dynamicity in the malaria blood stage development in the human host. To globally map m6A modifications in P. falciparum, we used DART-seq technology to directly determine m6A sites at a single-nucleotide resolution. Additionally, we have developed an m6A mapping and detection pipeline using Oxford Nanopore-based direct RNA sequencing. To gain insight into m6A’s functional significance, we investigated m6A epitranscriptomic changes in response to a variety of stresses. These studies should shed light on the extent and dynamicity of this intrinsic RNA modification in malaria and determine m6A’s potential role in stress responses in the human host.
Keywords: m6A, RNA modifications
28. Investigating the mechanisms of cell-type-specific alternative polyadenylation
Anthony Caputo (Case Western Genetics and Genome Sciences), Ashleigh Schaffer (Case Western Genetics and Genome Sciences), Jingyi Liu (Case Western Genetics and Genome Sciences), Eric Wagner (Dept. of Biochemistry and Biophysics, Univ. of Rochester Sch. of Med. and Dentistry)
Abstract:
A necessary step for the functional maturity of nearly all eukaryotic pre-mRNAs is 3’ cleavage and polyadenylation. Interestingly, over 70% of human transcripts have more than one site at which polyadenylation can occur, a phenomenon known as alternative polyadenylation (APA). APA produces mRNA isoforms that differ in stability, subcellular localization, translational efficiency, or even the encoded protein sequence. Poly(a) sites (PASs) are selected and processed by dynamic interactions of proteins known as the cleavage and polyadenylation (CPA) complex. Mutations in these proteins cause neurological disorders and are associated with poor cancer prognosis. Because PASs are present throughout gene bodies, aberrant APA caused by mutations of the CPA complex is thought to produce functionally diverse outcomes at the cellular level. Indeed, specialized RNA sequencing technologies have revealed dramatic alterations in PAS usage and significant impacts on cell viability when CPA complex proteins are perturbed.
Interestingly, PAS patterns across identical gene sets vary widely by cell type. Exploring the link between cell-type specific APA and CPA complex activity, we have identified distinct, cell-type-specific responses to genetic knockout of the canonical CPA constituent, CLP1. CLP1 is dispensable for human embryonic stem cell (hESC) viability but is essential for the viability of hESC-derived motor neurons. A potential explanation for these observations is that the composition of the CPA complex varies between cell types. To test this hypothesis, we will first identify differences in CPA complex composition between three isogenic cell types: hESCs, motor neurons, and skeletal muscle. Next, we will determine whether APA differences observed between cell types are due to variation in CPA complex composition. To supplement these findings, we will also explore the connection between selective vulnerability of neurons to CLP1 depletion and the composition of the CPA complex across different cell types.
Keywords: Alternative polyadenylation, stem cells, differentiation
29. Predicting the subcellular localization of RNA-binding proteins
Defne Ceyhan (Memorial Sloan Kettering Cancer Center), Jessica Lin (Memorial Sloan Kettering Cancer Center), Cyrus Tam (Memorial Sloan Kettering Cancer Center), Ilyes Baali (Memorial Sloan Kettering Cancer Center), Kaitlin Laverty (Memorial Sloan Kettering Cancer Center), Quaid Morris (Memorial Sloan Kettering Cancer Center)
Abstract:
RNA-binding proteins (RBPs) bind specific RNA sequences or structures to regulate various post-transcriptional processes, including splicing, nuclear export, localization, translation, and RNA degradation. The processes that an RBP is involved in are closely related to its subcellular localization — whether it binds to RNA in nuclear or cytoplasmic compartments. A number of experimental methods have been developed to assess RBP function. In particular, enhanced crosslinking and immunoprecipitation (eCLIP) assays have allowed for identification of an RBP’s RNA-binding sites [1]. Peakhood, a recently developed tool, has allowed for more accurate eCLIP analysis, enabling the determination of the RNA context in which the RBP binds, specifically whether the bound RNA is spliced or unspliced [2]. Using Peakhood, we derived the proportions of exonic sites, spliced-transcript-context sites, and exon border sites from 244 eCLIP experiment datasets representing 168 unique RBPs and two cell lines [3]. We then determined the proportion of eCLIP peak sites overlapping with a 3’UTR. In addition to these site statistics, we explored nanopore RNA-seq data from both chromatin and cytoplasmic compartments by mapping isoform-level quantifications against Peakhood-predicted RBP-bound transcripts [4]. Combining these features derived from eCLIP and RNA-seq data, we developed a machine learning model to predict the subcellular localization of RBPs. This classifier has the potential to provide additional insight into RBP cellular function. To further improve our approach, we intend to explore other methods for deriving “true” localization labels, as well as how filtering for eCLIP data quality and incorporating additional informative features impact classifier performance.
References:
1. Van Nostrand, E. L. et al. (2016). Nature Methods 13.
2. Uhl, M. et al. (2022). Bioinformatics 38(4).
3. Van Nostrand, E. L. et al. (2020). Nature
4. Ietswaart, R. et al. (2024). Molecular Cell 84(14).
Keywords: RNA-binding proteins, machine learning, subcellular localization
30. The NuRD chromatin remodeler as a novel regulator of RNA polymerase III and the small RNA universe
Ruiying Cheng (University of Illinois Urbana-Champaign), Rajendra K C (University of Illinois Urbana-Champaign), Sihang Zhou (University of Illinois Urbana-Champaign), Simon Lizarazo (University of Illinois Urbana-Champaign), Kevin Van Bortle (University of Illinois Urbana-Champaign)
Abstract:
RNA polymerase (Pol) III is centrally involved in shaping the small RNA universe, producing diverse noncoding RNAs (ncRNA) such as tRNA, 5S rRNA, and U6 small nuclear RNA (snRNA) integral for translation, RNA processing, and other processes critical for cell growth. However, despite being a major determinant of small RNA abundance in a cell, Pol III transcription and the mechanisms mediating its dynamic and tissue-specific activities remain unresolved. To address this gap, we used an unbiased proteomic approach to identify Pol III protein-protein interactions and thereby predict novel regulators of Pol III transcription. Notably, we discover 14 out of 15 NuRD (Nucleosome Remodeling and Deacetylase) complex subunits in our survey, which together mediate histone deacetylation and ATP-dependent chromatin remodeling. We therefore further explored the global landscape of NuRD occupancy using an innovative large-scale genomic approach that combines all available NuRD-binding data in humans. Our genomic survey identifies widespread NuRD occupancy at most Pol III-transcribed gene classes, further supporting NuRD as a putative Pol III regulator. Finally, we test this hypothesis using a combination of small molecule inhibition and other functional genomic methods to investigate the contribution of NuRD activity on Pol III transcription. Our preliminary data suggest the NuRD complex’s histone deacetylation activity negatively regulates Pol III transcription, consistent with its established role in regulating non-Pol III transcribed genes. Taken together, our study points to the NuRD chromatin remodeling complex as a novel modulator of Pol III transcription with important implications for the small RNA universe.
Keywords: Expression Regulation, Small Non-Coding RNA, RNA Polymerase III
31. XPO1 and topoisomerase-1 inhibitors synergistically sensitize XPO1-mutant colorectal cancers
Wannasiri Chiraphapphaiboon (Masonic Cancer Center, Department of Pharmacology, University of Minnesota), Tulasigeri M. Totiger (Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine), Justin Taylor (Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine), Hai Dang Nguyen (Masonic Cancer Center, Department of Pharmacology, University of Minnesota)
Abstract:
Exportin 1 (XPO1) is a major export transporter that is responsible for nuclear-cytoplasmic transport of proteins and RNAs. XPO1 is commonly overexpressed in different cancers, highlighting an attractive therapeutic target. In our current study, pan-cancer analysis revealed that XPO1 is mutated (p.R749Q) in colorectal cancers. We used CRISPR-Cas9 to generate XPO1-R749Q mutation at the endogenous locus in HCT116 and LS174T colorectal cell lines. Intriguingly, XPO1-R749Q mutant cells are more resistant to standard chemotherapeutic and DNA-damaging agents. Mechanistically, we found that XPO1-R749Q mutant cells displayed lower DNA damage levels compared to wild-type cells following camptothecin (CPT) exposure. Interestingly, albeit lower DNA damage levels, CPT-treated XPO1-R749Q mutants induced higher RPA phosphorylation in γH2AX-positive cells. These results suggest that XPO1-R749Q cells hyper-activate RPA phosphorylation to prevent DNA damage. In line with this notion, we found that selinexor, an inhibitor that targets XPO1, reduced CPT-induced RPA phosphorylation. Moreover, selinexor treatment combined with the standard of care agent, irinotecan synergistically sensitized XPO1-R749Q mutant cells. Taken together, our results suggest that increased RPA phosphorylation could mediate resistance to DNA-damaging therapies in XPO1-R749Q mutant cells. We further demonstrate that impairing RPA phosphorylation by selinexor may provide a preclinical rationale to treat colorectal cancer patients with XPO1 mutations to overcome resistance to chemotherapeutic agents.
Keywords: Exportin 1, Chemotherapeutic resistance, Colorectal cancers
32. Dynamic expression of the nuclear poly(A) binding protein N1 is critical for postnatal Heart development and disease.
Chorghade S,Joe Seimetz,Subhashish Natua, Bo Zhang,Chaitali Misra (Department of Biochemistry,University of Illinois Urbana-Champaign), Qinyu Hao, Kananganattu V. Prasanth (Department of Cell and Developmental Biology,Department of Biochemistry,University of Illinois Urbana-Champaign), Jiwang Chen (Department of Medicine, University of Illinois Chicago), Xander Wehrens (Department of Molecular Physiology and Biophysics, Baylor College of Medicine), Auinash Kalsotra (Department of Biochemistry, University of Illinois Urbana-Champaign)
Abstract:
The nuclear poly(A) binding protein (PABPN1) is a ubiquitously expressed, evolutionarily conserved protein that controls many steps of the mRNA life cycle, including 3’-poly(A) tail elongation and alternative polyadenylation (ApA). Here we report that PABPN1 protein expression is post-transcriptionally silenced during postnatal heart development in humans, mice and rats; and this silencing is key for achieving adult, cardiac-specific gene expression profiles. Combining single-molecule RNA imaging and nucleo-cytoplasmic fractionations, we demonstrate that PABPN1 silencing during development occurs through a drastic shift in its mRNA localization: the mRNA is cytoplasmic in neonatal cardiomyocytes but becomes partially spliced, nuclear-retained, and translation-inaccessible in adult cardiomyocytes.
In contrast, PABPN1 protein levels are elevated in failing human hearts and mouse models of hypertrophy and heart failure. Moreover, we demonstrated that acute stress-related stimuli triggered the complete splicing and translocation of nuclear-sequestered PABPN1 mRNA to the cytoplasm. Additionally, cardiomyocyte-specific conditional loss- and gain-of-function studies revealed that premature elimination or persistent expression of PABPN1 in the mouse myocardium provokes major structural and functional abnormalities, resulting in heart failure and death. Using nanopore-based direct RNA and high-resolution Illumina sequencing, we identify widespread changes in ApA and poly(A) tail lengths of cardiac transcripts during development, a significant portion of which are dependent on the postnatal silencing of PABPN1. Remarkably, forced re-expression of PABPN1 leads to a global reversal of poly(A) site usage, tail length, and translation for transcripts directly associated with maladaptive cardiac cell growth. Taken together, these results indicate that developmental silencing of PABPN1 through its regulated mRNA splicing and localization is critical for postnatal maturation and function of the heart.
Keywords: Heart, Nuclear sequestration, PABPN1
33. Binding Preferences amongst Three Saccharomyces cerevisiae tRNA Primary Nuclear Exportins
Sophie Cihan (Department of Molecular Genetics, The Ohio State University), Jackson Hastings (Department of Molecular Genetics, The Ohio State University), Anita K. Hopper (Department of Molecular Genetics, The Ohio State University)
Abstract:
tRNAs are diverse, highly modified molecules. Disruptions in tRNA biogenesis are linked to human diseases, including neuronal disorders, neurodegenerative disorders, and cancer. tRNAs must be trafficked out of the nucleus to perform their iterative function in the cytoplasm by a pathway termed primary nuclear export. There are 42 tRNA gene families in Saccharomyces cerevisiae, 10 of which contain introns which are spliced in the cytoplasm. Primary tRNA nuclear export employs at least three nuclear exportins: Los1, Mex67-Mtr2, and Crm1. However, the binding preferences these three exportins have for different intron-containing tRNAs is not understood.
We aim to uncover new insights regarding pre-tRNA and nuclear exportin preferences in primary nuclear export. Two approaches offer different perspectives into this pathway. First, we are currently purifying intron-containing pre-tRNAs via pulldowns using biotinylated oligonucleotides complementary to their introns. The relative abundances of co-purifying exporters will be identified via Mass Spectrometry. To ensure the specificity of the methods, biotinylated tRNA pulldowns were optimized using purified small RNA preparations. Second, we utilized qRT-PCR to measure relative abundances of intron-containing tRNAs between Wild-Type and los1∆ strains. A given tRNA’s dependence on Los1 for export can then be inferred by the relative difference between that tRNA’s abundance in los1∆ compared to Wild-Type strains.
References:
1. Phizicky EM, Hopper AK. The life and times of a tRNA. RNA. 2023 Jul;29(7):898-957.
2. Huang HY, Hopper AK. Multiple Layers of Stress-Induced Regulation in tRNA Biology. Life (Basel). 2016 Mar 23;6(2):16.
3. Chatterjee K, Nostramo RT, Wan Y, Hopper AK. tRNA dynamics between the nucleus, cytoplasm and mitochondrial surface: Location, location, location. Biochim Biophys Acta Gene Regul Mech. 2018 Apr;1861(4):373-386.
4. Chatterjee K, Marshall WA, Hopper AK. Three tRNA nuclear exporters in S. cerevisiae: parallel pathways, preferences, and precision. Nucleic Acids Res. 2022 Sep 23;50(17):10140-10152.
Keywords: tRNA, nuclear trafficking, S cerevisiae
34. Investigation of Pus4 Substrate Selection
Amelia Cochran (Chemistry, University of Michigan), Jacqueline Anthenien (Chemistry, University of Michigan), Kristin Koutmou (Chemistry, University of Michigan), Markos Koutmos (Chemistry, University of Michigan)
Abstract:
Pseudouridine synthase (Pus) enzymes catalyze the isomerization of uridine (U) to pseudouridine (Ψ), a common modification known to impact RNA stability and structure. Originally characterized as modifiers of non-coding RNAs, some Pus enzymes are now also known to target mRNA. Because Ψ has been shown to impact mRNA processing, translation, and stability, these mRNA-modifying Pus enzymes may participate in regulation of gene expression. But how and why specific mRNA targets are selected is not yet fully understood. Pus4, which modifies U55 in the T-loop of most tRNAs, is one of the major mRNA-modifying Pus enzymes. Though it has been proposed that Pus4 only modifies mRNAs with similarities to its native tRNA targets, recent studies suggest that Pus4 can bind to and modify RNAs that differ from these native substrates. To fully understand how RNA features impact Pus4 substrate selection, we are working to characterize Pus4’s ability to bind to and modify RNAs of different sequences and structures in vitro. Additionally, we aim to understand how Pus4’s structural features contribute to its substrate selection, and how structural differences between prokaryotic and eukaryotic Pus4 homologs influence apparent differences in specificity. Our initial findings show a lack of selectivity in RNA binding in eukaryotic Pus4, pointing to the possibility of enzyme promiscuity and reliance on factors outside of protein-RNA interaction for mRNA target selection.
Keywords: Pseudouridine, RNA modifications , RNA binding proteins
35. The role of hAGO1 variants in neurodevelopmental disorders
Mariana Correia (The Ohio State University), Nassim Meziane (Sorbonne University), Mira Brazane (Sorbonne University), Amelie Piton (Institute of Genetics, Molecular, and Cellular Biology), Laure Teysset (Sorbonne University), Clement Carre (Sorbonne University)
Abstract:
Neurodevelopmental disorders (NDDs) are defined as impairments in brain function resulting from developmental abnormalities. De novo coding variants in the human AGO genes AGO1 and AGO2 cause NDDs with intellectual disability (ID), referred to as Argonaute syndromes. AGO proteins are central to RNA silencing by associating with small non-coding RNAs (sncRNAs), such as microRNA (miRNAs) and small interfering (siRNAs), to form the RNA-induced silencing complex (RISC). Once assembled, RISC directs AGO to bind target mRNAs, facilitating gene expression regulation through mRNA degradation or translational repression. The project aims to model two NDD-associated hAGO1 variants, F180∆ and G199S in Drosophila melanogaster AGO1 (dAGO1). As a first experimental approach, the host lab generated a Drosophila miRNA overexpression sensor line (mir7-OE) using the inducible expression UAS/GAL4 system. This sensor provides direct insights into dAGO1 dysfunction through its associated wing notch phenotype. We began characterizing the mir7-OE line by crossing it with different UAS RNAi (Knock Down) fly lines for the genes white, Dicer-2, and AGO2, which are not involved in the miRNA-mediated silencing pathway. Various UAS RNAi AGO1 lines were also tested against this tool. The findings concluded the need for further refinement in UAS RNAi AGO1 models to better understand their role in NDDs. In parallel, two human variants were engineered by introducing the corresponding mutations into the fly’s endogenous AGO1 gene using CRISPR/Cas9, a process that is currently ongoing. In continuation of this research, I have joined Dr. Hopper’s Ohio State University laboratory to investigate the newest member of the class of sncRNAs, tRNA introns, using Saccharomyces cerevisiae an organism deficient in RNAi. This research may reveal how tRNA introns evolved as an early form of RNAi, broadening understanding of RNA regulation.
Keywords: neurodevelopmental disorders, argonaute proteins, RNAi
36. Exon 10 skipping of ATXN3 as a potential therapy for Spinocerebellar Ataxia type 3 (SCA3)
Allan Victor Cortes (Biology, Lewis University), Ava Artz (Biology, Lewis University), Joseph Owens (Biology, Lewis University), Ana Botros (Biology, Lewis University), Aleksandra Borek (Biology, Lewis University)
Abstract:
Spinocerebellar Ataxia type 3 (SCA3), or Machado Joseph Disease, is an autosomal dominant disorder caused by excessive CAG repeats within exon 10 of the Ataxin-3 gene. As a result, the polyglutamine in the subsequent protein aggregates triggering neurodegeneration. Symptoms arise when CAG repeats exceed 44. Currently, there is no cure for the disease. Inducing exon 10 skipping using steric binging antisense oligonucleotides (ASO) removes the excessive repeats and leaves the binding domain and C-terminus intact. To determine the most efficient ASO chemistry, and sequence, and the therapeutic viability of skipping exon 10, C. elegans lacking endogenous ataxin gene, and patient fibroblasts were used. C. elegans were transfected with the human version of the ATXN3 containing 28 CAG repeats, 84 CAG repeats, or with exon 10 removed. Lifespan and locomotive functions were monitored. ASOs of various sequences, with 2’MOE modifications with and without phosphothioate modifications were tested in patient fibroblasts.
Keywords: SCA3, Spinocerebellar Ataxia, Machado Joseph
37. Determining the role of the tRNA methyltransferase Trm7:Trm734 in repression of TY1 elements in yeast
Natalie Creech (Department of Chemistry and Biochemistry, Northern Kentucky University), Ruofei Ding (Department of Chemistry and Biochemistry, Northern Kentucky University), Holly M. Funk (Department of Chemistry and Biochemistry, Northern Kentucky University), Emma Nasipova (Department of Chemistry and Biochemistry, Northern Kentucky University), Faith Meghrian (Department of Chemistry and Biochemistry, Northern Kentucky University), Michael P. Guy (Department of Chemistry and Biochemistry, Northern Kentucky University)
Abstract:
Post-transcriptional tRNA modifications are required for efficient protein translation. In yeast, the Trm7 methyltransferase forms a complex with Trm734 to modify tRNA at position 34. In humans, mutations in the TRM7 homolog FTSJ1 cause intellectual disability. In yeast, retrotransposons replicate through reverse transcription of a region of RNA that can then integrate into new sites throughout the genome. Increased TY1 transposition can lead to damage of once-functional genes. In a previous screen, TRM7 and TRM734 were identified as being involved in repression of TY1 transposition, although it is unclear whether this effect is direct or indirect. We hypothesize that loss of TRM7 or TRM734 leads to increased TY1 transposition because of lowered expression due to defects in the translation of other genes involved in limiting TY1 transposition. We analyzed the codon content of genes known to suppress TY1 transposition and selected four genes which had higher than expected Phe codon content, becauseTrm7 and Trm734 are important for post-transcriptional modification of the tRNAPhe anti-codon loop. We are testing genetic interactions to determine if TRM7 and TRM734 are directly or indirectly involved in TY1 transposition. In a parallel approach, to test expression levels in yeast, tagged proteins of interest will be transformed into strains lacking TRM7 and TRM734. Expression of the four TY1-associated genes will then be compared to that in wild type strains. By performing these experiments, we will better understand the role of Trm7 and Trm734 in the translation of Phe-rich genes.
Keywords: tRNA, yeast, modifications
38. Imaging lncRNA TERRA using γPNA probes
Ruwani Madushika Dalath (Department of Biological Sciences, Carnegie Mellon University ), Mackenzie Riley (Department of Chemistry, Carnegie Mellon University ), Meng Xu ( Department of Biological Sciences, Carnegie Mellon University ), Nisha Hasija (Department of Chemistry, Carnegie Mellon University), Bruce Armitage (Department of Chemistry, Carnegie Mellon University ), Huaiying Zhang (Department of Biological Sciences, Carnegie Mellon University )
Abstract:
Telomeric repeat-containing RNA (TERRA) is a long, noncoding RNA transcribed from subtelomeric and telomeric regions of chromosomes (Azzalin et al., 2007). Decades of research conducted on TERRA has established its functional roles in both cancerous and noncancerous cells. However, the mechanistic understanding of TERRA functions remains elusive due to the unavailability of a proper and reliable imaging tool. This work introduces a novel Gamma Peptide Nucleic Acid (γPNA) based fluorescent imaging tool to track endogenous TERRA in cells. Considering γPNA's superior binding affinity, enhanced water solubility, and extended cellular half-life due to its resistance to enzymatic degradation, we identified it as the most promising candidate for TERRA imaging (Andrasi et al., 2006). So far, we have designed and evaluated five different lengths of γPNA (ranging from 8-12 bases), two different backbone structures (γSerine and γMethoxy), and a modified version of γPNA with an attached miniPEG sequence between the backbone and the fluorescent tag to enhance signal intensity. Our findings revealed a length-dependent increase in TERRA puncta detected per nucleus and a rise in total signal intensity, though selectivity toward RNA diminished beyond ten bases. The specificity of the designed probes was further validated by knocking down TERRA using LNA Gapmers. In our efforts to optimize a compatible RNA Fluorescent in situ hybridization (FISH) protocol for the designed gamma PNA, we realized that our TERRA probes could provide a maximum detection at a Formamide concentration of 20% in the hybridization buffer within just a 2-hour incubation time, significantly improving the efficiency compared to the prevailing protocols. After successfully implementing the modified tool for fixed cell imaging, we demonstrated its sensitivity by showing reduced TERRA levels in Promyelocytic leukemia (PML) knockout cells, underscoring its robustness and potential as a transformative tool for TERRA research.
References:
Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., & Lingner, J. (2007). Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science (New York, N.Y.), 318(5851), 798–801.
Dragulescu-Andrasi, A., Rapireddy, S., Frezza, B. M., Gayathri, C., Gil, R. R., & Ly, D. H. (2006). A simple gamma-backbone modification preorganizes peptide nucleic acid into a helical structure. Journal of the American Chemical Society, 128(31), 10258–10267
Keywords: TERRA, PNA, PML
39. Dysregulated RNA splicing network drives regeneration failure causing hepatic decompensation in alcohol-associated liver disease
Diptatanu Das (Department of Biochemistry, University of Illinois, Urbana-Champaign), Anuprova D. Bhowmik (Department of Biochemistry, University of Illinois, Urbana-Champaign), Arnab K. Roy (Department of Biochemistry, University of Illinois, Urbana-Champaign), Rajesh Dutta (Division of Gastroenterology, Department of Medicine, Duke University Health System), Anna M. Diehl (Division of Gastroenterology, Department of Medicine, Duke University Health System), Auinash Kalsotra (Department of Biochemistry, University of Illinois, Urbana-Champaign)
Abstract:
Alcohol-associated liver diseases (ALDs) are the primary cause of liver-related mortality worldwide, accounting for ~50% of all liver transplants. ALD progresses from alcohol-associated fatty liver (AFL) to steatohepatitis (SAH)—characterized by inflammation and acute-on chronic hepatocellular injury. While a normal liver is renowned for its ability to regenerate, why it fails in ALD remains unclear. Here, we performed comprehensive multi-omic profiling of healthy and diseased human livers using bulk and single-nucleus RNA- plus ATAC-seq. We report that hepatic immune milieu alterations in ALD prevent hepatocytes from transitioning to a proliferative progenitor-like state, trapping them into an unproductive intermediate state. We found reduced expression of adult transcription factors (TFs), in agreement with their chromatin states, but the expression of fetal TFs, in contrast to their chromatin accessibility, did not increase in ALD. This discordance between gene expression and chromatin accessibility motivated us to reason that post-transcriptional mechanisms might be dominant in driving altered activity of key genes responsible for cellular transitions. We found striking changes in RNA binding protein (RBP) expression, particularly ESRP, PTBP, and SR families, that cause misregulation of developmentally controlled RNA splicing in ALD. Our data pinpoint ESRP2 as a pivotal disease-sensitive RBP and support a causal role for its deficiency in ALD pathogenesis. We discovered that ESRP2 downregulation decreased inclusion of nuclear localization signal (NLS)-encoding exons of two cellular state-associated proteins – SLK and Wnt effector TCF4. We found cytoplasmic enrichment of SLK and TCF4 in human SAH livers. Using antisense oligonucleotides (ASOs), we recapitulated the disease-linked missplicing of Slk and Tcf4 in hepatocyte cultures, and studied how defects in SLK and TCF4 nuclear localization disrupts WNT and Hippo signaling pathways, which are critical for normal liver regeneration. In summary, our results demonstrate that dysregulated RNA splicing impedes regeneration in ALD, eventually leading to hepatic decompensation and liver failure.
Keywords: Alternative Splicing, Alcohol-associated Liver Disease, nuclear localization
40. Exploring the role of a tRNA SNP in the molecular basis of dextrocardia, a laterality disorder
Moulisubhro Datta (MCDB, The Ohio State University), Ila A. Marathe (Chemistry and Biochemistry, The Ohio State University), Lauren Levesque (Department of Molecular Genetics, The Ohio State University), Vicki H. Wysocki (Chemistry and Biochemistry, The Ohio State University), Susan E. Cole (Department of Molecular Genetics, The Ohio State University), Venkat Gopalan (Chemistry and Biochemistry, The Ohio State University)
Abstract:
Dextrocardia is a rare congenital condition in which the heart is misaligned in the thoracic cavity 1. A recent case study identified homozygosity for a rare SNP allele (chr 17) as a possible cause for dextrocardia and situs inversus2. This SNP lies in the 3' UTR of the Notch signaling gene HES 7. However, mutation of HES 7 is associated with skeletal and not laterality defects3,4. Interestingly, we noted that a tRNAArg UCU (n-Tr25) is encoded on the opposite strand from HES 7 and in the region that contains the SNP. Because several genes that dictate left-right symmetry have large numbers of AGA codons 5, we hypothesized that alterations to n-Tr25 levels from impaired 5' maturation (of the SNP-bearing mutant) by RNase P could lead to laterality defects.
Native PAGE experiments revealed that while the wildtype pre–n-Tr25 exhibited a Mg2+-dependent shift to folded states, the mutant appeared diffuse, reflective of structural heterogeneity. Native mass spectrometry and mass photometry confirmed that both RNAs existed as a monomer under the conditions tested. We also found that in vitro reconstituted Escherichia coli RNase P cleaves the mutant (with a presumed longer acceptor–T-stem stack) at a slower rate. This is consistent with previous studies that showed RNase P prefers a single-stranded region preceding the pre-tRNA cleavage site 6-8. We found that the laterality disorder-associated tRNA SNP dampens the rate of processing by RNase P but recognize the need to assess functional levels of this tRNA in vivo; reporter constructs in cultured cells and transgenic mice will better inform this scenario (ongoing studies). Preliminary data indicate that that the SNP leads to pre–n-Tr25 transcripts with longer 3' trailers presumably due to altered pol III termination. We are excited to add to the growing evidence that point mutations in tRNAs affect their structure and maturation to culminate in disease and highlight the unexpected ripple effects of SNPs.
References:
1. Deng et al. (2015) Expert Rev. Mol. Med. 16: e19.
2. Netravathi et al. (2015) BMC Med. Gen. 16: 1-8.
3. Sparrow et al. (2010) Eur. J. Hum. Genet. 18: 674-679;
4. Luo et al. (2016) Sci. Rep. 6: 31583.
5. Orellana et al. (2021) Mol Cell 81: 3323-3338.
6. Kirsebom & Svärd (1992) Nucleic Acid Res. 20: 425-432.
7. Lee et al. (1997) RNA 3: 175-185.
8. Torabi et al. (2021) RNA 27: 1140-1147.
Keywords: tRNA, mutation, disease
41. Engineering Aminoacyl-tRNA Synthetases (aaRS)/tRNA Pairs to Recognize Cysteine-Reactive Unnatural Amino Acids to Generate Bacterial-Displayed Cyclic Peptides
Olabode Dawodu (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana), Caitlin Specht (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana), Alejandro Tapia (Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana), Jennifer Flora (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana), Jeffery M. Tharp (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana)
Abstract:
Bacteria surface display is a direct evolution technique that allows screening of large peptide libraries to identify novel peptides that bind to a protein of interest. Unlike traditional peptide screening, the peptides are genetically encoded, allowing for the ease of peptide hit determination via sequencing. To form the cyclic peptide, we genetically encoded an unnatural amino acid (uAA) in our peptide that will react with cysteine to form the cyclized peptide. We use an engineered amino-acyl tRNA synthetase (aaRS) that will encode the uAA at TAG stop codons. To confirm installment of the unnatural amino acid at the TAG codon, we utilize our streptavidin model by constructing a plasmid containing a streptavidin-binding peptide that is suppressed by a TAG stop codon. Installment of the uAA was confirmed by flow cytometry by comparing the two cell populations (+/- uAA) that were labeled with streptavidin-PE. Cells supplemented with the uAA, had an increased PE signal compared to cells that were not supplemented with the uAA. These findings gave us support that we can develop a cyclic peptide library to our target protein, programmed cell death ligand 1 (PD-L1); an immune checkpoint protein overexpressed in many cancer cells.
As a starting point, we developed a cyclic peptide library based on a previously reported anti-PD-L1 linear peptide, CLP003. Using fluorescently-activated cell sorting (FACS), we identified four peptides hits that bind to fluorescently-labeled PD-L1. Our results suggest that engineered aaRS/tRNAs can be used to generate and screen novel cyclic peptides to PD-L1.
References:
1.Claudio Zambaldo, Xiaozhou Luo, Angad P. Mehta, and Peter G. Schultz. Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids. Journal of the American Chemical Society 2017;139 (34), 11646-11649.
2.Liu H, Zhao Z, Zhang L, Li Y, Jain A, Barve A, Jin W, Liu Y, Fetse J, Cheng K. Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy. J Immunother Cancer. 2019 Oct 22;7(1):270. doi: 10.1186/s40425-019-0705-y.
3.Tejas Navaratna, Lydia Atangcho, Mukesh Mahajan, Vivekanandan Subramanian, Marshall Case, Andrew Min, Daniel Tresnak, and Greg M. Thurber. Directed Evolution Using Stabilized Bacterial Peptide Display. Journal of the American Chemical Society 2020;142 (4), 1882-1894
Keywords: Aminoacyl-tRNA synthetases, Bacteria display, Cyclic
42. Multi-Color Single-Molecule Tracking of Stop-Codon Readthrough on the Vitamin D Receptor mRNA in Live Cells
Kara Delbridge (Carnegie Mellon University, Department of Biology, Mellon College of Science, Pittsburgh, PA, USA; Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA), Kelsey E. Bettridge (Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA), Agnes Karasik (Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA), Nicholas R. Guydosh (Laboratory of Biochemistry and Genetics, NIDDK, Bethesda, MD, USA)
Abstract:
Faithful translation of the genetic code by ribosomes requires efficient termination at the end of a coding sequence. However, ribosomes can occasionally insert an amino acid at stop codons and continue translating the 3’ untranslated region (3’ UTR) of the mRNA, leading to the release of a protein with a C-terminal extension. Such readthrough (RT) events can be modulated by the sequence context surrounding the stop codon; several conserved cases with very high RT have been identified and are referred to as programmed RT. One example is the Vitamin D Receptor (VDR), a gene that encodes a master transcription factor that controls the cell’s response to vitamin D and undergoes RT ~7% of the time. To understand the mechanism of programmed RT, we adopted an approach using single-molecule microscopy and a multi-color fluorophore system to observe differences in RT kinetics between mRNAs on a single mRNA. Our system reveals single RT events and that they occurred stochastically about 1% of the time for a normal stop codon, as expected. Surprisingly, we found that programmed RT due to the sequence context from VDR occurred in bursts: one RT event was generally followed by multiple RT events on a given mRNA. This burst process could arise if ribosomes form queues at stop codons and the interactions between ribosomes in the queue change the efficiency of termination. Since ribosome queueing can be altered by stress, such a mechanism could couple stress to programmed RT and regulate the cell’s sensitivity to vitamin D.
Keywords: Microscopy, Ribosome, Readthrough
43. Dynamic Expression of Epithelial Splicing Regulatory Protein 2 Promotes Cell Migration and Proliferation to Enable Regeneration Following Drug-Induced, Acute Liver Injury
Jessica M. Derham (Department of Biochemistry, University of Illinois Urbana-Champaign), Esha Kulkarni (Department of Bioengineering, University of Illinois Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, University of Illinois Urbana-Champaign)
Abstract not available online - please check the booklet.
44. Investigating the Interaction Between Yeast tRNA Modification Proteins Trm7 and Trm734
Alisha Detmer (Chemistry and Biochemistry, Northern Kentucky University ), Julia Verhoff (Chemistry and Biochemistry, Northern Kentucky University), Holly Funk (Chemistry and Biochemistry, Northern Kentucky University), Michael P. Guy (Chemistry and Biochemistry, Northern Kentucky University)
Abstract:
In the anticodon loop of tRNAPhe, the modification of nucleotide G34 is fundamental for translation in Saccharomyces cerevisiae, humans, and other eukaryotes. In S. cerevisiae, the 2'-O-methylation of G34 is done by the Trm7:Trm734 complex. We study this complex because it is not fully understood why Trm7 needs Trm734 to function. The TRM7 human homolog is FTSJ1, and the TRM734 homolog is WDR6. Loss of function mutations in FTSJ1 that cause loss of Gm34 modification in patients result in non-syndromic x-linked intellectual disability. Through sequence alignment of Trm7 homologs in a variety of species and analysis of the crystal structure of the yeast Trm7:734 complex, Trm7 variants were made by changing amino acids which were conserved in different species, or that had a salt bridge, hydrogen bond, or high buried surface area with Trm734. A growth test that forces Trm7 to interact with only Trm734 or only its other binding partner Trm732 in yeast cells showed that some of the chosen amino acids were important for the Trm7:Trm734 interaction, but not for the Trm7:Trm732 interaction. Subsequent immunoprecipitation experiments further showed that these Trm7 residues were important only for Trm734 interactions, although addition of a tag to Trm7 appeared to result in some interference with Trm734 binding.
Keywords: tRNA , methylation, yeast
45. Combines loss of ADARs and RDE-4 provides resistance to an opportunistic human pathogen
Alfa Dhakal (Indiana University), Heather A. Hundley (Indiana University)
Abstract:
All organisms must be able to fight infection to survive. To defend against infections, cells have equipped machinery to activate signaling pathways and secrete effector molecules. My project focuses on the role of ADARs in the pathogen response of C. elegans. ADARs are RNA binding proteins that regulate gene expression by either binding or chemically modifying RNA. Since these RBPs act on double-stranded RNA which mimics the viral genome, most ADAR studies have focused on understanding the mechanism of viral infection and how a host responds. However, the roles of ADARs in bacterial infection and host-defense mechanisms have been less studied.
Using a standard slow killing assay with Pseudomonas aeruginosa as a pathogen, we observed that C. elegans lacking ADARs have increased susceptibility to infection. Interestingly, when another dsRNA binding protein, RDE-4, is removed along with ADARs, the worms become resistant to infection. RDE-4 has been mostly studied for its role in the RNAi pathway, where it recruits Dicer to dsRNA to be cleaved into small interfering RNA (siRNA). Since the substrate for both ADARs and RDE-4 is dsRNA, the RBPs could compete for substrates, which may influence the level of small RNA and thereby influence gene expression. Behavioral assays show that adr;rde-4 animals are not resistant because they can avoid the lawn of bacteria more efficiently but have significantly lower proliferation of PA14 in the intestine. Upon looking at the gene expression changes in these animals we found no distinctive ontology. Ongoing experiments are focused on finding the tissue specific contributions of ADARs and RDE-4 in pathogen resistance using auxin inducible degron (AID). These investigations will help us understand the interaction between two different families of RNA binding proteins and how this interaction helps animals respond to infection.
Keywords: Innate immunity, ADAR, dsRNA binding protein
46. Dissecting the role of protein arginine methylation on the function of Snp1, the yeast homolog of human U1-70K
Trevor Doiron (Department of Biological Sciences, University at Buffalo), Michael C. Yu (Department of Biological Sciences, University at Buffalo)
Abstract:
Unlike mammalian systems, Saccharomyces cerevisiae expresses a single type I PRMT, Hmt1. Previous studies have shown that Hmt1 catalyzes arginine methylation on the U1 snRNP subunit, Snp1. This protein, along with its human homolog U1-70K, is involved in the association of the U1 snRNP with the 5’ splice site (SS). However, how arginine methylation affects Snp1 function remains largely unknown. In this study, we aim to dissect the functional role of specific methylation sites on Snp1 by analyzing the phenotypic consequence of arginine substitution mutants. Additionally, we use a genetic approach to investigate the effect of these mutations on the U1 snRNA association with the 5’SS. Two classes of Snp1 methylarginine substitution mutants were generated: arginine-to-lysine mutants, which preserves the amino acid charge, and arginine-to-alanine mutants. By assessing the combined effects of Snp1 methylarginine mutations and specific U1 snRNA point mutants on yeast growth phenotypes, we aim to gain insight into how Snp1 arginine methylation affects U1 association with pre-mRNA. Our findings indicate that methylation at these specific arginine sites does not significantly impact yeast growth phenotypes. However, when combined with specific U1 snRNA mutations, Snp1 methylarginine mutations do impact growth phenotype compared to either mutation alone. Current efforts are focused on uncovering the molecular basis underlying this observed interaction.
Keywords: Protein arginine methylation, Spliceosome, Snp1
47. Investigation of the ability of 5-methylcytosine to modulate RNA-protein interactions in humans
Chathurani Ekanayake (Department of Chemistry and Biochemistry, Kent State University, Kent OH), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University, Kent OH)
Abstract:
During the epitranscriptomic regulation of gene expression, post-transcriptional modifications such as m5C, m7G, m1A, and Ψ play crucial roles in functional RNAs. Dysregulation of mRNA nucleotide modifications is associated with various human diseases, including cancer, diabetes, and neurodevelopmental disorders. Despite their prevalence, the specific mechanisms underlying RNA modification recognition and their role in disease pathogenesis remain incompletely understood. This study aims to investigate the impact of RNA sequence on m5C recognition by proteins. Phage display experiments were conducted using several m5C containing model RNAs with diverse sequences, including consensus sequences for NSUN2 and NSUN6 methyltransferases. The impact of RNA sequence on m5C recognition by proteins was examined, particularly in the breast cancer cell line MDA-MB 231. RNA pull-down assays targeting NSUN6, NSUN6 M5C, NSUN2, and NSUN2 M5C were performed, followed by LC-MS/MS analysis to identify associated proteins. Peptide alignment analysis revealed specific interactions between peptides and protein structures, suggesting a potential methyl-reading function. Results indicated enrichment of RNA-binding proteins, highlighting a complex regulatory network involved in m5C recognition. Protein Network analyses provided insights into functional implications and pathways influenced by the identified proteins. These findings enhance our understanding of RNA modification recognition mechanisms and their dysregulation in breast cancer. Future research will focus on the functional characterization of identified proteins and their implications in disease pathogenesis, potentially leading to targeted therapeutic interventions. Overall, this study contributes to elucidating the molecular mechanisms underlying gene expression regulation and offers insights into potential therapeutic targets in breast cancer.
References:
1.Abedeera, S. M., Jayalath, K. S., Xie, J., Rauff, R. M. & Abeysirigunawardena, S. C. Pseudouridine Synthase RsuA Confers a Survival Advantage to Bacteria under Streptomycin Stress. Antibiotics 12, 1447 (2023).
2.Khan, K., Baleanu-Gogonea, C., Willard, B., Gogonea, V. and Fox, P.L., 2020.3-Dimensional architecture of the human multi-tRNA synthetase complex. Nucleic Acids Research, 48(15), pp.8740-8754.
3.Marine, J.C. and Lozano, G., 2010. Mdm2-mediated ubiquitylation: p53 and beyond. Cell Death & Differentiation, 17(1), pp.93-102.
4.Rauff, R., Abedeera, S. M., Schmocker, S., Xie, J. & Abeysirigunawardena, S. C. Peptides Targeting RNA m6 A Methylations Influence the Viability of Cancer Cells. ChemMedChem 18, e202200549 (2023).
5.Wang, T., Kong, S., Tao, M. & Ju, S. The potential role of RNA N6-methyladenosine in Cancer progression. Mol. Cancer 19, 88 (2020).
Keywords: RNA-protein interactions, m5C methylation, Breast cancer
48. Pleiotropic Effects of Spliceosome Disruption Include Upregulation of Nonsense-Mediated mRNA Decay Targets
Caleb Embree (Department of Molecular Genetics, Center for RNA Biology, Ohio State University), Andreas Stephanou (Department of Molecular Genetics, Ohio State University), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, Ohio State University)
Abstract:
Pre-mRNA splicing, carried out by a large ribonucleoprotein machine known as the spliceosome, sculpts mRNAs by splicing out introns and decorating mRNA exon junctions with the exon junction complex (EJC). Both functions are critical for proper mRNA expression as they impact mRNA open reading frames and monitoring for premature translation termination by the nonsense mediated mRNA decay (NMD) pathway. Recently, genetic screens in human cell lines have identified spliceosome components as putative NMD factors. Using publicly available RNA-seq datasets from knockdowns of 18 different spliceosome components in K562 human cell lines, we have investigated the effect of spliceosome disruption on NMD efficiency by quantifying changes in abundance of NMD target and non-target mRNA isoforms. While we find that NMD targeted isoforms are upregulated when members of the catalytic spliceosome are knocked down, so are other non-canonical isoforms. After accounting for novel isoforms produced through missplicing, non-canonical isoforms remain upregulated compared to canonical isoforms, which are largely downregulated. Similarly, the canonical isoforms encoding NMD factors are downregulated , contrary to expectation, as NMD factor mRNAs are subjected to auto-regulatory feedback upon strong NMD inhibition. These results indicate that depletion of spliceosome components broadly changes the transcriptome, resulting in upregulation of NMD-targeted transcripts through mis-splicing or a minor reduction in NMD efficiency, or both. Interestingly, depletion of one spliceosomal component, CDC40, like the knockdown of EJC core protein EIF4A3, causes higher upregulation of NMD targeted isoforms as compared to other non-canonical isoforms, suggesting that its effects on NMD could be more direct. All together, we have shown that disruptions of pre-mRNA splicing have pleotropic effects on gene expression that also results in increased expression of NMD targeted transcripts. Although the precise reason of this effect on NMD targets remains unresolved, we have also observed increased abundance of NMD targeted transcripts in cells derived from retinitis pigmentosa patients with mutations in PRPF8 and PRPF31. Thus, altered levels of NMD regulated genes may contribute to the molecular phenotypes in spliceosomopathies.
Keywords: Splicing, NMD, mRNA Regulation
49. ADR-2 regulates the germline transcriptome and affects fertility and oocyte fate
Emily A. Erdmann (Biology Department, Indiana University), Melanie Forbes (Biology Department, Indiana University ), Margaret Becker (Medical Sciences Department, Indiana University School of Medicine), Sarina Perez (Biology Department, Indiana University), Heather A. Hundley (Biology Department, Indiana University)
Abstract:
RNA-binding proteins (RBPs) play essential roles in coordinating proper gene expression and development in all organisms. Adenosine DeAminases that act on RNA (ADARs) are a class of RBPs that regulate cellular processes at the RNA level by binding to double-stranded RNA (dsRNA) and catalyzing the deamination of Adenosine to Inosine, known as A-to-I RNA editing. Previous work from our lab has shown that ADR-2, the sole A-to-I editing enzyme in Caenorhabditis elegans, can target unique RNAs and produce unique cellular outcomes in different life stages, tissues, and environmental conditions. However, the molecular factors influencing ADR-2 function in these different environments are largely unknown. Using a high-throughput reverse genetics screen, we recently identified a molecular interaction between ADR-2 and SQD-1, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Using microscopy, reproductive assays, and transcriptomic analysis, we uncovered that SQD-1 and ADR-2 act in the same pathway to regulate fertility and oogenesis in C. elegans. As ADARs have primarily been studied in neural tissues, this discovery has prompted investigation into the roles of ADR-2 in regulating the germline transcriptome. High-throughput RNA sequencing analysis of isolated germline tissue has revealed extensive A-to-I editing of germline transcripts (unpublished). Ongoing investigations are aimed at understanding the molecular and biological consequences of germline RNA editing.
Keywords: ADAR, germline, RNA editing
50. Role of the sarcin-ricin loop of 23S rRNA in biogenesis of the 50S ribosomal subunit
Sepideh Fakhretaha Aval (Ohio state Biochemistry program, The Ohio State University, Columbus, OH 43210, USA.), Amal Seffouh (Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada), Kyung-Mee Moon (Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.), Leonard J. Foster (Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.), Joaquin Ortega (Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada.), Kurt Fredrick (Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.)
Abstract:
The sarcin-ricin loop (SRL) is one of the most conserved segments of the large subunit ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, interact with the SRL, and this interaction is essential for GTP hydrolysis and factor function. Cleavage and modification of the SRL by cytotoxins α-sarcin and ricin disrupt GTPase-ribosome interaction, leading to reduced protein synthesis and cell death. However, the full role of the SRL remains unclear. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in E. coli. When ΔSRL particles were purified from cells, they were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, block 4 of the subunit, which includes the peptidyl transferase center, remained unfolded. Here, we explore the basis of this assembly defect. We show that 23S rRNA extracted from ΔSRL subunits can be efficiently reconstituted into 50S subunits, and these reconstituted ΔSRL particles exhibit full peptidyl transferase activity. We also further characterize ΔSRL particles purified from cells, using cryo-EM and proteomic methods. These particles lack density for rRNA and r-proteins of block 4, consistent with earlier chemical probing data. Incubation of these particles with excess total r-protein of the large subunit (TP50) fails to restore substantial peptidyl transferase activity. Interestingly, proteomic analysis of control and mutant particles shows an overrepresentation of multiple assembly factors in the ΔSRL case. We propose that one or more GTPases normally act to release assembly factors, and this function is blocked in the absence of the SRL.
Keywords: Ribosome assembly, Sarcin-ricin loop, Ribosomal RNA
51. NUCLEOBASE protonation in an RNA Thermometer
Opeyemi O. Fatunbi (Department of Chemistry & Biochemistry, Ohio University), Md. Ismail Hossain (Department of Chemistry & Biochemistry, Ohio University), Erin R. Murphy (Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University), Jennifer V. Hines (Department of Chemistry & Biochemistry, Ohio University)
Abstract:
Infectious diseases have claimed more than 15 million out of the over 57 million annual global deaths,1 and the second leading cause of global death has been associated with bacteria.2 Many pathogens responsible for these infections have RNA as their genetic material. Many pathogenic RNAs have non-canonical base pairs, which may be protonated with perturbed pKa towards neutrality. The pH dependence and base pair protonation have been reported to influence various biological processes, such as miRNA processing and viral frameshifting.3 Bacteria such as Shigella dysenteriae, which causes Shigellosis and accounts for about 1.1 million annual deaths, have the Shigella heme uptake (shu) system, such as shuT.4 An RNA thermometer (RNAT) is located within the 5’- UTR of shuT. Structurally, the shuT RNAT has non-canonical base pairs (C●U and U●C) that may influence its structure and function. Hence, to fully understand the structural and functional relevance of the non-canonical base pairs of the shuT RNAT, it is pertinent to determine their possible pH dependence, pKa shift, and protonation state.
Herein, we employed UV spectroscopy and fluorescence spectroscopy to measure the pKa of the shuT RNAT. We observed a clear pH dependence and one ionization event in the shuT RNAT. In addition, we observed a pKa shift towards neutrality, and we were able to identify the non-canonical base pair that may be protonated, responsible for the pH dependence and upward pKa shift. These findings suggest that the protonation of one of the non-canonical base pairs may be pertinent in the structural stability, function, and interactions of the shuT RNAT.
Understanding the pH-dependent behavior of RNAs is vital in the drug discovery process that targets RNA molecules. pH environment in which RNA protonation occurs should be considered when designing small molecules capable of interacting with RNAs. This knowledge can guide the development of more effective RNA-targeted drugs, thereby advancing the field of RNA-based drug discovery.
References:
(1) Gliddon, H. D.; Herberg, J. A.; Levin, M.; Kaforou, M. Genome-Wide Host RNA Signatures of Infectious Diseases: Discovery and Clinical Translation. Immunology 2018, 153 (2), 171–178.
(2) Zhang, C.; Fu, X.; Liu, Y.; Zhao, H.; Wang, G. Burden of Infectious Diseases and Bacterial Antimicrobial Resistance in China: A Systematic Analysis for the Global Burden of Disease Study 2019. The Lancet Regional Health - Western Pacific 2024, 43, 100972.
(3) Wilcox, J. L.; Bevilacqua, P. C. A Simple Fluorescence Method for pK(a) Determination in RNA and DNA Reveals Highly Shifted pK(a)’s. J Am Chem Soc 2013, 135 (20), 7390–7393.
(4) Wei, Y.; Kouse, A. B.; Murphy, E. R. Transcriptional and Posttranscriptional Regulation of Shigella shuT in Response to Host-Associated Iron Availability and Temperature. Microbiologyopen 2017, 6 (3), e00442.
Keywords: Shigella dysenteriae, RNA thermometer, pH dependence
52. When does splicing do more than remove introns?
Tulika Sharma (West Virginia University), Jen Gallagher (West Virginia University)
Abstract:
All organisms have evolved robust mechanisms for responding to changing cellular environments. Response to nutrient deprivation is coordinated by rapidly changing transcription and translation to quickly alter metabolic pathways to better equip the cells for changing nutrient conditions, and yet our understanding of how the molecular mechanisms that span across multiple processes remain obscure. Glyphosate, the active ingredient in the herbicide Roundup (GBH), inhibits aromatic amino synthesis, creating a starvation signal similar but distinct to exposure to rapamycin. From quantitative trait loci analysis in yeast differentially sensitive to GBH (both a lab and agricultural isolates), we identified variants in the MUD1 gene, which encodes a nonessential splicing factor in the U1 snRNP. Loss of no other non-essential U1 protein confers resistance to amino acid starvation while loss of TRAMP complex increases sensitivity to starvation. The TRAMP complex degrades polyA transcripts in the nucleus and starvation sensitivity is repressed by also knocking out Mud1. The most downregulated transcripts in starvation encode ribosomal protein genes (RPGs) and RPGs contain most of the introns in the S. cerevisiae genome. The spliceosome is limiting in the cell and one of the ways non-essential energy-intensive processes are regulated during starvation is by inefficient splicing. The splicing index for the transcriptome was calculated when yeast were starved with glyphosate, depleting nutrients in the cell. The U3 snoRNA is an intron-containing Box C/D snoRNA required for processing the pre-rRNA into the 18S rRNA of the small ribosomal subunit. There are two paralogs that are expressed at different levels and have many polymorphisms in the intron. Splicing of the minor allele (U3b) is reduced to a greater extent during starvation. The minor allele has numerous SNPs and it is unknown if U3b functions differently than the higher expressed major allele. Regulation of splicing during starvation allows cells to rapidly reallocate resources away from energy-intensive ribosome biogenesis when resources are limited.
Keywords: splicing, starvation, U3 snoRNA
53. Post-Transcriptional Regulation of AR Variant 7 in Castration-Resistant Prostate Cancer
Samantha Gargasz (Cleveland State University, Department of Biological, Geological, and Environmental Sciences), Luca Argentieri (Cleveland State University, Department of Biological, Geological, and Environmental Sciences), Girish C. Shukla (Cleveland State University, Center For Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Science)
Abstract:
Prostate cancer (PCa) is the most commonly diagnosed cancer in males in the United States and the second leading cause of cancer-related death, with up to 50% of cases progressing to a metastatic, therapy-resistant stage of disease. Thus, the discovery of novel molecules involved in the progression of this disease is needed to uncover potential therapeutic strategies to combat tumors resistant to current treatment options. The long-noncoding RNA, ARLNC1, was first discovered due to its prostate-tumor-specific expression and interaction with the androgen receptor (AR), a major driver of PCa growth and progression. In PCa, ARLNC1 binds within the 3’ untranslated region (UTR) of the full-length AR mRNA, resulting in AR overexpression. This causes accelerated tumor growth and development. ARLNC1 has been demonstrated to regulate AR in a positive feedback loop. Recently, potential interactions between ARLNC1 and AR-V7, a splice variant of AR that arises in response to certain PCa treatments, have been uncovered through in silico sequence analysis. These putative interactions have been supported by in vitro studies suggesting that certain regions of the AR-V7 3’UTR could provide an interaction site for ARLNC1. The below experiments explore these interactions and propose a mechanism by which ARLNC1 also regulates AR-V7 expression in castration-resistant prostate cancer, though the details of this interaction have yet to be uncovered. The post-transcriptional landscape of castration-resistant prostate tumors expressing AR-V7 has not yet been investigated. We hypothesize that AR-V7 mRNA is controlled by a diverse post-transcriptional landscape of non-coding RNAs. Exploring the role of ARLNC1 and its role in the regulation of AR-V7 expression and activity will uncover novel molecular mechanisms contributing to PCa progression, providing potential therapeutic targets for the development of treatment strategies to combat castration-resistant disease.
References:
1. Siegel RL, Giaquinto AN, Jemal A. 2024. Cancer statistics, 2024. CA Cancer J Clin. 74(1):12-49.
2. Teo MY, Rathkopf DE, Kantoff P. 2019. Treatment of advanced prostate cancer. Annu Rev Med. 70:479-499.
3. Scher HI, Sawyers CL. 2005. Biology of progressive, castration-resistant prostate cancer: Directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol. 23(32):8253-8261.
Keywords: Prostate Cancer, non-coding RNA, 3UTR
54. Bacterial translation initiation factor IF-2’s N-terminal IDR promotes cold-induced phase separation
Aishwarya Ghosh (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI), Vidhyadhar Nandana (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI), Kaveendya Mallikaarachchi (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI), Jared M. Schrader (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI)
Abstract:
Bacterial cells, face challenges in organizing biochemical pathways because they generally lack membrane-bound organelles and have limited space. Biomolecular condensates (BMCs) are non-membrane bound organelles often assembled by phase-separation and offer a potential solution to organize their biochemical reactions in bacteria. BMCs composed of the machinery for DNA replication (1), transcription (2), and mRNA decay (3) have been identified. However, no BMCs for the mRNA translation machinery have been documented in bacteria. Our bioinformatic analyses of the bacterial translation machinery revealed that translation initiation factor 2 (IF-2) possesses a large intrinsically disordered region (IDR), suggesting it likely forms a BMC. To investigate the hypothesis that bacterial IF-2 can phase separate, we studied IF-2 from Escherichia coli and Caulobacter crescentus using both in vivo imaging and in vitro reconstitution approaches. Immunofluorescence results revealed that IF-2 localizes in foci within the cells of both species. We investigated the ability of IF-2 to phase-separate by purifying the IF-2 protein and conducting in vitro droplet formation assays. Our results demonstrate that IF-2 can phase separate in an RNA-dependent manner and that deleting the IDR strongly reduces phase separation. The deletion of the IDR in E.coli IF-2 was previously shown to yield a cold sensitive phenotype (4), but the source of cold sensitivity was not established. To investigate the role of IF-2 phase separation in E. coli cold sensitivity, we examined how temperature affects IF-2 phase separation. Our in vitro reconstitution experiments demonstrated that E. coli IF-2 phase separation is enhanced at lower temperatures and the cold-enhancement requires the IDR, suggesting that reduced phase separation of the IDR deletion may be the cause of cold sensitive growth. In conclusion, IF-2 phase separation provides the first evidence that bacteria possess translation machinery BMCs. Given the essential nature of central dogma processes in bacteria, IF-2 BMCs represent a potential new target for next-generation antibiotics against pathogenic E.coli that survive in refrigerated food.
References:
1. Harami, Gábor M., et al. "Phase separation by ssDNA binding protein controlled via protein− protein and protein− DNA interactions." Proceedings of the National Academy of Sciences 117.42 (2020): 26206-26217.
2. Ladouceur, Anne-Marie, et al. "Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation." Proceedings of the National Academy of Sciences 117.31 (2020): 18540-18549.
3. Al-Husini, Nadra, et al. "α-Proteobacterial RNA degradosomes assemble liquid-liquid phase-separated RNP bodies." Molecular cell 71.6 (2018): 1027-1039.
4. Brandi, Anna, et al. "Translation initiation factor IF2 contributes to ribosome assembly and maturation during cold adaptation." Nucleic Acids Research 47.9 (2019): 4652-4662.
Keywords: Phase separation, Biomolecular condensate, Translation initiation factor IF-2
55. Structural elucidation of an RNA triple helix in complex with a small molecule
Madeline M. Glennon (Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN), Martina Zafferani (Department of Chemistry, Duke University, Durham, NC), Conner J. Langeberg (Innovative Genomics Institute, University of California, Berkeley, CA), Jeffery S. Kieft (New York Structural Biology Center, New York, NY), Amanda E. Hargrove (Department of Chemistry, Duke University, Durham, NC), Krishna M. Shivakumar, Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN)
Abstract:
Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA with a 3′-terminal triple helix, which stabilizes and protects the RNA from degradation. This stabilization contributes to MALAT1 overaccumulation, promoting cancer and disease. The unique structure and function of the MALAT1 triple helix makes it an ideal target for small molecule intervention. Yet, structural details regarding the interactions between the MALAT1 triple helix and a small molecule drug remain unclear. Herein, I aim to solve a 3D structure of the MALAT1 triple helix in complex with a diminazene (DMZ) small molecule: DMZp8. Single-particle cryo-electron microscopy (cryo-EM) is a technique most suitable for solving large macromolecular structures, yet can be applied to solve structures of small RNAs (<50 kDa). Visualization of small RNAs is limited by the signal-to-noise ratio, hindering global resolution. To overcome these limitations, RNA scaffolding techniques graft an RNA-of-interest onto a larger, well-structured RNA scaffold. Herein, we grafted the MALAT1 triple helix onto two established RNA scaffolds: TTR-3 (PDB ID: 6WLK) and a circularly permuted version of the Tetrahymena ribozyme (TetP6b) (PDB ID: 8TJX). Thus far, we have solved the unbound 3D structures of the MALAT1 triple helix-TTR-3 (5.39 Å) and the MALAT1 triple helix-dTetP6b (3.11 Å). Additionally, optimal single-particle density and distribution was observed for the MALAT1 triple helix-TTR-3:DMZp8 complex at a 1:4 ratio. Promising single-particle conditions for the MALAT1 triple helix-dTetP6b scaffold were achieved using a 1:25 ratio of the MALAT1 triple helix-dTetP6b:DMZp8. A high-resolution structure of a small molecule bound to the MALAT1 triple helix will advance the rational design of small molecules selective for disease-promoting RNAs.
Keywords: RNA triple helix, cryo-EM, small molecule
56. Characterization and quantification of nucleic acid methylation catalyzed by the MTR1 ribozyme
Maddie Goodman (Denison University, Department of Chemistry/Biochemistry ), Rachel Mitton-Fry (Denison University, Department of Chemistry/Biochemistry )
Abstract:
Nucleic acid modification, including methylation, occurs frequently in biological systems as a means of genetic regulation. Ribozymes, or catalytic RNAs, typically engage in processes like RNA splicing and protein translation. Methyltransferase Ribozyme 1 (MTR1) was recently selected to catalyze the transfer of a methyl group from the small cofactor O6-methylguanine to a specific adenine on a target RNA strand. Previous studies have shown that MTR1 can methylate a variety of RNA substrates and use different cofactors. The aim of this research project was to further study MTR1, including in reactions with a DNA substrate and with fluorescent cofactors such as ATTO488-BG. The transfer of this fluorophore is helpful for quantifying reaction progress. Methylation was successful on a DNA substrate, which was observed to react faster than the RNA substrate. This finding was further supported quantitatively by the reactions where the fluorescent cofactor was used. Continuing to gain insights into the capabilities of MTR1 is valuable as it will allow for an understanding of its variability in terms of the substrates and cofactors it will interact with.
Keywords: ribozyme , methyltransferase
57. Simplifying eukaryotic transfer RNA Modification Mapping via FPLC
Aastha Gyawali (Department of Chemistry, University of Cincinnati), Scott Abernathy (Department of Chemistry, University of Cincinnati), Patrick Limbach (Department of Chemistry, University of Cincinnati)
Abstract:
Transfer RNA (tRNA) undergoes post-transcriptional modifications and these modified residues play important roles in RNA structure formation and stability. Moreover, tRNA modifications are known to have correlated and causal effects with a variety of human diseases. Eukaryotes can transcribe hundreds or even thousands of different tRNA sequences. Out of 150 total RNA modifications discovered, 93 modifications are found in tRNAs. Organisms typically express around 30-50 tRNA genes. However, many eukaryotic organisms may express hundreds or even thousands of different tRNA genes - even if some of these tRNAs only differ from one another by a single nucleotide change. Current LC-MS/MS approaches are unable to differentiate this complexity of tRNAs in the sample. Given the complexity of tRNA expression patterns, mass spectrometry-based RNA modification mapping, demonstrated for bacterial and archaeal organisms, can be complex. Thus, the goal of this project is to reduce the complexity of eukaryotic tRNA mixtures in a fashion that would mimic bacterial tRNAs. We selected FPLC as a fast, efficient, and reproducible fractionation method that can handle relatively large sample amounts using a strong anion exchange column with mobile phase A(20mM tris HCl, pH 8) and mobile phase B(1M NaCl + 20mM tris HCl) and weak anion exchange column with mobile phase A(20mM HEPES-KOH pH7.5) and mobile phase B (1MNaCl +20mM HEPES-KOH pH7.5). The standard sample used was 90 micrograms of total yeast tRNA for all studies. We investigated a variety of different mobile phase gradients - including both starting and ending %B - to identify those conditions that lead to appropriate and reproducible fractionation of the tRNA mixture. For each fraction, LC-MS/MS was used to identify the constituent tRNAs in each fraction, and LC-MS/MS sequencing using RNase T1 will be used in further study to verify the predictions of different yeast tRNAs present in each fraction.
Keywords: tRNA, FPLC, AEX
58. Identification and characterization of proteins that interact with the oncomiR-1 NPSL2 hairpin which may regulate miR-92a biogenesis through directing RNA conformational change
Cade T. Harkner (Department of Chemistry, University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109, USA), Sarah C. Keane (Department of Chemistry and Program in Biophysics, University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109, USA)
Abstract:
MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. To maintain proper microRNA expression levels, the enzymatic processing of primary (pri-) and precursor (pre-) microRNA elements must be strictly controlled. However, the molecular determinants underlying this strict regulation of microRNA biogenesis are not fully understood. RNA-binding proteins (RBPs) play important regulatory functions in the microRNA biogenesis pathway. We sought to identify proteins that could modulate RNA structures within the oncogenic polycistronic microRNA cluster miR-17~92a (oncomiR-1). The 3ʹ-domain of oncomiR-1 contains a four-way junction which is comprised of pri-miR-19b, pri-miR-92a, the non-precursor stem-loop element NPSL2, and helix50, a short helix linking back to the 5ʹ-domain. In the 3ʹ-domain, formation of the NPSL2 stem-loop structure sequesters the Drosha cleavage sites for pri-miR-92a within the junction, preventing formation of a pri-miR-92a basal helix, a structure essential for Drosha binding and processing. However, if the NPSL2 stem loop were unfolded and base paired with downstream sequence elements, the pri-miR-92a Drosha cleavage sites would be liberated and a basal helix could form. This structural rearrangement may function to regulate biogenesis of pre-miR-92a. We hypothesize that such rearrangement is protein-mediated, and thus performed proteomics-based screening to identify protein binding partners for the NPSL2 hairpin. Among our top hits are the DEAD-box ATPase DDX24 and hnRNPLL. In this work, we focused on hnRNPLL, a RBP with four RNA-recognition motifs (RRMs) in a 2x2 arrangement. We show that hnRNPLL does bind the NPSL2 stem-loop in vitro. We show that isolated dual RRMs bind NPSL2 with low affinity, but a construct containing all four RRMs shows moderate (low micromolar) binding. Follow-up work will investigate whether hnRNPLL can induce NPSL2 remodeling in vitro. This work continues to highlight the importance of RNA-protein interactions on the biogenesis of microRNAs, particularly from structurally complex polycistronic clusters.
Keywords: miRNA, hnRNP, RBPs
59. Quantification of RNA nanoparticles in the eye tissues using LC-MS
Mohammed Hassan (Chemistry Department / University of Cincinnati), Kevin Li (College of Pharmacy / University of Cincinnati), Patrick Limbach (Chemistry Department / University of Cincinnati)
Abstract:
Effective ocular drug delivery for treatment chronic posterior eye diseases remains a challenging task. RNA nanoparticles, with defined size and shape, could be used as vectors for enhancement of delivery for RNA-based therapeutics. Moreover, they can be fabricated to allow simultaneous targeting and/or delivery of multiple therapeutic and targeting ligands. Consequently, this could be used to achieve synergistic effects for drug delivery and therapy. On the other hand, quantification of small RNA molecules in a complex sample is challenging as well. However, PCR is the gold standard for RNA quantification, it doesn’t work with small RNA. Here, This first report for LC-MS assay for quantification of RNA nanoparticles in the murine eye tissues after subconjunctival injection in the mouse eye. Our results show that the limit of detection is in femtomolar range. This assay would open the door for starting the pre-clinical studies on the delivery of RNA nanoparticles to the eye.
References:
1. Urtti A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv Drug Deliv Rev. 2006;58:1131–1135.
2. Lee SS, Robinson MR. Novel drug delivery systems for retinal diseases. A review. Ophthalmic Res. 2009;41:124–135
3. Khisamutdinov EF, Li H, Jasinski DL, Chen J, Fu J, Guo P. Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles. Nucleic Acids Res. 2014;42:9996–10004.
4. Jasinski DL, Khisamutdinov EF, Lyubchenko YL, Guo P. Physicochemically tunable polyfunctionalized RNA square architecture with fluorogenic and ribozymatic properties. ACS Nano. 2014;8:7620–7629.
Keywords: LC-MS, Quantification , RNA
60. The role of IDRs in the assembly pathway of pre-ribosomes
Stefanie A. Hedayati (Department of Biological Sciences, Carnegie Mellon University), Collin Bachert (Department of Biological Sciences, Carnegie Mellon University), John L. Woolford (Department of Biological Sciences, Carnegie Mellon University)
Abstract:
Eukaryotic ribosome biogenesis occurs in a step-wise fashion in the nucleolus, nucleoplasm and cytoplasm. About 200 assembly factors (AFs) are required for the maturation of both the large and small subunit. Cryo-EM structures of co-transcriptional to cytoplasmic pre-ribosome intermediates have provided structural information about the composition and conformation of assembly factors, ribosomal proteins and compacted rRNA in these complexes. However, many AFs contain intrinsically disordered regions that are not visualized by cryo-EM. Interestingly, a major fraction of IDR-containing AFs is released right when pre-ribosomes transit from the nucleolus to the nucleoplasm. We hypothesize that these IDRs contribute to form the nucleolar condensate, a phase-separated non-membrane bound compartment, via trans interactions between pre-ribosomal particles, and that the release of these IDR-containing AFs triggers the nucleolar exit of pre-ribosomes. Consistent with this idea, mutations that block the release of these factors prevent the nucleolar exit of pre-ribosomes. To more specifically assess the roles of these IDRs in ribosome assembly as well as ribosome flux through the nucleolar and nuclear compartments, we deleted the non-visible IDRs in select AFs and assessed growth, polysome profiles and pre-ribosome composition. Certain IDR deletions led to slow growth or temperature-sensitive phenotypes, a decrease in 60S subunits, altered composition of pre-ribosomes and sensitivity to the mistranslation-inducing antibiotic paromomycin. Similarly to IDRs, non-compacted rRNA is also often poorly resolved in early pre-ribosomal intermediates. Hence, it is possible that this non-compacted rRNA is involved in trans interactions between pre-ribosomal particles. Thus, we also want to determine the contribution of rRNA to nucleolar phase separation and release of pre-ribosomes from the nucleolar condensate.
Keywords: ribosome assembly, nucleolus, intrinsically disordered
61. Ribosome association excludes stress-induced gene mRNAs from stress granules
Noah S. Helton (Department of Human Genetics, University of Michigan ), Ben Dodd (Department of Human Genetics, University of Michigan ), Stephanie Moon (Department of Human Genetics, University of Michigan )
Abstract:
Cells respond to stress via the integrated stress response (ISR), which causes translation suppression, stress-induced gene expression, and assembly of biomolecular condensates called stress granules. Despite the temporal connection between stress-induced gene expression and stress granules, little is known about how these two ISR branches interface. No direct, quantitative, and mechanistic analysis of the relationship between stress-induced gene mRNAs and stress granules has been performed. Stress granules sequester non-translating mRNAs away from the translation machinery and stress-induced genes evade translational suppression via mechanisms including upstream open reading frames (uORFs). These genes include the transcription factor ATF4 and the translation depressor GADD34, which are crucial for remodeling gene expression in response to stress. Therefore, we hypothesized that stress-induced gene mRNAs would be excluded from stress granules to enable their expression. Using a panel of small-molecule translation inhibitors and single-molecule imaging, our data reveal that the translationally upregulated mRNAs, ATF4 and GADD34, are excluded from stress granules in part by their association with one or more ribosomes. This suggests a role for uORFs in preventing mRNA condensation during stress. Further, we show that trapping a single ribosome on mRNAs is sufficient to inhibit stress granule formation, supporting that translation complexes prevent mRNA assembly into stress granules independently of ribosomal translocation or occupancy across the open reading frame. We also observed reduced ATF4 and GADD34 expression and cellular resilience to stress in a stress granule deficient G3BP1/2 knock-out cell line. Together, we provide evidence that the major stress-induced genes that reprogram gene expression evade stress granule sequestration through ribosome association and G3BP1/2 promote their expression during the recovery from stress.
Keywords: Translation, Stress Granules, Integrated stress response
62. CHARACTERIZATION of Transcription Product Outcomes in T-box Riboswitch Modulation
Danushika Herath (Chemistry and Biochemistry, Ohio University), Jennifer V. Hines (Chemistry and Biochemistry, Ohio University)
Abstract:
Developing new antibacterial compounds and finding novel targets is crucial as a solution for antibacterial drug resistance. Non-coding RNA such as T-box riboswitches can be used as efficient drug targets against antibacterial resistance because they regulate amino-acid-related genes essential for bacterial survival. The main objective of this project is to characterize and quantify transcription product outcomes for modulating the T-box riboswitch function. It would provide insights into the RNA structure-function relationships in finding novel drugs against antibacterial resistance. Grundy et al., 2002, discovered that tRNA plays a key role in T-box riboswitch antitermination and this tRNA-dependent antitermination can be studied in vitro in bacterial transcription systems such as Bacillus subtilis by performing in vitro transcription antitermination (IVT) assays. We designed four variants with modified glyQS T-box antiterminator sequences, by mutating the 100% conserved 10th nucleotide position of the 5’- bulge in the glyQS T-box antiterminator of Bacillus subtilis, which is found to be involved in the stacking interactions with the corresponding tRNA. Changes in the transcription antitermination as a result of the specific modifications were assessed through the transcriptional read-through by performing IVT assays. Through these approaches, the role of the specific structural elements in glyQS T-box riboswitch regulation can be elucidated.
Keywords: T-box, Antiterminator, Antibacterial
63. Investigating the role of kinetochore component HCP-1 perinuclear condensates for successful cell division in C. elegans
Hannah L. Hertz (Biological Chemistry and Pharmacology, The Ohio State University), Ian F. Price (Biological Chemistry and Pharmacology, The Ohio State University), Wen Tang (Biological Chemistry and Pharmacology, The Ohio State University)
Abstract:
In C. elegans, HCP-1 and HCP-2 are key components of the kinetochore which initiates central spindle formation and promotes proper chromosome segregation during mitosis. Similar to their mammalian counterpart, HCP-1/2 exhibit dynamic localization patterns, transitioning from the nuclear envelope during prophase to kinetochores during cell division. Although the localization of HCP-1 at kinetochores is well studied, less is known why HCP-1 localizes to the nuclear envelope. Here we seek to characterize this dual localization of HCP-1 throughout the cell cycle. We found prior to cell division HCP-1 is stored in condensate-like foci at the nuclear envelope along with nucleoporins. Notably, perinuclear localization of HCP-1 is dependent on the nucleoporin NPP-14. By disrupting perinuclear HCP-1 localization in npp-14 mutants, our project seeks to elucidate whether perinuclear HCP-1 is essential for its subsequent recruitment to kinetochores and the proper assembly of the central spindle. Moreover, we seek to address whether proper perinuclear localization of HCP-1 in adults is required for proper partitioning of HCP-1 and successful mitosis in their progeny.
Keywords: Condensates, Kinetochore
64. High-throughput molecular evolution function exploration of the fluoride riboswitch
Laura Marie Hertz (Department of Chemical and Biological Engineering, Northwestern University), Elena Rivas (Department of Molecular and Cellular Biology, Harvard University), Julius B Lucks (Department of Chemical and Biological Engineering, Northwestern University)
Abstract:
RNA structure remains elusive to biophysical techniques due to its flexible backbone and exploration of different ensemble states. However, since the 90s comparative genomics has demonstrated how nucleotides covary within genetic sequences to reveal RNA structures. Here, we expand upon these computational techniques to explore the complete evolution of riboswitches, which are typically cis-regulator RNA elements in the 5` UTR of genes in response to ligand binding. Covariation analysis has only revealed half the structure of a riboswitch, the ligand binding half, due to high variability of structure and function of the gene expression half. We address this gap and then bring comparative genomics out of the computer by developing a high-throughput RNA-sequencing assay to test every unique occurrence of the fluoride riboswitch. We compare the accuracy of computational predictions and validate hits of interest. These findings have led to development of new hypothesizes around the evolution of the RNA sequence-structure-function relationship with implications for biotechnological innovation.
Keywords: riboswitch, structure, high-throughput
65. The Role of YHR020W 5’ UTR Isoforms in Stress Tolerance
Audrey Hoelscher (University of Michigan, Department of Biological Chemistry), Charlotte Clark-Slakey (University of Michigan, Department of Biological Chemistry), Rachel Niederer (University of Michigan, Department of Biological Chemistry)
Abstract:
Translational control of gene expression influences cellular diversity, stress response, and disease. While the extent of mRNA features that control protein output are not fully characterized, evidence suggests that 5’ untranslated regions (UTRs) contain potent features which regulate protein output and control gene expression. Importantly, many genes utilize 5’ UTR isoforms that contain distinct regulatory elements. For example, previous work has shown that some 5’ UTR isoforms in yeast can drive >30-fold differences in ribosome recruitment. Here, we were interested in whether expression of 5′-UTR isoforms known to differentially recruit ribosomes can drive phenotypic differences in yeast. To test this, we have ectopically expressed either the long or short 5’ UTR isoforms from the gene YHR0202W in a knockout background. The knockout cells exhibit altered stress tolerance, which can be rescued by expression of the gene with the long 5’ UTR isoform. Next, to determine the exact location of the functionally relevant features we are generating scanning mutations in the unshared sequence of the long isoform. Luciferase output of the mutants will be measured and compared to the original isoform protein output. The insights found on 5’UTR regulatory features will provide a better understanding of the role of translational control elements in human diseases.
Keywords: translational control, 5UTR
66. Comprehensive analysis of SARS-CoV-2 5’ UTR functional elements that facilitate evasion of Nsp1-mediated translational shutdown
Jacob S. Horn (Cellular and Molecular Biology Program, University of Michigan), Rachel Niederer (Department of Biological Chemistry, University of Michigan)
Abstract:
SARS-CoV-2 protein Nsp1 induces a global translation shutdown in host cells upon infection. The viral genome can escape the translational shutdown via secondary structure in its 5’ untranslated region (UTR). The first hairpin structure, stem-loop 1 (SL1), has been identified as necessary and sufficient to evade Nsp1-mediated translation shutdown. Despite proven functional roles within the 5’ UTR, other elements remain understudied. We wondered if the 5’ UTR has other functional regions that might influence translational control and evasion of the translational shutdown. We will use a recently developed method called direct analysis of ribosome targeting (DART), a high throughput method that tests the ribosome recruitment ability of thousands of 5’ UTRs. We will generate a diverse pool of sequences that will allow thorough examination of each region of the 5’ UTR and its role in evasion. The pool will include all known natural, scanning, structural, and compensatory mutations. Completing DART with and without Nsp1 will elucidate what elements facilitate the evasion of Nsp1-mediated translational shutdown.
Keywords: 5 UTR, Translation, SARS-CoV-2
67. Pseudouridine synthase 1 becomes localized more to the cytoplasm during stress in C. albicans
Michael R. Hrabak (Department of Biology Ball state), Douglas A. Bernstein (Department of Biology Ball state)
Abstract:
Pseudouridine is a crucial RNA modification found in all domains of life. Pseudouridine synthases (Pus) carry out this modification. Most investigations into these enzymes in fungi have been completed in the model S. cerevisiae. Relatively little is known about this process in the opportunistic pathogen C. albicans. C. albicans is a diploid fungus and thus has two alleles for almost all genes, including PUS1, a pseudouridine synthase. In S. cerevisiae, Pus1 modifies several sites on tRNA. S. cerevisiae Pus1 also plays a role in the nuclear export of tRNAs; thus, Pus1 localizes to the nucleus. PUS1 is nonessential for growth under optimal growth conditions. However, the loss of PUS1 displays a growth delay at elevated temperatures. Other Pus enzymes have also demonstrated sensitivity to elevated temperatures and other stress conditions, indicating an importance of these enzymes for cellular health during stress conditions. Here, we analyzed the localization of the two C. albicans Pus1 proteins under stress conditions caused by heat, cold, and hydrogen peroxide. We also analyzed the localization of the two proteins during exposure to metals such as lead, chromium, and zinc. We tagged Pus1a or Pus1b with GFP and measured the fluorescent intensities inside the nucleus and the cytoplasm, generating a cytoplasmic-to-nuclear ratio. We used this ratio to compare the localization of the proteins during different stress responses. Our results demonstrated increased cytoplasmic localization during heat stress for Pus1a and Pus1b. Our results demonstrated decreased cytoplasmic localization for both Pus1a and Pus1b during chromium exposure. Interestingly, the two proteins differed in cytoplasmic localization levels when treated with hydrogen peroxide, lead nitrate, and zinc, suggesting distinct roles for Pus1a and Pus1b.
Keywords: Pseudouridine synthase, C albicans
68. Calculations of pKa values for canonical nucleosides and nucleoside analogs
Sun Jeong Im (Department of Chemistry, Wayne State University), Evan L. Jones (Department of Chemistry, Wayne State University), Alan J. Mlotkowski (Department of Chemistry, Wayne State University), H. Bernhard Schlegel (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)
Abstract:
Nucleosides play diverse structural and functional roles in biological systems. Understanding fundamental properties of the nucleosides is important for their applications as chemical probes of nucleic acid structure and function. As the nucleosides are tuned to modify their binding properties, their physical properties such the pKa values may also change. Unlike canonical nucleosides, modified nucleosides are often not well understood in terms of acid-base properties. Previously, theoretical pKa values of canonical and aza-/deaza-modified nucleobases were determined.1, 2 In this study, the theoretical pKa values for canonical nucleosides and nucleoside analogs such as antiviral compounds were calculated by using an ab initio quantum mechanical method and B3LYP density functional with 6-31+G(d,p) basis set along with an implicit-explicit solvation model. The results of these computations will be presented along with comparisons to known experimental pKa values. The ability to estimate theoretical pKa values will be beneficial for further development of modified nucleosides and their applications.
References:
1. Jones, E. L.; Mlotkowski, A. J.; Hebert, S. P.; Schlegel, H. B.; Chow, C. S., Calculations of pKa Values for a Series of Naturally Occurring Modified Nucleobases. The Journal of Physical Chemistry A 2022, 126 (9), 1518-1529.
2. Mlotkowski, A. J.; Schlegel, H. B.; Chow, C. S., Calculated pKa Values for a Series of Aza- and Deaza-Modified Nucleobases. The journal of physical chemistry. A 2023, 127 (15), 3526-3534.
Keywords: pKa calculations, nucleosides, nucleoside analogs
69. ADAR1 Suppresses PKR-Mediated Stress Granule Oscillations in Interferon-Stimulated Cells
Amra Ismail (Department of Biochemistry and Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Saad Badat (Department of Biochemistry and Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Joseph M. Luna (Department of Biochemistry and Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)
Abstract:
Stress granules (SGs) are cytoplasmic condensates traditionally viewed as stable indicators of cellular stress. Our study identifies previously unobserved dynamic behaviors of SGs and suggests an important role for ADAR1 in regulating stress responses. We demonstrate that in ADAR1-depleted cells, interferon stimulation induces periodic formation and dissolution of SGs. This oscillatory behavior is mediated by protein kinase R (PKR) activation in response to double-stranded RNA (dsRNA) accumulation. In wild-type cells, ADAR1 suppresses this oscillation by editing dsRNA, preventing PKR activation. Using high-resolution live-cell imaging and machine-learning classification, we quantified SG dynamics for up to 3 days. In ADAR1-knockout cells, we observed initial synchronized SG oscillations followed by unsynchronized patterns. These oscillations persisted after interferon removal and required an intact interferon signaling pathway. Mechanistically, interferon treatment of ADAR1-depleted cells establishes an auto-activation loop coupled with negative feedback from PKR activation, enabling self-perpetuating SG oscillation. Conversely, PKR knockout resulted in loss of SGs and reduced interferon levels, further confirming PKR's central role in this process. Our findings reveal a previously unknown function of ADAR1 in suppressing stress granule oscillations and provide insights into the interplay between RNA editing, innate immunity, and cellular stress pathways. This work offers a new perspective on stress granule dynamics and their potential role in antiviral responses, laying the groundwork for understanding how cells fine-tune their response to persistent stressors.
Keywords: Stress Granules, RNA-binding proteins
70. Investigating functions outside of tRNA for a bacterial 3'-5' reverse polymerase from the Thg1/TLP superfamily
Brandon Iwaniec (Chemistry and Biochemistry, The Ohio State University), Susanne Mueller (Department of Microbiology and Immunology, Medical College of Wisconsin), John. R. Kirby (Department of Microbiology and Immunology, Medical College of Wisconsin), Jane Jackman (Chemistry and Biochemistry, The Ohio State University)
Abstract:
tRNAHis guanylyltransferase (Thg1)-like proteins (TLPs) catalyze nucleotide addition to RNA substrates in the opposite direction (3'-5') of canonical RNA polymerases. This biochemical activity is seen in vitro with bacterial TLPs, which can act on the 5'-ends of tRNA substrates, similar to the known biological functions of the two characterized eukaryotic TLPs. However, no physiologically relevant substrates for any bacterial TLPs have been identified to date and there is no known requirement to repair tRNA in these species that would make use of the observed in vitro activity. Here we report the first investigation of the biological function of the 3'-5' RNA polymerase in the bacterium Myxococcus xanthus. When the M. xanthus TLP (MxTLP) gene is deleted, the cells exhibit defects in starvation-induced fruiting body formation and sporulation, suggesting that this enzyme plays a role in maturation or maintenance of one or more biologically relevant RNAs. We used the Δmxtlp strain to rule out a function for MxTLP in adding of an essential G-1 nucleotide to the 5'-end of tRNAHis, which is an important function of some eukaryotic members of the Thg1 family. These results are consistent with the existence of an alternative RNase P-dependent pathway for incorporation of G-1 into tRNAHis in bacteria like M. xanthus, whereby this residue is encoded in the tRNA gene and not necessary to be added post-transcriptionally. Instead, biochemical studies with purified MxTLP demonstrate that the enzyme can incorporate labeled nucleotides into several larger RNA species isolated from vegetative and developing cells, consistent with a function for the enzyme involving substrates other than tRNA. Efforts are ongoing to identify and test RNA targets for MxTLP activity using enhanced CLIP (eCLIP) that would be leading candidates as substrates of MxTLP. These results will be used to help elucidate the first biological function of a TLP in any bacterial system.
Keywords: Reverse polymerization , tRNA, Myxococcus xanthus
71. The Role of 5’ UTR in gene expression regulation
Zeqing Jiang (Department of Biological Sciences, Carnegie Mellon University), Gemma May (Department of Biological Sciences, Carnegie Mellon University), Cassia Williams-Rogers (Washington University in St. Louis), Haoran Wang (Department of Biological Sciences, Carnegie Mellon University), Dr. Joel Mcmanus (Department of Biological Sciences, Carnegie Mellon University)
Abstract:
Protein synthesis is a vital process in all living organisms. Titin (TTN), the largest protein found in the human body, serves a critical function in muscle contraction and the operation of sarcomeres. The mutation in the titin protein can cause the development of typical cardiac diseases like dilated cardiomyopathy and heart failure. Notably, the Titin 5’ UTR is among the most conserved in humans. We performed DMS-Map to model the structure of the Titin 5’ UTR. This revealed three RNA stem loops, which are also conserved. The 5’ UTR also harbors a tandem upstream open reading frame (uORF). We used luciferase reporter assays to evaluate the influence of these stem loops and uORFs on titin gene expression. Our results shows that disruption of the stem-loop structures and uORFs increased reporter expression in both HEK293T cells and C2C12 myoblast cells. Interestingly, Titin stemloop 2 has been lost in two clades of South American bats and rodents. However, the Titin UTRs from two of these species appear to repress translation as well as the human 5’ UTR. This work suggests that highly conserved sequences in the Titin 5’ UTR collectively reduce ribosome loading, which may be beneficial for Titin expression in heart and skeletal muscle tissue.
References:
1. Shadrin, I Y et al. (2016). Striated muscle function, regeneration, and repair. Cellular and molecular life sciences: CMLS vol. 73,22: 4175-4202.
Keywords: Titin, 5 UTR, RNA secondary structure
72. Distal elements in the MDM2 gene facilitate an eight-exon splicing event by coordinating multiple splice regulatory proteins
Whitney D. Jimenez (The Ohio State University College of Medicine, Biomedical Sciences), Zac LaRocca-Stravalle (Abigail Wexner Research Institute at Nationwide Childrens Hospital), Aishwarya G. Jacob (University of Cambridge), Ravi K. Singh (University of Houston), Dawn S. Chandler (The Ohio State University )
Abstract:
Alternative splicing is a key post-transcriptional mechanism that diversifies biological processes. Recent advances in high-throughput sequencing have uncovered many alternatively spliced variants involved in tumorigenesis, highlighting the role of splicing regulation in cancer. Our lab focuses on MDM2-Alt1, a spliced variant of the oncogene MDM2, a negative regulator of the tumor suppressor p53. In response to cancer or genotoxic stress, MDM2 undergoes alternative splicing to form MDM2-Alt1, consisting of exons 3 and 12. This variant is upregulated in tumors such as breast, lung, and rhabdomyosarcomas (RMS), but little is known about the regulators of its splicing. We hypothesize that MDM2-Alt1 splicing is driven by trans factors and cis elements on exons 4 and 11 of MDM2 pre-mRNA, orchestrating an 8-exon splice event.
Using RNA affinity chromatography and mass spectrometry, we identified several trans factors binding cis elements on intron 11 under damaged conditions. Notably, RNA-binding proteins FUBP1, MATR3, and PTBP1 were confirmed to interact with intron 11 of the MDM2 minigene. Our lab demonstrated that FUBP1 and PTBP1 positively regulate MDM2 splicing, while MATR3 promotes MDM2-Alt1 formation. Chimeric minigene experiments also highlighted exons 4 and 11 as critical for the damage response. We identified cis elements in intron 11 and exon 11 and the trans factors governing MDM2 splicing. FUBP1 and PTBP1 support canonical splicing, while MATR3 drives the formation of MDM2-Alt1. This work emphasizes the importance of exons 4 and 11 in genotoxic stress response and elucidates the dynamic interaction between cis elements and trans factors. Our findings advance the understanding of MDM2 alternative splicing and its regulatory balance. This research opens avenues for targeting alternative splicing in cancer therapy, with potential therapeutic strategies aimed at modulating MDM2-Alt1 splicing in tumors where it is implicated.
Keywords: MDM2, Splicing, RNA-Binding Proteins
73. Translational control of a unique bicistronic gene linked to Prader-Willi Syndrome
Lucia Johnson (Biological Sciences Department, Carnegie Mellon University), Gemma May (Biological Sciences Department, Carnegie Mellon University), Robert Nicholls (Pediatrics Department, UPMC), Joel McManus (Biological Sciences Department, Carnegie Mellon University)
Abstract:
Prader-Willi Syndrome (PWS) is a developmental disorder characterized by low-muscle tone, developmental delays, intellectual impairment, and hyperphagia (1). Diagnosis is determined by detection of genomic abnormalities in chromosome 15 (15q11.2-q13), including deletions or epigenetic errors in the paternal chromosome, or inheritance of two maternal copies and no paternal copy of the chromosome (maternal parental disomy; 2). Within this region lies a unique bicistronic gene, SNURF-SNRPN, that encodes the SNURF (SNRPN Upstream Reading Frame) and SNRPN (Small Nuclear Riboprotein N) genes (3). SNRPN encodes SmN, a neuronal-specific spliceosomal snRNP protein, but the function of SNURF remains to be determined (1). Because eukaryotic translation typically initiates via directional scanning, SNURF’s presence should prevent translation of SNRPN. How, then, is SNRPN translated? To investigate this, we transfected tissue culture cells with luciferase reporter mRNAs. Our results show that translation of SNRPN requires cap-dependent directional scanning, indicating SNRPN does not have an Internal Ribosome Entry Site. Intriguingly, we found a highly conserved upstream open reading frame (uORF) upstream of SNURF that promotes SNRPN translation. Mutation of the uORF start codon decreases translation initiation at SNRPN in a SNURF-dependent manner. These results suggest that a uORF, not an IRES, is responsible for efficient translation of SNURF-SNRPN, likely due to delayed reinitiation. We are carrying out additional experiments to further test this model and evaluate the potential for translational regulation under stress in a variety of PWS relevant cell types.
References:
1. Chung, Michael S et al. “Prader-Willi syndrome: reflections on seminal studies and future therapies.” Open biology vol. 10,9 (2020): 200195. doi:10.1098/rsob.200195
2. Driscoll DJ, Miller JL, Cassidy SB. Prader-Willi Syndrome. 1998 Oct 6 [Updated 2023 Nov 2]. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2024. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1330/#
3. Gray, T A et al. “An imprinted, mammalian bicistronic transcript encodes two independent proteins.” Proceedings of the National Academy of Sciences of the United States of America vol. 96,10 (1999): 5616-21. doi:10.1073/pnas.96.10.561
Keywords: translation
74. Studying the Mechanism for Ribosome-Inactivating Protein-Induced Apoptosis
Daniel M. Judge (West Virginia University), Jennifer E.G. Gallagher (West Virginia University)
Abstract:
Apoptosis, a conserved process of programmed cell death, can be activated through diverse cellular mechanisms, many of which remain poorly understood. This study focuses on a specific mechanism triggered by ribosome-inactivating proteins (RIP), a category of potent toxins such as ricin and sarcin. Ricin halts translation by irreversibly damaging large ribosomal subunit rRNA through an adenine N-glycosidic cleavage referred to as depurination. While the role of the catalytic subunit of ricin, known as ricin toxin A chain (RTA), is well-established, the precise process that initiates apoptosis in RTA-compromised cells remains unclear. Importantly, while ricin induces apoptosis, translation inhibitors employing mechanisms other than depurination, such as the peptidyl transferase inhibitor anisomycin, often only induce cell cycle arrest. Ribosome depurination may be a critical step in initiating apoptosis. We hypothesize that ribosomes translating on the same mRNA would collide with the stalled depurinated ribosome resulting in improper recycling when translation terminates. The resulting physical damage to ribosomes would activate ribosome quality control mechanisms, ultimately leading to apoptosis. In this study, we will utilize an in vivo ricin reporter plasmid to modulate RTA gene expression and examine the apoptotic response in Saccharomyces cerevisiae. We will closely monitor ribosomal biogenesis, localization, and degradation before and during apoptosis and compare the effects of wildtype RTA to catalytically dead mutants such as E177K, S215F, and P95L, which do not induce apoptosis. Genetic screens will be conducted to identify the essential elements required for ricin-induced apoptosis, and in-lab evolution experiments will be carried out to identify mutations that confer resistance to ricin-induced apoptosis. Long-term, this research aims to provide a comprehensive understanding of the cellular pathways affected after exposure to RTA and establish a controlled method for reliably inducing apoptosis in specific target cells. The insights gained from this study are likely to have broader implications in the fields of cancer biology, embryology, and immunology, offering new avenues for exploring cellular homeostasis and understanding how cells respond to stress.
Keywords: Stress, Ribosomes, Apoptosis
75. Combined CRISPR-activation and CRISPR-interference capabilities using dCas9 and G-quadruplex Structures
Mohammad Lutful Kabir (Chemistry and Biochemistry,Kent State University ), Sineth G. Kodikara (Physics,Kent State University), Mohammed Enamul Hoque (Chemistry and Biochemistry,Kent State University ), Soumitra Basu (Chemistry and Biochemistry,Kent State University ), Hamza Balci (Physics,Kent State University)
Abstract:
Catalytically dead Cas9 (dCas9) has been employed in CRISPR-interference (CRISPRi) and CRISPR-activation (CRISPRa) to modulate transcription (gene expression). Altering the stability of G-quadruplex structures located in promoters is also known to modulate transcription (gene expression). We demonstrate that both CRISPRi and CRISPRa are feasible within the same system by targeting the vicinity of a putative GQ sequence in c-Myc promoter with CRISPR-dCas9. The achieved suppression and activation levels are at or beyond above those reported with alternative approaches, such as drug like small molecules which lack sequence specificity or site-directed mutations which are permanent. In addition to demonstrating up or down regulation of c-Myc in Burkitt's Lymphoma cell line (at both RNA and protein levels) and the resulting impact on cell viability, we demonstrate extensive in vitro studies probing the underlying mechanism. Our studies illustrate dCas9 to be more likely to block RNAP progression and suppress transcription when the non-template strand is targeted, the orientation in which the GQ is also stabilized. Targeting multiple sites in the non-template strand simultaneously with dCas9 further suppressed transcription (~2-fold for single site and ~5-fold for dual site targeting). When the template strand was targeted, CRISPR-dCas9 destabilized the GQ and enhanced transcription (~2-fold). Our study demonstrates the promising potential of targeting PQS with CRISPR-dCas9 as a means to differentially regulate transcription in a simple system that combines both CRISPRa and CRISPRi capabilities.
Keywords: CRISPR,dCas9,G-quadruplex, Transcription Regulation, , RNA Polymerase
76. Translational control of HIV-1 unspliced RNA is regulated at the level of transcription through an RNA structure-guided mechanism
Joseph G Kanlong (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University, Columbus, OH), Zetao Cheng, Saiful Islam, Olga A. Nikolaitchik, Wei-Shau Hu (Viral Recombination Section, HIV DRP, NCI, Frederick, MD), Crystal Stackhouse, Joseph D. Puglisi, Elisabetta Viani Puglisi, (Department of Structural Biology, Stanford University School of Medicine, Stanford, CA), Vinay K. Pathak (Viral Mutation Section, HIV DRP, NCI, Frederick, MD), Michael G. Kearse (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus OH), Madeline Sheppard, Heewon Seo, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH)
Abstract:
The 9.2 kb unspliced viral RNA of Human Immunodeficiency type 1 serves a dual purpose; it functions as the viral genome, packaged into new viral particles as a dimer, and as the mRNA template for the synthesis of the essential structural proteins Gag and Gag-Pol. During transcription of HIV-1 RNA by host cell RNA polymerase II, various transcription start sites are used resulting in heterogeneous RNAs containing either one (1G), two, or three (3G) 5ʹ guanosines and a modified 5ʹ cap. Despite 3G being the predominant species in the cell, 1G is selectively packaged as genomic RNA (gRNA). We have shown previously that the 5ʹ UTR of 1G and 3G RNAs adopt distinct conformational ensembles that favor selective 1G packaging by exposing structural elements required for efficient gRNA dimerization, Gag binding, and Gag multimerization. Additionally, mutations that eliminate structural differences between the 1G and 3G 5ʹ UTR abolish selective 1G packaging. Our recent in vitro and cell-based assays demonstrated that 3G RNAs are translated more efficiently than 1G RNAs because of their distinct 5’ UTR conformational ensembles. However, in vitro assays indicated that both the 1G and 3G 5’ UTR structures are generally inhibitory to translation when compared to unstructured controls, consistent with a requirement for additional host factors in cells. While we have shown that these two species of RNA are translated with varying efficiencies, the underlying mechanisms that govern HIV-1 translation initiation are not well understood. Electrophoretic mobility shift assays and single-molecule FRET experiments revealed that both 1G and 3G RNAs are bound and unwound similarly by eukaryotic initiation factor (eIF) 4F, the heterotrimeric complex responsible for mRNA activation, and load 43S ribosomes with similar efficiencies. Future work will test the hypothesis that distinct 5ʹ UTR conformational ensembles result in altered host factor binding and ribosome scanning efficiencies.
Keywords: HIV-1, Translation, RNA structure
77. Investigating tRNA maturation by Protein-Only RNase P (PRORP) & the Elucidating the Role of Interacting Methyltransferases in Arabidopsis thaliana
Abigail Kelly (Chemistry, University of Michigan)
Abstract:
Ribonuclease Proteins (RNase Ps) are a class of enzymes responsible for cleaving the 5' leader sequence of premature tRNAs (pre-tRNA). In 1998, a subclass of RNase P was discovered in the human mitochondria, termed protein-only RNase Ps (PRORPs), which were not catalytically driven by RNA. The complex consisted of PRORP, the catalytic center, and two accessory proteins required for efficient cleavage- TRMT10C, a methyltransferase, and SDR5C1, a dehydrogenase. Later, single subunit PRORPs were discovered in non-metazoan eukaryotes. Much of what we know about these enzymes comes from studies in Arabidopsis thaliana. There are three isoforms of PRORPs in this model organism, with At-PRORP1 localizing in the mitochondria and chloroplasts, and At-PRORP2 and At-PRORP3 localizing in the nucleus. While these single subunit PRORPs do not contain accessory proteins like metazoan mitochondrial (mt) RNase P, it was recently discovered that At-PRORP2 interacts with methyltransferases At-TRM1A and At-TRM1B. Knockout of both methyltransferases were found to be lethal to plant growth and pre-tRNA processing. I am currently conducting assays to determine 5' cleavage rates for different At-tRNAs to discover if the enzyme has a sequence or structural preference for cleavage. Additionally, I am working on cloning and purifying At-TRM1A and At-TRM1B to see what effect they have on cleavage rates. It has been shown that miscleavage occurs in vitro for specific tRNAs such as At-tRNACys. I will be investigating which At-tRNAs are prone to miscleavage and if TRM1A and TRM1B can rescue these miscleavage events. My project is aimed to better elucidate the PRORP interactome, as the enzyme was previously thought to act independently.
References:
Arrivé, M. et. al, (2023). A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei. Nature Plants, 9, 2031–2041.
Howard, M. J., et. al, (2016). Differential substrate recognition by isozymes of plant protein-only Ribonuclease P. RNA, 22(5), 782–792.
Rossmanith, W., & Karwan, R. M. (1998a). Characterization of human mitochondrial RNase P: Novel aspects in tRNA processing. Biochemical and Biophysical Research Communications, 247(2), 234–241.
Wilhelm, C. et. al, (2024).The protein-only RNase Ps, endonucleases that cleave pre-tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes. WIRES RNA, 15(2), e1836.
Keywords: RNase P, tRNA modifying enzyme, Cleavage kinetics
78. The evolutionarily conserved polyadenosine RNA binding protein Nab2 regulates mRNA splicing in the developing Drosophila melanogaster nervous system
Seth M. Kelly (Department of Biology and Program in Neuroscience The College of Wooster), Allison Paschack, Gargi Mishra, Katie Shelmidine (Program in Biochemistry and Molecular Biology, The College of Wooster), Jinsoo Ahn, Kichoon Lee (Department of Animal Sciences, The Ohio State University), Emma Smith (Department of Biology, The College of Wooster), Young Ji (Program in Neuroscience, The College of Wooster)
Abstract:
Due to the interconnected nature of mRNA processing events, mutations in the genes encoding RNA binding proteins can lead to pleiotropic disease phenotypes. Thus, understanding how RNA binding proteins individually and collectively post-transcriptionally regulate gene expression can provide a comprehensive understanding of RNA processing and underlying mechanisms of disease. For example, mutations in the vertebrate ZC3H14 gene, which encodes an evolutionarily conserved RNA binding protein, cause alterations in transcript abundance, extended poly(A) tails, and changes to alternative mRNA splicing. The yeast and Drosophila orthologs of ZC3H14, both called Nab2, have likewise been implicated in multiple aspects of RNA metabolism, including poly(A) tail length control, alternative splicing, and RNA methylation. Importantly, mutations in ZC3H14 and Nab2 are linked to changes in nervous system function and development, suggesting that these proteins are especially critical for RNA processing in neurons.
To better understand the network of interactions that ZC3H14 and Nab2 participate in, we investigated functional interactions between Drosophila Nab2 and RNA processing components. We show here that Nab2 genetically interacts with multiple components of the spliceosome, supporting a functional connection between Nab2 and mRNA processing. In support of this connection, we have also demonstrated that Nab2 is required for control of alternative splicing and prevention of intron retention during fly brain development. Finally, long-read RNA sequencing data suggests that during development, changes in splicing occur independent of changes in poly(A) tail length. Together, these data suggest that Nab2 may function as a regulator of alternative splicing during brain development. The alterations in RNA processing that we have observed may also contribute to the developmental and behavioral alterations observed in Nab2 null flies and ZC3H14 null mice.
Keywords: RNA Binding proteins , splicing, polyadenylation
79. Discovery of novel FMN Riboswitch Binding Partners using an Interdisciplinary Approach
Ingrid R. Kilde (Program in Chemical Biology, University of Michigan - Ann Arbor), Dr. Elizabeth D. Tidwell (Life Sciences Institute, University of Michigan - Ann Arbor), Anna Anders, Dr. Brandon T. Ruotolo (Department of Chemistry, University of Michigan - Ann Arbor), Dr. Aaron T. Frank (Arrakis Therapeutics), Zhijian Hu, Dr. Kevin Wood (Department of Physics and Department of Biophysics, University of Michigan - Ann Arbor), Dr. Markos Koutmos (Department of Chemistry and Department of Biophysics, University of Michigan - Ann Arbor)
Abstract:
Prokaryotic flavin homeostasis (import and biosynthesis) is regulated via flavin mononucleotide riboswitches (FMN-RS) in most bacterial species groups, including 41 of the 49 highest priority pathogens identified by the World Health Organization. Riboswitches are structured RNA elements capable of altering gene expression at the transcriptional or translational level in response to specific binding of an essential metabolite ligand such as FMN. While dysregulation of flavin metabolism can be deleterious for bacterial robustness, investigation into antibacterials targeting the FMN riboswitch has been complicated by the off-target effects inherent to flavin analogues, and the lack of whole-cell potency in current lead compounds. To overcome these challenges, we aim to identify potent small molecule (SM) inhibitors of the FMN-RS using an interdisciplinary pipeline incorporating computational, analytical, biochemical, and biophysical techniques. We have developed a robust label-free and cell-free high throughput fluorescent equilibrium displacement (FLED) assay for monitoring the impact of SM on the FMN:FMNRS complex. In collaboration with UM Center for Chemical Genomics, we utilized this assay to screen over 20K compounds from diverse libraries including traditional unbiased libraries, commercial libraries curated for RNA targets, and libraries we assembled through computational prescreening using either docking (rDock) or ligand-based chemical and structural overlay (ROCS). For rigorous hit characterization, we are using ion mobility-mass spectrometry (IM-MS) to quantify binding capacity and collision-induced unfolding (CIU) to determine the structural impact (stabilization or destabilization) of SM binding. Furthermore, we have optimized a FMNRS-specific transcription termination assay and are in the process of implementing a high throughput in vivo growth inhibition assays for functional validation. Results from the SM characterization will inform future in silico prescreening for RNA:SM interactions, with the goal of iterative improvement of virtual screening for RNA bioactive compounds and further development of RNA drug discovery pipelines.
Keywords: FMN Riboswitch, Drug discovery, High throughput
80. Offline HPLC fractionation and LC-MS/MS characterization of tRNAs
Jennifer A. Kist (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Cassandra Herbert (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Scott Abernathy (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati)
Abstract:
Transfer ribonucleic acids (tRNAs) are adapter molecules acting as a carrier of amino acids to the growing polypeptide chain. Among all RNAs, tRNAs exhibit the largest number and the widest variety of modifications to ensure proper structure and function. Mass spectrometry methods are becoming increasingly important for the characterization of post-transcriptional modifications on ribonucleic acids. RNA Modification Mapping by LC-MS/MS is applicable to mixtures of RNAs (in particular, tRNAs) from bacterial and archaeal organisms. However, many organisms, especially eukaryotes, can transcribe hundreds or more individual tRNA sequences within the cell. Such mixtures are inherently difficult to accurately map to particular sequences given current technologies. In this work, a reverse phase (RP) HPLC method for fractionating tRNAs compatible with RNA modification mapping was developed. Yeast tRNA as well as tRNA from WM793 primary melanoma cells were both successfully fractionated into 4 fractions. The fractions were collected, washed, and ran on LC-MS for nucleoside analysis.
Keywords: tRNA, LC-MSMS, RP-HPLC
81. Investigation of telomerase reverse transcriptase (TERT) ubiquitination and telomerase regulation in the parasitic protozoan, Trypanosoma brucei
Kaitlin Klotz (Department of Biological Science; University of North Carolina at Charlotte), Justin Davis (Department of Biological Sciences; University of North Carolina at Charlotte), Tiffany Barwell (Department of Biological Sciences; University of North Carolina at Charlotte), Nate Tomaszycki (Department of Biological Sciences; University of North Carolina at Charlotte), Bibo Li (Center for Gene Regulation in Health and Disease GRHD; Cleveland State University), Kausik Chakrabarti (Department of Biological Sciences; University of North Carolina at Charlotte)
Abstract:
Telomerase is a ribonucleoprotein complex that creates and extends caps of repetitive, non-coding DNA at the chromosome ends. It is minimally composed of a telomerase RNA (TR) which is the template for making repeats and a telomerase reverse transcriptase (TERT) which catalyzes the addition of telomeric repeats. The TR and TERT must interact to achieve telomerase activity. The interaction between TR and TERT can be regulated at both the RNA and protein levels. One such regulatory mechanism is TERT turnover. Ubiquitination marks old, damaged, or too concentrated proteins for degradation in the proteasome. In many eukaryotes such as Tetrahymena, Saccharomyces, and humans, TERT is ubiquitinated and processed through the proteasome. Previously, evidence of TERT ubiquitination in Trypanosoma brucei, the parasitic protozoan that causes African sleeping sickness had not been demonstrated. We hypothesized that like other eukaryotes, TbTERT was ubiquitinated, that the absence of TbTR could influence levels of ubiquitination, and that TbTERT was degraded through the proteasome. TbTERT ubiquitination was identified through anti-TbTERT immunoprecipitation followed by an anti-ubiquitin western blot in bloodstream form (BF) and procyclic form (PF) T. brucei. We performed a western blot to determine the polyubiquitin linkage and uncovered K48 polyubiquitination in BF parasites which supports our hypothesis of proteasomal TbTERT degradation. Finally, we administered MG-132 to BF and PF T. brucei to inhibit proteasomal degradation and measured FLAG-tagged TbTERT build-up via western blot. Preliminary data suggests BF and PF T. brucei are ubiquitinated and degraded in the proteasome. Our results indicate that TbTERT ubiquitination likely plays a role in regulating T. brucei telomerase activity which could have implications in cell proliferation rate and disease progression.
References:
1. Büscher, P., Cecchi, G., Jamonneau, V., & Priotto, G. (2017). Human African trypanosomiasis. The Lancet, 390(10110), 2397-2409.
2. Blackburn, E. H., & Collins, K. (2011). Telomerase: an RNP enzyme synthesizes DNA. Cold Spring Harbor perspectives in biology, 3(5), a003558.
3. Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annual review of biochemistry, 70(1), 503-533.
4. Walsh, I., Di Domenico, T., & Tosatto, S. C. (2014). RUBI: rapid proteomic-scale prediction of lysine ubiquitination and factors influencing predictor performance. Amino Acids, 46, 853-862.
5. Davis, J. A., & Chakrabarti, K. (2022). Telomerase ribonucleoprotein and genome integrity—An emerging connection in protozoan parasites. Wiley Interdisciplinary Reviews: RNA, 13(5), e1710.
Keywords: Telomerase , Ubiquitination, Regulation
82. Protection of the Telomeric Junction by the Shelterin Complex
Sineth G Kodikara (Department of Physics, Kent State University), Sajad Shiekh (Department of Physics, Kent State University), Amanda Jack (Department of Molecular and Cell Biology, University of California), Janan Alfehaid (Department of Physics, Kent State University), Ahmet Yildiz (Department of Molecular and Cell Biology, University of California), Hamza Balci (Department of Physics, Kent State University)
Abstract:
Shelterin plays a vital role in preventing inappropriate DNA damage repair mechanisms at telomeres. The junction between double-stranded telomeric regions (dsTEL) and the single-stranded telomeric overhang (ssTEL) represents the most accessible part of telomeric DNA. Shelterin comprises proteins that bind to both dsTEL and ssTEL, allowing it to protect this junction by bridging the ssTEL and dsTEL segments. To explore this function, we examined shelterin binding to telomeric DNA substrates with varying lengths of ssTEL and dsTEL and assessed its effect on telomere accessibility using single-molecule fluorescence microscopy in vitro. Our findings reveal that the first dsTEL repeat closest to the junction serves as the primary binding site for shelterin to form the bridge. Shelterin requires at least two ssTEL repeats for binding, whereas the POT1 subunit, which specifically binds ssTEL, necessitates longer ssTEL tracts for stable association and effective protection of the junction. The protective function of POT1 at the junction is significantly improved by the presence of a 5′-phosphate group. Overall, our results demonstrate that shelterin enhances the binding stability of POT1 to ssTEL and provides superior protection by bridging single- and double-stranded telomeric regions, compared to POT1 alone.
Keywords: Telomere , Shelterin, FRET-PAINT
83. Orthogonal Organic Phase Separation (OOPS) Reveals Cellular RNA Binding Proteome Changes in Response to Flavivirus Infections
Vidusha Kothwela (Case Western Reserve University, School of Medicine, Biochemistry Department, Cleveland, OH), Joseph Luna (Case Western Reserve University, School of Medicine, Biochemistry Department, Cleveland, OH)
Abstract:
RNA viruses in the Flaviviridae family, such as Zika virus (ZIKV) and Hepatitis C virus (HCV), heavily rely on host RNA-binding proteins (RBPs) throughout their life cycle. These viruses present a significant global health challenge, with effective antiviral therapies remaining elusive. Our limited understanding of how cellular RBPs influence ZIKV and HCV infections hinders progress toward effective treatments. By investigating RBPome remodeling during these infections, we aim to uncover crucial insights into how RBPs regulate viral replication and immune evasion. We used Orthogonal Organic Phase Separation (OOPS) to profile the RNA-bound proteome during ZIKV and HCV infections. Our analysis revealed virus-specific alterations in RNA-binding activity, with 115 RBPs identified in ZIKV-infected cells and 135 RBPs in HCV-infected cells. These RBPs were distinct from those previously reported for both viruses, suggesting a specialized and dynamic role for RBPs during infection. Only five RBPs exhibited increased RNA-binding activity in both infections, and these did not follow a discernible pattern of functionality, underscoring the specificity of the RBP response to each virus. Among the RBPs identified, the ATP-binding RNA helicase DHX38 showed a marked increase in RNA binding upon ZIKV and HCV infections. Known for its role in pre-mRNA splicing as part of the spliceosome, DHX38 did not exhibit transcriptional activation during infection, suggesting a novel, non-canonical function in viral infection. These findings demonstrate OOPS's robust and broadly applicable nature in uncovering novel RNA-protein interactions during RNA virus infections. More importantly, they provide new insights into how ZIKV and HCV remodel the RBPome, offering potential targets for therapeutic intervention.
Keywords: OOPS, RBPome, Flavivirus
84. Rif-seq reveals C. crescentus mRNA decay is globally coordinated with transcription and translation
Hiranthi Kulathungage (Department of Biological Sciences, Wayne State University, Detroit, MI, USA), James R. Aretakis (Department of Biological Sciences, Wayne State University, Detroit, MI, USA), Kavya Vaidya (Department of Physics, University of Illinois, Urbana-Champaign, IL, USA), Nadra Al-Husini, Nisansala S. Muthunayake (Department of Biological Sciences, Wayne State University, Detroit, MI, USA), Sangjin J. Kim (Department of Physics, University of Illinois, Urbana-Champaign, IL, USA), Jared M. Schrader (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI, USA)
Abstract:
mRNA decay is an essential and highly regulated step of gene expression that acts as an off switch1,2. To examine the global regulation of mRNA decay in the bacterium C. crescentus, we utilized Rif-seq, a method which allows for global measurement of mRNA lifetimes. We observed about 1/3 of the mRNAs are cotranscriptionally degraded and many mRNAs in different gene functional categories have variable turnover rates. To examine the role of translation on mRNA stability, we mapped the global RNA decay sites containing a 5’ P end sequencing. Across the transcriptome, we identified a majority of mRNA cut sites in 5’ UTRs, the translation initiation regions, and in coding sequences. Using ribosome profiling, we observed that half-life is correlated with translation efficiency and mRNA 5’ P cleavage sites are enriched in regions that are poorly occupied by ribosomes. To distinguish the individual roles of translation initiation and elongation on mRNA half-life, we conducted Rif-seq experiments on cells treated with initiation inhibitor retapamulin and elongation inhibitor chloramphenicol, which showed that retapamulin led to a slight stabilization of mRNA half-life, while chloramphenicol led to a near total stabilization of the entire transcriptome. In line with this observation that mRNA decay rates are strongly influenced by the translation elongation rate, we examined the codon adaptation index (CAI) and surprisingly, observed a strong negative correlation between CAI and global mRNA half-lives (R2 = 0.66). To systematically alter the rates of translation initiation and elongation and test their impact on mRNA decay, we designed synthetic YFP mRNAs with weak and strong initiation regions (TIRs) and combined these with codon usage ranging from highly suboptimal to highly optimal. Across the synthetic mRNAs we observed an inverse correlation between mRNA half-lives and CAI, while we observed a stabilizing impact from the TIR strength. While initiation and elongation influence the overall translation efficiency, TIR and CAI appear to work in an antagonistic manner to determine the overall mRNA half-life.
References:
1Presnyak, V., Alhusaini, N., Chen, Y.-H., Martin, S., Morris, N., Kline, N., Olson, S., Weinberg, D., Baker, K. E., Graveley, B. R., & Coller, J. (2015). Codon optimality is a major determinant of mrna stability. Cell, 160(6), 1111–1124.
2Chan, L. Y., Mugler, C. F., Heinrich, S., Vallotton, P., & Weis, K. (2018). Non-invasive measurement of mrna decay reveals translation initiation as the major determinant of mrna stability. eLife, 7.
Keywords: mRNA decay, translation initiation and elongation, Rif-seq, Ribosome profiling
85. Defining a co-regulatory relationship between RNA binding proteins ADR-2 and TDP-1 in C. elegans nervous systems
Emma Lamb (Indiana University Department of Biology), Heather Hundley (Indiana University Department of Biology )
Abstract:
Editing of RNA not only contributes to molecular diversity of transcripts within a cell, but also mediates RNA silencing, innate immune activation, and numerous developmental processes (Erdmann et al., 2021). ADARs (Adenosine Deaminases that Act on RNA) are enzymes that bind double-stranded RNA catalyzing the deamination of adenosine to inosine (A-to-I editing). As RNA polymerase II is polymerizing a transcript, the nascent RNA can act as a binding site for proteins like ADARs that bind dsRNA (Yu et al., 2023). One such RNA binding protein is the human ortholog TAR-DNA binding protein, TDP-1. This protein binds RNA and promotes its nuclear localization for several processes including small RNA degradation pathways, translation, and stress granule formation. The loss of TDP-1 promotes ADAR editing of intron-containing regions of RNA by repressing cytoplasmic localization of dsRNA (Saldi et al. 2014). RNA sequencing data comparing first larval stage worms and mature worms indicated that in neurons there is a shift in the targets of A-to-I editing from mostly 3’ UTRs to mostly introns (Rajendran 2021). This is of interest as the activity of ADARs has been linked to neurological phenotypes in mammals as well as C. elegans such as impaired chemotaxis capability and motor coordination (Yang et al., 2021). These findings pose further questions about the implications of TDP-1 antagonism of the intronic editing by ADARs in neuronal health and development which would inform the work connecting both ADAR and TDP-1 activity to human neurodegenerative diseases like Alzheimer’s Disease and ALS. Here, I detail my plans to address these questions over the course of my degree.
References:
Erdmann, E. A. et al. (2021). To protect and modify double-stranded RNA – the critical roles of ADARs in development, immunity, and oncogenesis. Critical Reviews in Biochemistry and Molecular Biology, 56(1), 54–87.
Rajendren, S. et al. (2021). Profiling neural editomes reveals a molecular mechanism to regulate RNA editing during development. Genome
Research, 31(1), 27–39.
Saldi, T. K. et al. (2014). TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA. The EMBO Journal, 33(24),
2947–2966.
Yu, G. et al. (2023).Genome-wide probing of eukaryotic nascent RNA structure elucidates cotranscriptional folding and its antimutagenic effect. Nature Communications, 14(1), 5853.
Keywords: ADAR, neuron, RBP
86. Unveiling the Alternative Splicing Landscape in Hepatoblastoma: Enhancing Prognostic and Therapeutic Approaches
Zac LaRocca-Stravalle (Center for Childhood Cancer at Nationwide Childrens Hospital), Safiya Khurshid (Center for Childhood Cancer at Nationwide Childrens Hospital), Dawn Chandler (Center for Childhood Cancer at Nationwide Childrens Hospital)
Abstract:
Objectives
Hepatoblastoma (HB), the most common pediatric liver cancer, has a 3-year event-free survival rate above 80% with chemotherapy and surgery, yet outcomes for advanced tumors remain poor. While molecular stratifications based on genomic, transcriptomic, and epigenomic data have been proposed to predict prognosis, the alternative splicing (AS) landscape in HB remains unexplored. Since pediatric cancers, including HB, typically have a low mutational burden but are enriched for AS events, investigating AS could reveal new biomarkers and therapeutic targets.
Methods
We used RNA-sequencing data from 30 matched tumor-normal HB patients (GEO: GSE133039) to perform differential splicing analysis with rMATS. Significant genes were further analyzed using R, and unsupervised hierarchical clustering of patients and their PSI values was conducted with correlation distanced. Differential gene expression was assessed using edgeR, and key AS events were validated in HepG2 cells and normal tissue using RT-PCR.
Results
We identified 1,876 significant AS events, including 55% skipped exons (SE), 8% retained introns, 24% mutually exclusive exons, 6% alternative 5’ splice sites, and 7% alternative 3’ splice sites. Hierarchical clustering revealed two tumor subgroups. One cluster, comprising 5 out of 30 patients, was characterized by the skipping of exon 11 in INSR, which is linked to tumor growth and angiogenesis. This cluster also exhibited elevated levels of splicing factors CELF5, PTBP1, and FUS, while the other cluster showed upregulation of MBNL1, MBNL2, and CELF2.
Conclusion
These findings suggest that HB tumors can be stratified based on AS profiles, with distinct tumor clusters showing differential splicing factor expression. Further research is needed to explore the clinical implications of these clusters, including associations with metastasis, PRETEXT stage, histology, and survival. This study opens new pathways for the molecular characterization of HB, providing insights into AS mechanisms that could lead to novel biomarkers and therapies.
References:
Carrillo-Reixach, J., et al. (2020). Epigenetic footprint enables molecular risk stratification of hepatoblastoma with clinical implications. Journal of Hepatology, 73(2), 328–341. https://doi.org/10.1016/j.jhep.2020.03.025
Hooks, K. B., et al. (2018). New insights into diagnosis and therapeutic options for proliferative hepatoblastoma. Hepatology, 68(1), 89–102. https://doi.org/10.1002/hep.29672
Venkataramany, A.S., et al. (2022). Alternative RNA splicing defects in pediatric cancers: New insights in tumorigenesis and potential therapeutic vulnerabilities. Annals of Oncology, 33(6), 578–592. https://doi.org/10.1016/j.annonc.2022.03.011
Keywords: Alternative Splicing , Hepatoblastoma , RNA therapeutics
87. Caulobacter RppH and NudC have Direct and Indirect Effects on mRNAs
Vincent A. Lawal (Departments of Biological Sciences and Chemistry, Wayne State University, Detroit, MI), Imalka W. Ratyhnayaka-Mudiyanselage (Departments of Biological Sciences and Chemistry, Wayne State University, Detroit, MI), Hadi Yassine (Departments of Biological Sciences and Chemistry, Wayne State University, Detroit, MI), Vidhyadhar Nandana (Departments of Biological Sciences and Chemistry, Wayne State University, Detroit, MI), Jeremy G. Bird (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI), Jared M. Schrader (Departments of Biological Sciences and Chemistry, Wayne State University, Detroit, MI)
Abstract:
In eukaryotic cells, the 5’ m7G “cap” protects cellular mRNAs from degradation and promotes their translation. In bacteria, 5’ PPP or 5’ NAD/H “cap”-like modifications have been shown to protect mRNAs from degradation (Deana et al., 2008; Bird et al., 2016), yet their global roles in mRNA decay are poorly understood. To investigate the roles of 5’ PPP or 5’ NAD/H modifications on bacterial mRNA metabolism, we deleted the 5’ PPP decapping enzyme (rppH) or 5’ NAD/H decapping enzyme (nudC) from the Caulobacter crescentus genome and performed rif-seq experiments to measure mRNA lifetimes. We found only a small subset of mRNAs were stabilized in either of the decapping mutant strains, suggesting that these 5’ modifications have little impact on the decay of modified mRNAs in bacteria. To investigate other functions of the 5’ modifications, we investigated whether decapping mutants impact the formation of BR-bodies, which are biomolecular condensates known to organize mRNA decay machinery (Muthunayake et al., 2020). We observed that decapping enzyme deletions significantly reduce BR-bodies, suggesting that decapped RNA may promote BR-body phase separation. Finally, we observed that decapping mutants show no phenotype in the exponential growth of C. crescentus cells and discovered that they are more strongly expressed in the stationary phase, where we see they lead to significant defects in growth out of the stationary phase. Overall, this suggests that 5’ RNA “cap” modifications in bacteria have more specialized functions compared to the well-established roles in eukaryotes.
References:
Deana, A., Celesnik, H., & Belasco, J.G. (2008). The bacterial enzyme RppH triggers messenger RNA degradation by 5' pyrophosphate removal. Nature, 451(7176), 355-358. https://doi.org/10.1038/nature06475.
Bird, J.G., Zhang, Y., Tian, Y., Panova, N., Barvík, I., Greene, L., Liu, M., Buckley, B., Krásný, L., Lee, J.K., Kaplan, C.D., Ebright, R.H., Nickels, B.E. (2016). The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA. Nature, 535(7612), 444–447. https://doi.org/10.1038/nature18622.
Muthunayake, N.S., Tomares, D.T., Childers, W.S., & Schrader, J.M. (2020). Phase-separated bacterial ribonucleoprotein bodies organize mRNA decay. Wiley Interdisciplinary Reviews: RNA, 11(6), e1599. https://doi.org/10.1002/wrna.1599.
Keywords: Decapping enzymes, BR-bodies, RNA decay, Rifampicin-sequencing
88. Uncovering how lncRNA MALAT1 controls gene expression in cancer metastasis
Kayla Lenshoek (University of Michigan, Cellular and Molecular Biology), Brittany Bowman (University of Michigan, Biological Chemistry), Chase Weidmann (University of Michigan, Biological Chemistry )
Abstract:
Long noncoding RNA Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is linked to the progression of multiple cancer types. Upregulation of MALAT1 is often correlated with aggressive metastasis, and depletion of MALAT1 in cancer cells decreases metastatic potential. Interestingly, in healthy cells and wild-type mouse models, loss of MALAT1 has no effect on viability. The mechanisms by which MALAT1 promotes metastatic activity exclusively in cancer cells is not well understood, and MALAT1’s function in healthy cells is also unknown. Here we aim to uncover the mechanisms by which MALAT1 alters gene regulatory programs to promote metastasis in a lung cancer cell model. We create and validate A549 lung adenocarcinoma cell lines where CRISPR-based gene deletions and insertions alter MALAT1 expression or destabilize the MALAT1 transcript. We find that MALAT1 accumulates at high levels in nuclear speckles, but loss of MALAT1 does not strongly alter nuclear speckle formation. Increased MALAT1 expression promotes a mesenchymal phenotype, whereas reduction in MALAT1 expression promotes a more epithelial cell state. Further, we find that MALAT1 expression is inversely correlated with levels of EZH2 protein, the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). Consequently, levels of the PRC2 epigenetic mark, Histone H3 Lysine 27 tri- methylation (H3K27me3) are inversely correlated with MALAT1 levels. In line with MALAT1’s pro-metastatic activity, we observe an upregulation in genes and pathways associated with cell adhesion, mobility, and mesenchymal cell identity in MALAT1 overexpression lines. These results are consistent with a model wherein MALAT1 reprograms cancer cells through reorganization of epigenetic marks to promote metastasis through mesenchymal gene programs.
Keywords: lncRNA, MALAT1, Epigenetics
89. Identification of mediators of NOTCH-dependent mRNA degradation during vertebrate segmentation
Lauren Levesque (Molecular Genetics, The Ohio State University), Kara Braunreiter (Molecular Genetics, The Ohio State University), Susan Cole (Molecular Genetics, The Ohio State University)
Abstract:
Somitogenesis produces the embryonic precursors of the ribs and vertebrae. Oscillation of the transcriptional repressor HES7 in the presomitic mesoderm (PSM), is central to regulating somitogenesis in mammals. Notch signaling activates Hes7 transcription, and the HES7 protein then represses its own expression, creating a negative feedback loop necessary for cyclic expression. To maintain proper HES7 oscillation, the production and degradation of the mRNA must be tightly regulated1,2. In Notch-deficient PSMs, there is a reduction in Hes7 transcription coupled with stabilization of Hes7 mRNA, indicating that in addition to activating Hes7 transcription, Notch promotes decay of Hes7 through an unknown mechanism3,4.
The mouse PSM produces three Hes7 isoforms that differ in the length of their 3′ untranslated region (3′UTR). RNA ISH revealed that all Hes7 isoforms exhibit oscillatory expression in the PSM. Analysis by qRT-PCR shows all three isoforms are stabilized in Notch-deficient PSMs, but it is difficult to quantify the effect as Notch influences both transcription and mRNA stability. To uncouple transcription from Notch, I will use cell-culture based reporter assays to determine which isoforms have Notch-dependent decay, and which cis-region confers this dependency.
To identify Notch-dependent trans-acting factors that may promote mRNA decay, I analyzed publicly available transcriptomic and ChIP-seq data5–9. I searched for genes involved in mRNA processing that had a binding site in the Hes7 3’UTR, exhibit cyclic expression in mouse PSMs, and/or are potential Notch targets. The analysis yielded potential candidates such as: Hnrnpl, Pum2, and Rbm4. Future directions involve qRT-PCR, RNA in-situ hybridization, and cell-culture assays to characterize candidate expression patterns and test their ability to promote Hes7-3′UTR reporter decay.
References:
References
1. Wahi, K., Bochter, M.S., and Cole, S.E. (2016). The many roles of Notch signaling during vertebrate somitogenesis. Semin. Cell Dev. Biol. 49, 68–75. https://doi.org/10.1016/j.semcdb.2014.11.010.
2. Blatnik, M.C., Gallagher, T.L., and Amacher, S.L. (2022). Keeping development on time: Insights into post-transcriptional mechanisms driving oscillatory gene expression during vertebrate segmentation. Wiley Interdiscip. Rev. RNA, e1751. https://doi.org/10.1002/wrna.1751.
3. Williams, D.R., Shifley, E.T., Braunreiter, K.M., and Cole, S.E. (2016). Disruption of somitogenesis by a novel dominant allele of Lfng suggests important roles for protein processing and secretion. Development 143, 822–830. https://doi.org/10.1242/dev.128538.
4. Bochter, M.S., Servello, D., Kakuda, S., D’Amico, R., Ebetino, M.F., Haltiwanger, R.S., and Cole, S.E. (2022). Lfng and Dll3 cooperate to modulate protein interactions in cis and coordinate oscillatory Notch pathway activation in the segmentation clock. Dev. Biol. 487, 42–56. https://doi.org/10.1016/j.ydbio.2022.04.004.
5. Sewell, W., Sparrow, D.B., Smith, A.J., Gonzalez, D.M., Rappaport, E.F., Dunwoodie, S.L., and Kusumi, K. (2009). Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis. Dev. Biol. 329, 400–409. https://doi.org/10.1016/j.ydbio.2009.02.023.
6. Matsuda, M., Yamanaka, Y., Uemura, M., Osawa, M., Saito, M.K., Nagahashi, A., Nishio, M., Guo, L., Ikegawa, S., Sakurai, S., et al. (2020). Recapitulating the human segmentation clock with pluripotent stem cells. Nature 580, 124–129. https://doi.org/10.1038/s41586-020-2144-9.
7. Fongang, B., and Kudlicki, A. (2013). The precise timeline of transcriptional regulation reveals causation in mouse somitogenesis network. BMC Dev. Biol. 13, 42. https://doi.org/10.1186/1471-213X-13-42.
8. Oki, S., Ohta, T., Shioi, G., Hatanaka, H., Ogasawara, O., Okuda, Y., Kawaji, H., Nakaki, R., Sese, J., and Meno, C. (2018). ChIP‐Atlas: a data‐mining suite powered by full integration of public ChIP‐seq data. EMBO Rep. 19, e46255. https://doi.org/10.15252/embr.201846255.
9. Zou, Z., Ohta, T., Miura, F., and Oki, S. (2022). ChIP-Atlas 2021 update: a data-mining suite for exploring epigenomic landscapes by fully integrating ChIP-seq, ATAC-seq and Bisulfite-seq data. Nucleic Acids Res. 50, W175–W182. https://doi.org/10.1093/nar/gkac199.
Keywords: mRNA degradation, development, NOTCH
90. Dissecting the role of CLP1 in RNA maturation and alternative polyadenylation through the R140H mutation
Shaokun Li (Department of Neurosciences, Case Western Reserve University), Ashleigh Schaffer (Department of Genetics and Genome Sciences, Case Western Reserve University)
Abstract:
CLP1 is a multifunctional RNA kinase essential for the maturation of both tRNA and mRNA. The CLP1 R140H mutation causes pontocerebellar hypoplasia type 10 (PCH10), a severe neurodegenerative disorder. Patients with PCH10 exhibit mild atrophy of the cerebellum, pons, and corpus callosum, along with progressive microcephaly and severe axonal sensorimotor neuropathies. Previous studies have demonstrated that the R140H mutation in CLP1 impairs its kinase activity and its interactions with the TSEN complex, which is involved in tRNA maturation, as well as the CPA complex, which regulates mRNA alternative polyadenylation (APA). However, only defects in mRNA processing have been observed in motor neurons derived from PCH10 patients. Specifically, mRNAs in these neurons preferentially use distal polyadenylation sites, generating longer isoforms, which is opposite of the pattern observed in CLP1 KO motor neurons. Thus, we hypothesize that the CLP1 R140H mutation confers a gain-of-function effect, resulting in an APA shift toward distal sites to cause the pathogenesis of PCH10. Our data indicates that overexpression of CLP1R140H, but not wild-type CLP1, in motor neuron progenitors recapitulated the phenotypes observed in motor neurons derived from PCH10 patients. To further investigate the underlying mechanism of PCH10, we propose to utilize CLIP-seq and BioID/AP-mass spectrometry to characterize the RNA and protein interactomes of both wild-type CLP1 and the CLP1R140H. Additionally, we will perform transient transcriptome sequencing to identify the primary RNA targets of CLP1 and CLP1R140H. The outcomes of our research will provide profound insight into the mechanism by which the cleavage site is selected from multiple possible elements.
Keywords: alternative polyadenylation, neurodegenerative disorders, motor neurons
91. Identifying the mechanism of PUS7 relocalization upon stress
Xiaoyan Li (University of Michigan, Department of Chemistry), Kristin S. Koutmou (University of Michigan, Department of Chemistry)
Abstract:
Pseudouridine is one of the most abundant post-transcriptional modifications found across all RNA types. This modification modulates RNA structure, dynamics and RNA-protein interactions to influence gene expression and protein synthesis. In eukaryotes, pseudouridine synthase 7 (PUS7) is an enzyme responsible for the catalysis of pseudouridine incorporation into crucial substrates for protein synthesis, tRNA, and mRNA. Dysregulation and mutations in PUS7 have been linked to various diseases, including intellectual disabilities and cancers. Our lab recently demonstrated that upon stress exposure, PUS7 translocates from the nucleus to the cytoplasm, resulting in increased mRNA pseudouridylation in both yeast and human lung cells. Cytoplasmic accumulation of PUS7 enhances stress tolerance and induces proteomic alterations. However, the precise molecular mechanisms governing PUS7 relocalization remain unclear. In this study, we aim to elucidate the protein-protein interactions that mediate PUS7's nuclear-cytoplasmic transport and to identify its nuclear export and localization sequences. These objectives will be addressed through proximity labeling, co-immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS), and site-directed mutagenesis.
References:
1. Roundtree, I. A., Evans, M. E., Pan, T. & He, C. Dynamic RNA Modifications in Gene Expression Regulation. Cell 169, 1187–1200 (2017).
2. Li, J., Ma, X., Chakravarti, D., Shalapour, S. & DePinho, R. A. Genetic and biological hallmarks of colorectal cancer. Genes Dev 35, 787–820 (2021).
3. Gilbert, W. V. & Nachtergaele, S. mRNA Regulation by RNA Modifications. Annual Review of Biochemistry 92, 175–198 (2023).
4. Cui, Q. et al. Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis. Nat Cancer 2, 932–949 (2021).
5. Kierzek, E. et al. The contribution of pseudouridine to stabilities and structure of RNAs. Nucleic Acids Research 42, 3492–3501 (2014).
6. de Brouwer, A. P. M. et al. Variants in PUS7 Cause Intellectual Disability with Speech Delay, Microcephaly, Short Stature, and Aggressive Behavior. Am J Hum Genet 103, 1045–1052 (2018).
Keywords: pseudouridine synthase 7, RNA modification, protein protein interaction
92. Determining the role of Trm7, Trm732, and Trm734 in tRNA binding
Anabel Lillie (Chemistry and Biochemistry, Northern Kentucky University), Ashton Davey (Chemistry and Biochemistry, Northern Kentucky University), Holly Funk (Chemistry and Biochemistry, Northern Kentucky University), Micheal Guy (Chemistry and Biochemistry, Northern Kentucky University)
Abstract:
Modifications of the tRNA anticodon loop are important to translation. Proteins Trm7, Trm732, and Trm734 work together to modify the anticodon loop of tRNAPhe. Trm7 plays a catalytic role in methylation activity, and Trm732 and Trm734 are predicted to bind and position tRNAPhe for methylation at nucleotides C32 and G34 in Saccharomyces cerevisiae. Lack of Trm7 causes a sick phenotype in S.cerevisiae, and mutations in the TRM7 human ortholog, FTSJI, cause non-syndromic X-linked intellectual disability. We are working to determine whether individual Trm7, Trm732, and Trm734 proteins can bind to tRNAPhe, or whether Trm7:Trm732 and Trm7:Trm734 complexes are required for binding. Tagged proteins of interest are pulled down and the RNA bound to the proteins is analyzed by Northern Blot. This approach will also allow us to determine if our previously identified non-functional variants of Trm732 and Trm734 are still able to bind to tRNAPhe. Due to the high conservation between the yeast and human proteins, the results will likely be applicable to the orthologous proteins in humans.
Keywords: tRNA , methylation, yeast
93. Involvement of the plant polyadenylation factors FIP1 and CPSF30 in immune responses
Huazhen Liu (Department of Plant Pathology, University of Kentucky), Lichun Zhu (Department of Plant and Soil Sciences, University of Kentucky), Pradeep Kachroo (Department of Plant Pathology, University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky)
Abstract:
In plants, Systemic Acquired Resistance (SAR) is a form of systemic immunity that protects uninfected parts of a pathogen-inoculated plant against further disease. In a previous study, it was shown that a novel plant polyadenylation factor subunit, CPSF30, is required for resistance against a bacterial pathogen (1). The Arabidopsis thaliana ortholog of the yeast polyadenylation complex subunit Fip1p, FIP1, associates with the Arabidopsis ortholog of CPSF30 in vitro (2, 3). This association raises the possibility that the two proteins may work as a unit, and thus that Arabidopsis FIP1 may also play roles in defense responses. To test this in the context of SAR, we treated a set of mutant and complemented Arabidopsis lines with an avirulent strain of Pseudomonas syringae to induce SAR. The set of lines included a mutant (fip1-1) that does not express FIP1, a line in which the FIP1 gene is over-expressed in the fip1-1 mutant background, an additional mutant (oxt6) that does not express CPSF30, and a line that expresses CPSF30 in the oxt6 background. The results show that lines that express FIP1 or CPSF30 can establish an SAR, but lines that do not express FIP1 or CPSF30 are deficient in SAR. All of the wild type, mutant, and transgenic plants have similar transcriptional responses in the inoculated leaf to the avirulent pathogen. However, they differ in the (immune) distal leaves, and the differences relate to the well-established transcriptional response in SAR. We conclude that FIP1 and CPSF30 act downstream from the initial challenge by the avirulent pathogen and outside of the inoculated leaf. Interestingly, the fip1-1 and oxt6 mutants show altered poly(A) site usage of non-coding RNAs encoded by the TAS3a locus. The non-coding RNA TAS3a is a precursor for tasi-RNAs D7 and D8, which are early mobile signals for SAR (4). TAS3a transcripts undergo alternative polyadenylation, and poly(A) site choice affects the ability to generate the D7 and D8 tasi-RNAs. The observed changes in TAS3a poly(A) site usage in the fip1-1 and oxt6 mutants preclude production of the D7 and D8 tasi-RNAs. We propose that FIP1/CPSF30-dependent usage of the distal TAS3a poly(A) site is the mechanism that links FIP1/CPSF30 with SAR. Taken together, these studies provide new insights into the connections between mRNA polyadenylation and the plant immune system.
References:
1. Q. Bruggeman et al., The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis. Plant Physiol 165, 732-746 (2014).
2. B. Addepalli, A. G. Hunt, A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor. Nucleic Acids Res 35, 4453-4463 (2007).
3. K. P. Forbes, B. Addepalli, A. G. Hunt, An Arabidopsis Fip1 homolog interacts with RNA and provides conceptual links with a number of other polyadenylation factor subunits. J Biol Chem 281, 176-186 (2006).
4. M. B. Shine et al., Phased small RNA-mediated systemic signaling in plants. Sci Adv 8, eabm8791 (2022).
Keywords: Systemic Acquired Resistance, alternative polyadenylation
94. Structural elucidation of the 3’ oncomiR-1 RNA by NMR spectroscopy
Yaping Liu (Biophysics Program), Somaye Badieyan (Department of Biological Chemistry), Michael Cianfrocco (Department of Biological Chemistry), Sarah C. Keane (Biophysics Program; Department of Chemistry)
Abstract:
MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. To maintain proper microRNA expression levels, the enzymatic processing of primary and precursor microRNA elements must be strictly controlled. However, the molecular determinants underlying this strict regulation of microRNA biogenesis are not fully understood. We are investigating the differential processing of oncomiR-1, a polycistronic primary microRNA that encodes six individual miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-20a) and is overexpressed in many cancers.
Chemical probing of oncomiR-1 revealed that the Drosha cleavage sites of pri-miR-92a are sequestered in a four-way junction. NPSL2, an independent stem loop element, is positioned just upstream of pri-miR-92a and sequesters a crucial part of the sequence that constitutes the basal helix of pri-miR-92a. Disruption of the NPSL2 hairpin structure could promote the formation of a pri-miR-92a basal helix that is primed for processing by Drosha. Thus, NPSL2 is predicted to function as a structural switch, regulating pri-miR-92a processing.
Studying the structure of the 3’ domain of oncomiR-1 is crucial for understanding the molecular basis of miR-92a biogenesis. 3’ oncomiR-1 is 234 nts long (~ 77 KDa), and therefore represents a significant technical challenge for structure elucidation by NMR.
By using a divide and conquer strategy along with deuterium editing approach, we were able to complete chemical shift assignments of 3’ oncomiR-1. To tackle the challenge of determining such a large RNA structure, we have also adopted a multidisciplinary approach combing NMR and cryo-EM. The future direction involves refining the 3’ oncomiR-1 structure using these hybrid methods.
References:
Ha, Kim Biogenesis 2014, Ketting et al. Genes and Development 2001, György et al. Science 2001, Hammond et al. Science 2001, Humphreys et al. PNAS 2005, Lim et al. Nature 2005
He et al., Nature 2005, O'Donnell et al. Nature 2005, Chaulk et al., RNA Biol. 2011, Chakraborty et al., RNA 2012, Chakraborty et al., NAR 2017
Keywords: miRNA, oncomiR-1, NMR
95. A Multi-tissue and cancer atlas of Pol III activity uncovers context-specific transcriptional expansion to genes implicated in disease progression
Simon Lizarazo (Molecular and Integrative Physiology, University of Illinois Urbana-Champaign), Sihang Zhou (Cell and Developmental Biology, University of Illinois Urbana-Champaign), Ruiying Cheng (Cell and Developmental Biology, University of Illinois Urbana-Champaign), Rajendra K C (Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign), Kevin Van Bortle (Cell and Developmental Biology, University of Illinois Urbana-Champaign)
Abstract:
RNA polymerase III (Pol III) produces a plethora of small noncoding RNA
involved in nearly all cellular processes, from transcription regulation and
splicing to RNA stability, translation, and protein turnover. Though Pol III
activity is broadly coupled with cellular demands for protein synthesis and
growth, a more precise understanding of gene-level dynamics and context-
specific expression patterns remain missing, in part due to multiple
challenges related to sequencing and mapping Pol III-derived small ncRNA
species. Here, we establish a multi-tissue map of Pol III activity across 19
tissues and 22 primary cancers by comprehensively profiling the chromatin
accessibility of Pol III-transcribed genes (e.g. tRNA, 5S rRNA, U6 snRNA,
7SL SRP RNA, etc.). Our framework relies on the unique relationship
between gene accessibility and Pol III transcription and defines a binary
gene “on” vs. “off” state across ~500 ATAC-seq datasets. Using an
information entropy method to characterize multi-context uniformity, we
provide a definition of the “core” Pol III transcriptome universally active in
all specialized tissues and catalog genes with varied levels of context
specificity. Notably, our genomic atlas points to variable levels of
restriction and expansion of Pol III activity across tissues, including sharp
contraction of the Pol III transcriptome in heart tissues and frequent
expansion across diverse cancers. We discover divergent epigenomic
regulatory features at universal and tissue-specific genes, including
evidence for multiple repressive mechanisms that likely contribute to the
silencing of context-specific Pol III-transcribed genes. These findings
provide a resource for better understanding Pol III dynamics in tissues and
the link between Pol III overactivity and small RNA biogenesis in cancer.
Keywords: RNA Polymerase III, Genomic Atlas
96. The kinase GSK-3 is a novel regulator of ADR-1 RNA recognition in the C. elegans nervous system
Ananya Mahapatra (Genome, Cellular and Developmental Biology, Indiana University, Bloomington), Heather A. Hundley (Department of Biology, Indiana University, Bloomington)
Abstract:
Previously, we have identified a unique post transcriptional mechanism in which under starvation conditions, a double stranded RNA binding protein, ADR-1, binds the mRNA of the transcription factor pqm-1 specifically in the nervous system. PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as promoting survival from hypoxia. The binding of ADR-1 pqm-1 also causes decreased expression of pqm-1 at the mRNA level in the nervous system. Decreased expression of the transcription factor further leads to decreased expression of the genes activated by pqm-1 in the nervous system as well as other tissues, suggesting the presence of inter-tissue signaling. Additionally, this neural specific regulation in the nervous system affects how animals respond in hypoxic conditions. However, the cellular factors and molecular signatures that cause this unique post transcriptional gene regulation of hypoxic survival were largely unknown. In these studies, an unbiased forward genetic screen was performed to identify the factors that can affect the post transcriptional regulatory mechanism in the nervous system. From the screening process, we have identified five unique regulators of this mechanism. Three out of these five candidates affect the post transcriptional mechanism by controlling ADR-1 binding to pqm-1. Using whole genome sequencing and variant identification, dsh-2 was identified as a potential regulator of the mechanism. On testing the mechanism by which DSH-2 affects ADR-1 binding, we identified kinase GSK-3 as the factor that is responsible for controlling ADR-1 binding to pqm-1.
Keywords: GSK-3, RNA recognition, C elegans
97. Identification of unique tRNA-specific effects of tRNA modifying enzymes on 5-fluorouracil toxicity in Saccharomyces cerevisiae
Nisha Makkar (The Ohio State University Chemistry and Biochemistry), Isobel Bowles (The Ohio State University Biochemistry Program), Jane Jackman (The Ohio State University Chemistry and Biochemistry)
Abstract not available online - please check the booklet.
98. Bacterial mRNA Decay Condensates are Broadly Conserved Across Bacterial Species and Play a Critical Role in Host Colonization
Kaveendya S. Mallikaarachchi (Wayne State University, Detroit, MI, USA 48202), Jason L. Huang, Shanmukha Madras, Rodrigo A. Cuellar, Zhenzhong Huang (San Francisco State University, San Francisco, CA, USA 94132), Vidhyadhar Nandana, Alisa Gega, Imalka Wanigasekara, Nadra Al-Husini (Wayne State University, Detroit, MI, USA 48202), Thomas Kim, Sean Crosson (Michigan State University, East Lansing, MI, USA 48824), Joseph C. Chen (San Francisco State University, San Francisco, CA, USA 94132), Jared M. Schrader (Wayne State University, Detroit, MI, USA 48202)
Abstract:
Bacteria typically lack membrane-bound organelles for organizing biochemical pathways. However, recent studies have revealed that they utilize biomolecular condensates as a mechanism for subcellular organization. These biomolecular condensates are phase-separated structures that organize multiple biochemical pathways without the use of a membrane. Bacterial Ribonucleoprotein Bodies (BR-Bodies) are the first biomolecular condensate discovered in bacterial cells and it compartmentalizes the bacterial RNA degradosome and substrate RNAs. In a limited number of bacteria tested so far, BR-bodies facilitate fast mRNA turnover and stress resistance, yet their phylogenetic breadth across bacteria and potential impacts on host colonization are not yet known. Here, we investigate the phylogenetic breadth of BR-bodies by recombinantly expressing RNA degradosome scaffolds from diverse bacterial species in E. coli. We observed that all RNA degradosome scaffolds tested form foci, indicating they can phase-separate into BR-bodies. This suggests that BR-bodies are likely utilized across a large majority of bacteria as a general organization mechanism. To examine the importance of BR-bodies in host colonization, we generated BR-body null mutants by truncating the C-terminal disordered region on RNase E in the plant-associated symbiont Sinorhizobium meliloti and the animal associated intracellular pathogen Brucella ovis. In both bacteria, we observed that BR-body null mutants have no detectable differences in growth in standard in vitro conditions, however both BR-body null mutants showed slower mRNA decay rates, increased stress sensitivity and marked reductions in plant or animal colonization respectively. Therefore BR-bodies that compartmentalize the mRNA decay machinery appear to be widespread across bacteria and have the capacity to overcome stress and promote host colonization, making BR-bodies a promising target for next-generation antibiotics.
Keywords: mRNA decay , BR-bodies, membrane-less organelles
99. x1 encodes a putative RNA-binding protein required for paramutation
Oliver J. Marchus (Department of Molecular Genetics and Centers for Applied Plant Sciences and RNA Biology, The Ohio State University, Columbus, OH, 43210), Jay B. Hollick (Department of Molecular Genetics and Centers for Applied Plant Sciences and RNA Biology, The Ohio State University, Columbus, OH, 43210)
Abstract:
Paramutations are defined by trans-homolog interactions that result in meiotically heritable gene silencing. In maize, paramutations occur between specific alleles at both the purple plant1 (pl1) and booster1 (b1) loci which encode transcription factors regulating anthocyanin biosynthesis. Relative to the highly expressed Pl-Rh and B-I reference states, the paramutant derivatives (Pl′ and B′ ) confer weak plant color and are exclusively transmitted from respective Pl-Rh / Pl′ and B-I / B′ heterozygotes in apparent violation of Mendelian segregation. While the nature of these interactions remains unclear, an Arabidopsis thaliana (A.t.)-based RNA-directed DNA methylation (RdDM)-like mechanism provides a working model. In forward genetic screens for factors required to maintain repression (rmr) of Pl′ states, ems-induced mutations have defined at least sixteen loci. All seven known RMR proteins facilitate RNA polymerase IV-derived 24 nucleotide (nt) RNA biogenesis, yet no RdDM-like 24nt RNA effector proteins have been identified. Here, we show the rmr17 locus is required to establish both pl1 and b1 paramutations. Bulk-segregant analysis putatively maps rmr17 to the x1 gene encoding the founding member of a plant-specific protein family with namesake XS domains responsible for binding double-stranded (ds) RNA. In A.t., three non-redundant X1-like proteins form heteromeric complexes required for RdDM. These complexes 1) bind 24nt RNA and RNA polymerase V-derived long non-coding RNA (lncRNA) duplexes, 2) interact with the SWI3B nucleosome remodeler, and 3) help recruit the DRM2 de novo cytosine methyltransferase. Our findings thus support a role for X1 in paramutation by recognizing 24nt RNA-lncRNA duplexes and recruiting proteins that facilitate meiotically-heritable repressive chromatin modifications.
Keywords: x1, paramutation
100. Mapping translation initiation sites using the bacterial toxin RelE
Cesar Martinez (Biological Chemistry, University of Michigan), Ross Kaufhold (Biological Chemistry, University of Michigan ), Dr. Jailson Brito Querido (Biological Chemistry, University of Michigan), Dr. Rachel Niederer (Biological Chemistry, University of Michigan)
Abstract:
Translational control of gene expression is critical for diverse cellular processes such as differentiation and stress tolerance. Translation regulation is achieved through a combination of cis and trans regulatory features within mRNA. However, the full extent of these features and their mechanisms of action remain poorly understood. To identify and delineate important regulatory elements our lab utilizes Direct Analysis of Ribosome Targeting (DART) (1) to measure ribosome recruitment to thousands of RNAs in parallel. We are interested in extending this method to instances where we expect changes in translation start site selection, such as RAN (repeat‐associated non‐AUG) translation. To gain insights into start site fidelity we have utilized the bacterial toxin, RelE. RelE cleaves ribosome‐bound mRNA in the ribosomal A site, leaving a record of precisely where the ribosome was bound. Addition of RelE dramatically reduces protein output from the in vitro translation assay, consistent with RelE actively cleaving RNA as expected. Future work will apply this method to investigate translation initiation start site selection in RAN translation substrates. This could potentially reveal how certain neurodegenerative diseases manifest in the human body.
References:
1 Niederer, R. O., Rojas‐Duran, M. F., Zinshteyn, B. & Gilbert, W. V. Direct analysis of ribosome targeting illuminates thousand‐fold regulation of translation initiation. Cell Syst 13, 256‐264 e253 (2022). https://doi.org:10.1016/j.cels.2021.12.002
Keywords: translation, initiation, RelE
101. Unveiling the sequence-specific recognition of N6-methyladenosine (m6A) in eukaryotic RNAs
Aftab Mollah (Kent State University), Dr. Rushdhi Rauf (Kent State University), Dr. Sanjaya Abeysirigunawardena (Kent State University)
Abstract:
The prevalence of N6-methyladenosine (m6A) as the primary nucleotide modification in eukaryotic RNAs underscores its pivotal role in post-transcriptional regulation of gene expression. However, the intricate molecular mechanisms governing m6A recognition by various readers remain elusive. To address this gap, we conducted phage display experiments aimed at deciphering the essential sequence requirements for m6A binding. Strikingly, the same peptide (MP1) was selected for several RNA constructs harboring m6A, emphasizing the significance of specific protein sequence motifs in m6A recognition. Complementary RNA pulldown assays coupled with mass spectrometry revealed proteins with that bind specifically to methylated RNA that showed sequence similarity to peptides enriched in phase display experiments suggesting the importance of protein sequence in m6A recognition. These enriched proteins shared sequence and structural similarities with established m6A readers. Further validation through calorimetric methods highlight the importance of distinctive sequence features of proteins that govern the recognition of N6-methyl modifications. Our study contributes valuable insights into the molecular basis of m6A recognition, with potential implications for understanding broader regulatory networks in gene expression.
Keywords: RNA-Protein Interaction, m6A, Epitranscriptomic Regulation
102. Investigating the transcriptomic changes in response to telomerase loss in Saccharomyces cerevisiae
Taizina Momtareen (West Virginia University), Jennifer Gallagher (West Virginia University)
Abstract:
Telomeres protect the genetic material at chromosome ends but progressively shorten with each cell division. In healthy cells, critically short telomeres trigger senescence and cell death, but cancerous cells use telomerase to elongate their telomeres and restart the cell cycle. ALT (Alternative Lengthening of Telomeres) cancer cells employ a recombination-based mechanism to extend their telomeres without telomerase. Similarly, some telomerase mutants of S. cerevisiae can bypass senescence through DNA recombination-based telomere elongation, forming post-senescence survivors. These survivor cells experience unregulated telomere lengthening, which leads to significant replication stress and upregulation of energy production genes (Nautiyal et al., 2002). While previous studies have examined growth rates and telomere lengths in these mutants, a detailed understanding of the factors contributing to replication stress and the cellular adaptations to overcome it remains incomplete. The aim of this study was to characterize the transcriptomic changes in tlc1- mutant yeast strains across multiple passages (P4, P7, and P10). We used RNA-seq to explore how tlc1- mutant yeast cells adapt to telomerase loss and replication stress over several generations. We identified key biological pathways such as mitochondrial function, ribosome biogenesis, stress response, and found that the regulation of these pathways varied depending on the passage. Several subtelomeric genes were also significantly differentially expressed across the passages, indicating the role of telomerase in maintaining subtelomeric stability. This study provides new insights into the molecular mechanisms of telomerase loss, with potential implications for understanding ALT and other age-related diseases in higher organisms.
References:
Nautiyal, S., DeRisi, J. L., & Blackburn, E. H. (2002). The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 99(14), 9316–9321. https://doi.org/10.1073/pnas.142162499
Keywords: transcriptome, telomere
103. Transcriptional start site usage in the HIV-1 genome dictates dimerization and selective packaging by modulating polyA stem stability
Michael R. Muccio (Ohio State University, Department of Chemistry and Biochemistry, Centers for RNA Biology and Retrovirus Research, Columbus, OH), Joseph G. Kanlong (Ohio State University, Department of Chemistry and Biochemistry, Centers for RNA Biology and Retrovirus Research, Columbus, OH), Jonathan P. Kitzrow, Olga Nikolaitchik (NCI, DRP, Viral Recombination Section, Frederick, MD), Vinay K. Pathak (NCI, DRP, Viral Mutation Section, Frederick, MD), Saiful Islam, Zetao Cheng, Akhil Chameettachall, Wei-Shau Hu (NCI, DRP, Viral Recombination Section, Frederick, MD), Karin Musier-Forsyth (Ohio State University, Department of Chemistry and Biochemistry, Centers for RNA Biology and Retrovirus Research, Columbus, OH)
Abstract:
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that encodes a 9.2 kb RNA genome. The HIV-1 unspliced (US) RNA serves as both the genomic RNA (gRNA) and as an mRNA template for translation of Gag and Gag-Pol polyproteins. The 5' untranslated region (UTR) of HIV-1 RNA is a major regulatory element and contains distinct structural motifs. These include the 5' terminal transactivation response element (TAR) and polyA hairpins, followed by three stem loops (SL1-SL3) that contain binding sites for Gag and a palindromic loop known as the dimerization initiation signal (DIS), which serves as the primary site for gRNA dimerization initiation. Recently, HIV-1 transcription start site (TSS) heterogeneity has been shown to produce major HIV-1 RNA transcripts with three guanosines (3G) or one guanosine (1G) at the 5' end. 3G RNAs are the major species of US RNA in the cytoplasm, whereas 1G RNAs are selectively packaged into new viral particles. 1G RNAs adopt a major conformation that stabilizes the polyA hairpin, exposing the DIS and Gag binding sites crucial for genome packaging, while 3G RNAs have an unstable polyA stem loop and adopt a conformation that sequesters these elements and favors mRNA translation. Thus, differences in the stability of the polyA hairpin appear to modulate the function of HIV-1 US RNA. Here, we performed UV thermal melting studies to investigate the stability of the polyA hairpins in various contexts. We first showed that the polyA hairpin was significantly less stable in the 3G HIV-1 TAR-PolyA RNA relative to the 1G construct. Viral packaging assays performed using a TSS mutant that produces RNAs with either a single 5' adenosine (1A) or a 5' GGA, showed that neither of the RNAs were selectively packaged; Tm studies showed that the differences in polyA hairpin stability were also eliminated. A compensatory mutant was identified that rescued selective 1A packaging and Tm studies showed that the polyA hairpin stability differences were also restored. We also performed native gel electrophoresis assays and RNA structure probing to observe how these mutants influenced the global 5’ UTR structure. Overall, these studies confirm a remarkable correlation between packaging selectively and differential polyA hairpin stability to inform new RNA-targeted therapeutic strategies.
Keywords: HIV-1, RNA Structure, Viral Packaging
104. Dissecting the Mechanism of IRE1 Activation with Orthogonal Control over Oligomerization and Phosphorylation
Swapnil Mukherjee (Department of Chemistry and Biochemistry, The Ohio State University), Vladislav Belyy (Department of Chemistry and Biochemistry, Center of RNA Biology, The Ohio State University)
Abstract:
Inositol-requiring enzyme 1 (IRE1) is an ER-localized bifunctional kinase/RNase membrane receptor that primarily senses ER stress. When activated, mammalian IRE1 cleaves a specific mRNA encoding the transcription factor X-box binding protein (XBP1), a key step in ER stress alleviation. The precise mechanism by which oligomerization activates IRE1 is still unknown, in part due to the substantial experimental challenges associated with studying subtle oligomeric transitions in low-affinity protein clusters. To overcome this problem, we engineered an optogenetic platform to study the consequence of oligomerization on the activity of IRE1. This system comprises the cytosolic domain of mammalian IRE1 fused to components of a light-inducible heterodimerization system. Phosphorylation or dephosphorylation of the protomers allows us to assemble dimers of well-defined phosphorylation states. We used in vitro RNA cleavage as a readout to study how the activity of IRE1 is modulated by change in protein state. In agreement with previous reports, we find that the activity of IRE1 increases on dimerization and phosphorylation. Surprisingly, our initial results suggest that while phosphorylated monomers exhibit higher activity than dephosphorylated monomers, dimeric forms of IRE1 exhibits similar rates of XBP1 cleavage, regardless of phosphorylation state. This suggests that the role of phosphorylation might be to primarily stabilize the active dimeric state of IRE1. Moreover, our preliminary results highlight unexpected kinetic differences between the processing of canonical substrates (XBP1) and non-canonical RNA substrates by distinct forms of IRE1. These findings are especially relevant in the context of IRE1’s additional proposed role in regulated degradation of a variety of ER-targeted mRNAs in response to stress. Taken together, our results build towards a model of how IRE1-dependent signaling is modulated in cells.
Keywords: IRE1, RNase, Optogenetics
105. Examining tRNA Quality Control in Yeast Cells Under Oxidative Stress Conditions
Hailey Mulvihill (Biochemistry and Molecular Biology, Wittenberg University), Kunal Chatterjee (Department of Biological and Environmental Sciences, Wittenberg University)
Abstract:
This study explores the response of transfer RNA (tRNA) in the yeast Saccharomyces cerevisiae to oxidative stress. In protein translation, tRNA attaches amino acids to a growing polypeptide chain at the ribosome. Extra nucleotide sequences present on the 3' end of tRNA, where amino acids attach, interfere with the ability of the tRNA to transport amino acids. Such aberrant tRNA is useless to the cell. Previous work has identified the presence of this end-extended, aberrant tRNA under standard growth conditions in yeast cells. However, there is little research into the quantity of yeast aberrant tRNA under atypical growth conditions. We were interested in exploring the effect of various 𝐇𝟐O𝟐 concentrations on yeast tRNA quality and aberrant tRNA quantity. We first investigated yeast growth in liquid media under 𝐇𝟐O𝟐 stress and determined that culture viability decreased after stress application. Through experimental methods, we then observed that under 𝐇𝟐O𝟐 stress, the aberrant tRNA quantity decreased. Whether aberrant tRNAs are being destroyed or repaired is still under investigation, however, it appears that at high 𝐇𝟐O𝟐 concentrations, transcription of tRNAs is hindered.
Keywords: tRNA , Oxidative Stress, Yeast
106. Ribosome pausing analysis in Cryptococcus neoformans
Rohan Munoth (Department of Biological Sciences,Carnegie Mellon University), Simon Chow, (Department of Biological Sciences,Carnegie Mellon University), Gemma May (Department of Biological Sciences,Carnegie Mellon University), Corey Knowles (Department of Microbiology and Immunology, University at Buffalo Medical school), John Panepinto (Department of Microbiology and Immunology, University at Buffalo Medical school), Joel McManus (Department of Biological Sciences,Carnegie Mellon University)
Abstract:
Cryptococcus neoformans is an opportunistic fungal pathogen that results in an estimated 625,000 deaths annually worldwide. Ribosome pausing during translation elongation is a critical regulatory mechanism influencing protein folding, targeting, and mRNA stability. Previous studies have identified that ribosome stalling can be induced by specific nascent peptides interacting with the ribosome exit tunnel, with factors such as peptide hydrophobicity, polarity, and charge contributing to this process. While it is well-established that certain amino acids, like proline, induce ribosome stalling in many eukaryotic species, the precise role of nascent peptide characteristics in influencing ribosome pausing in C. neoformans has not been determined. We used a novel analysis pipeline to investigate how nascent peptide properties affect ribosome pausing in C. neoformans. Consistent with prior findings, our results confirm that most pause sites occur when proline is in the ribosomal P site. In addition, our analysis revealed that the hydrophobicity and polarity of the nascent peptide increase significantly as it approaches the pause site, a trend that aligns with previous reports in other species. Furthermore, we observed that the charge at the ribosomal P-site is more positive in pause-site peptides compared to non-pause-site peptides, suggesting electrostatic interactions play a role in the stalling process. Our comparison with prior research corroborates the role of specific amino acids in pausing while highlighting additional peptide characteristics that may influence this phenomenon. In future work, we will apply our pipeline to analyze pausing in datasets from Saccharomyces cerevisiae and Candida albicans to determine the extent to which the trends observed in C. neoformans are conserved across species and to identify pause sites conserved across fungi.
References:
Stephen J Kiniry, Audrey M Michel, and Pavel V Baranov. Computational meth-
ods for ribosome profiling data analysis. Wiley Interdisciplinary Reviews: RNA,
11(3):e1577, 2020.
Carrie L Simms, Erica N Thomas, and Hani S Zaher. Ribosome-based quality control of mrna and nascent peptides. Wiley Interdisciplinary Reviews: RNA, 8(1):e1366, 2017.
Douglas R Tanner, Daniel A Cariello, Christopher J Woolstenhulme, Mark A Broadbent, and Allen R Buskirk. Genetic identification of nascent peptides that induce ribosome stalling. Journal of Biological Chemistry, 284(50):34809–34818, 2009.
Sai Zhang, Hailin Hu, Jingtian Zhou, Xuan He, Tao Jiang, and Jianyang Zeng. Analysis of ribosome stalling and translation elongation dynamics by deep learning. Cell systems, 5(3):212–220, 2017.
Keywords: Ribosome pausing, Cryptococcus neoformans
107. Why Bacteroidia ribosomes tuck in their tails
Fawwaz M. Naeem (Ohio State Biochemistry Program, Center for RNA Biology, OSU), Bappaditya Roy (Center for RNA Biology, Department of Microbiology, OSU), Kurt Fredrick (Ohio State Biochemistry Program, Center for RNA Biology, Department of Microbiology, OSU)
Abstract:
Certain groups of bacteria, including the Bacteroidia, generally lack Shine-Dalgarno (SD) sequences. Bacteroidia ribosomes contain the anti-Shine-Dalgarno (ASD) element but fail to recognize SD sequences, both in vivo and in vitro. A cryo-EM structure of the Flavobacterium johnsoniae ribosome shows that the 3’ tail of 16S rRNA is sequestered in a pocket formed by bS21, bS18, and bS6 on the 30S platform, explaining the basis of ASD inhibition (1). Interestingly, one gene in F. johnsoniae contains a strong SD sequence—rpsU, which encodes bS21. F. johnsoniae ribosomes lacking bS21 exhibit a liberated ASD and translate rpsU at a higher rate, which creates a feedback loop that autoregulates bS21 production (2). The platform pocket is conserved across members of Bacteroidia, even those with no apparent bS21 autoregulation, hinting at a broader role for ASD sequestration. Recently, we have systematically targeted the ASD of each 16S rRNA gene in F. johnsoniae, substituting the core element at 4 of 5 positions (CCUCC to GAAGC). This quadruple mutation (QM) should not only abolish SD recognition but also liberate the 3’ tail from the pocket. Consecutive replacement of each 16S gene with the QM allele had little effect on cell growth until the last gene was changed. The final strain, containing only QM ribosomes, grows very slowly, with a doubling time >4-fold larger than wild-type. This growth defect can be largely rescued by a plasmid-borne copy of rpsU with the SD-less leader of the EF-Tu gene (tuf). This underscores the importance of SD-ASD pairing for translation of endogenous rpsU in F. johnsoniae. However, re-wiring of rpsU translation cannot fully restore growth rate, indicating that the QM ribosomes are compromised in some other way. Currently, we are studying mutations in bS18 and bS6 that specifically target the platform pocket. Our initial findings suggest that initiation on SD-less mRNA depends on the integrity of the pocket. Thus, ASD sequestration appears to play both regulatory and housekeeping roles in the Bacteroidia.
References:
1. Jha, V., Roy, B., Jahagirdar, D., McNutt, Z.A., Shatoff, E.A., Boleratz, B.L., Watkins, D.E., Bundschuh, R., Basu, K., Ortega, J. et al. (2021) Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome. Nucleic Acids Res, 49, 547-567.
2. McNutt, Z.A., Roy, B., Gemler, B.T., Shatoff, E.A., Moon, K.M., Foster, L.J., Bundschuh, R. and Fredrick, K. (2023) Ribosomes lacking bS21 gain function to regulate protein synthesis in Flavobacterium johnsoniae. Nucleic Acids Res, 51, 1927-1942.
Keywords: Ribosome, Flavobacterium johnsoniae , bS21
108. Micropeptide-mediated regulation of mRNA translation during ER stress
Shyama Nandakumar (Cell Biology, University of Pittsburgh School of Medicine), Deepika Vasudevan (Cell Biology, University of Pittsburgh School of Medicine)
Abstract:
The eukaryotic transcription factor ATF4 regulates the Integrated Stress Response, which responds to various stressors including ER stress. When ISR is activated, global protein synthesis is swiftly dampened due to the phospho-inactivation of the initiator methionine complex, eIF2, by stress-responsive kinases. However, ATF4 translation is paradoxically induced due to its specialised 5' leader. Our work uncovers a novel trans-acting micropeptide, µP, that regulates translation of the ATF4 main ORF.
The ATF4 5’ leader contains multiple short upstream ORFs (uORFs), with the ultimate uORF (encoding µP) sequence overlapping the ATF4-encoding main ORF.µP. Under homeostasis, due to termination events at the uORF1 stop codon, the ribosome occasionally “reinitiates” translation at the uORF2 start codon to synthesize µP. However, under conditions of stress, reinitiation at µP decreases due to reduced initiator methionine, thus the ribosome can reinitiate at the ATF4 start codon implying a trade-off between synthesis of µP and ATF4, such that µP favored under homeostasis and ATF4 under stress.
To test if µP regulates Atf4 activity, we made fly and mammalian models with inducible, ectopic expression of µP. Our data show that expression of µP reduces ATF4 activity in both systems by reducing ATF4 protein. Using an ATF4-5’leader reporter, we find that µP regulates the translation ATF4 without affecting transcript levels. Further, the mechanism of µP action is not dependent on known regulators of translation reinitiation such as DENR. We aim to understand µP-mediated translation regulation of the ATF4 5’leader. Since ATF4 mRNA is ubiquitously transcribed in nearly all cells, there is near-constitutive expression of µP in homeostasis. Intriguingly, our data indicate µP may have independent physiological functions, since µP expression in fly adipocytes results in developmental acceleration and increased lipid reserves. Thus we present a novel modality of ATF4 regulation.
Keywords: ATF4, Stress, translation
109. Investigating the dynamic regulation of arginine methylation on the SR-/hnRNP-like protein Npl3
Paige Nati (Department of Biological Sciences, University at Buffalo), Joe Ricottone (Department of Biological Sciences, University at Buffalo), Tao Liu (Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center), Michael C. Yu (Department of Biological Sciences, University at Buffalo)
Abstract:
Npl3 is an RNA-binding protein in Saccharomyces cerevisiae involved in various cellular processes, such as translation, pre-mRNA splicing, and mRNA export. Npl3 undergoes arginine methylation by the protein arginine methyltransferase Hmt1 which facilitates its cellular localization and protein-protein interactions. Whether dynamic regulation of arginine methylation occurs in budding yeast remains an outstanding question. In mammalian cells, no bona fide arginine demethylase has been identified. Instead, a different class of enzyme, peptidyl arginine deiminases (PADs), can counteract the action of arginine methylation by converting a monomethylarginine to citrulline. Notably, no homolog of PAD has been identified in the budding yeast. We have recently observed changes in the level of Npl3 methylation across different growth stages, suggesting the presence of a putative mechanism that regulates Npl3 methylation. Specifically, we observed an increase in the ratio of methylated to total Npl3 in the fermentative growth phase of yeast, followed by a decrease in this ratio in the respiratory growth phase. Based on these observations, we hypothesize that a proteostasis mechanism exists that selectively degrades methyl-Npl3, thereby maintaining a specific ratio of methylated to non-methylated Npl3. Here, we will present our latest data on the selective degradation of methyl-Npl3 by investigating the two main degradation pathways: the ubiquitin-proteasome system and autophagy.
Keywords: Arginine methylation, proteostasis
110. Live-Cell Chromatin Looping by CRISPR and Engineered gRNAs
Yu-Chieh Chung (Department of Biological Chemistry and Pharmacology, The Ohio State University), Siou-Luan He (Department of Biological Chemistry and Pharmacology, The Ohio State University), Dilshodbek Nishonov (Department of Biological Chemistry and Pharmacology, The Ohio State University), Nevin Wise (Department of Biological Chemistry and Pharmacology, The Ohio State University), Li-Chun Tu (Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract:
Chromatin looping is critical in organizing the genome to allow for controlled regulation of transcriptional activity. However, the extent to which chromatin-chromatin interactions causally affect gene expression is unclear. Forced chromatin looping methods using CRISPR offer an additional avenue to study cause-effect relationships between chromatin state and transcription. Although there are existing CRISPR-based chromatin looping systems, limitations exist in their capability to form long-range looping interactions and in their efficiency. Here, we repurposed our existing CRISPR-Sirius system used for live cell imaging into CRISPR-CLIP, a novel method to physically manipulate chromatin folding at a kilobase and megabase scale. The transformation was achieved by fusing two CRISPR-Sirius guide RNAs (gRNAs) into a single CLIP gRNA to force loci pairs to localize into proximity. We successfully verified our system by targeting loci pairs within individual human chromosomes with varying genomic distances and by visualizing the colocalization of fluorescent signals from the MS2 and PP7 hairpins engineered in the CLIP gRNA scaffold. The colocalization was confirmed by measuring the physical distances between the loci pairs. Furthermore, we achieved large-scale chromosome contraction (~17 Mb) by targeting multiple CLIP gRNAs to the repetitive sequence within the chromosome 19 q arm. RT-PCR was performed to measure the changes in gene expression after the chromosome contraction. We did not observe significant changes in expression in response to the volume reduction, suggesting that chromatin volume reduction alone is not sufficient to repress gene expression. Overall, CRISPR-CLIP is a live-cell tool that can serve to answer questions about the role of chromatin organization at different genomic scales in gene expression and help elucidate how long-range interactions between cis-regulatory elements function.
References:
Ma, H., Tu, LC., Naseri, A. et al. CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods 15, 928–931 (2018). https://doi.org/10.1038/s41592-018-0174-0
Keywords: gRNA, chromatin, CRISPR
111. A Regulatory RNA:RNA interaction in Listeria monocytogenes
Martin OSteen (University of Michigan Department of Biophysics ), Aldrex Munsayac (University of Michigan Department of Chemistry), Jocelyn Chen (University of Michigan Department of Chemistry ), Sarah C. Keane (University of Michigan Department of Biophysics, University of Michigan Department of Chemistry)
Abstract:
Non-coding RNA elements found in the 5′ and 3′ untranslated regions (UTRs) of some
messenger RNA transcripts can alter gene expression in the absence of regulatory proteins.
These elements are tuned to respond to environmental changes, such as temperature (RNA
thermosensors) and metabolite concentration (riboswitches). Regulation by these elements is predominantly cis-acting, functioning on the downstream gene, and can occur at both the transcriptional and translational levels. Trans-regulation by these elements is rare, with few examples described in the literature. One such example is found in Listeria monocytogenes. L. monocytogenes is a Gram-positive bacterium commonly found in soil and decaying vegetation as a saprophyte. Upon ingestion, L. monocytogenes undergoes a virulence gene program to facilitate infection and intracellular spread, causing listeriosis. This transition is controlled by a transcriptional activator, positive regulatory factor A (PrfA). In turn, PrfA translation is regulated by an RNA thermosensor located in the 5′ UTR of the prfA transcript, which is tuned to allow translation at temperatures above 34 °C. Additional suppression of prfA translation is mediated by a series of S-adenosyl methionine (SAM)-sensitive riboswitches (sreA and sreB), demonstrating a complex regulatory network to prevent inadvertent PrfA expression. However, the sequence, molecular, and environmental determinants underlying this interaction are not well understood. Herein, we describe our efforts to characterize and elucidate the sequence and environmental determinants required for the sreA:prfA interaction. Our findings demonstrate that this interaction is sensitive to temperature and influenced by the chemical environment. Mutagenesis of the sreA riboswitch in functional translational assays and electromobility shift assays (EMSAs) revealed critical sequence determinants for both binding and translational inhibition of prfA.
Keywords: RNA Regulation, RNARNA Interactions, Riboswitch
112. Impact of Molecular Crowding Agents on RNA Aptamer Biosensors
Zachary L. Parada (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota), Katarzyna P. Adamala (Department of Genetics, Cell Biology, and Development), Aaron E. Engelhart (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota)
Abstract:
Molecular crowding agents replicate cellular environments in cell-free systems with poly-ethylene glycol (PEG) and Ficoll being two of the most commonly used crowding agents. PEG, a flexible, hydrophilic polymer, modulates solution viscosity, while Ficoll, a highly branched polymer, mimics osmotic conditions with minimal biomolecular interaction. Traditionally considered inert, recent studies suggest that PEG and Ficoll may influence biomolecules beyond spatial restriction.
This study investigates how varying concentrations and molecular weights of PEG (7.5k, 20k) and Ficoll (Ficoll-70, Ficoll-400) affect the binding affinity, stability, and fluorescence of SAM RNA aptamer-based biosensors (Corn and Pepper aptamers). We assessed the effects of these agents through fluorescence endpoint assays and UV-visible spectroscopy to measure dissociation constants (KD) and the optical properties of the cognate dyes (DFHO and HBC-620).
Our findings indicate that PEG treatments significantly increased KD values, reducing binding affinity at higher RNA aptamer concentrations. Ficoll-70 had a less pronounced effect, while Ficoll-400 at lower concentrations enhanced ligand interaction. Changes in the dyes' absorption spectra suggest potential interactions with crowding agents, influencing biosensor fluorescence. Importantly, in vitro transcription was unaffected, confirming that the crowding agents influenced binding properties without altering RNA production.
These results highlight the substantial impact of crowding agents on RNA aptamer biosensors, emphasizing the need for careful agent selection in biosensor design. By simulating crowded environments, the performance of RNA biosensors can be optimized, contributing to their potential use in biological and clinical applications.
Keywords: crowding agent, RNA aptamer, biosensor optimization
113. Determination of the mechanisms governing piRNA degradation
Benjamin Pastore (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University), Wen Tang (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University)
Abstract:
Organisms are constantly under attack by foreign nucleic acids from viruses and transposons that cause genome damage and an array of genetic diseases. To combat these foreign genetic elements organisms evolved RNA- and DNA- directed immune pathways such as RNA-interference (RNAi) and CRISPR/CAS. One of the most ancient and evolutionarily conserved RNA-directed immune pathways is the PIWI/piRNA pathway. In animal germlines piRNAs, together with effector PIWI proteins, suppress transposons and are indispensable for fertility. Since their discovery, piRNA biogenesis and function have been well studied, yet until recently the mechanisms governing piRNA degradation were largely unexplored. In a recent work, we identified a piRNA quality control mechanism that is mediated by 3' nontemplated nucleotide additions (3' tailing) of RNA nucleotides. Moreso, we found that piRNA::target interactions have the capacity to induce 3' uridine (U) tailing of piRNAs and their degradation. Using genetics, high-throughput RNA sequencing, and bioinformatics, we have expanded our understanding of piRNA degradation by identifying and characterizing the terminal nucleotidyltransferase (TENT) and exonuclease required for piRNA 3' U tailing and degradation. Additionally, to specifically interrogate factors mediating piRNA turnover, we designed an assay to measure global piRNA turnover rates by shutting of piRNA transcription via auxin inducible degron of the piRNA transcription factor TOFU-5. In all we find that piRNA 3' U tailing is concurrent, yet not required for decay. Additionally, we report that piRNAs have a broad range of half-lives ranging from less than 1 hour to greater than 16 hours. Intriguingly, we find that in general, piRNAs with 3' terminal U nts are turned over at rates faster than those with 3' G or C, suggesting 3' U nts make piRNAs susceptible to exonucleolytic degradation. In all this highly systematic work greatly expands our understanding of the mechanisms of piRNA decay by identifying the TENT, and exonuclease mediating piRNA tailing and degradation, as well as measuring, and determining factors that influence, piRNA half-life.
References:
Pastore, B., Hertz, H.L., Price, I.F., and Tang, W. (2021). pre-piRNA trimming and 2'-O-methylation protect piRNAs from 3' tailing and degradation in C. elegans. Cell Rep 36, 109640. 10.1016/j.celrep.2021.109640.
Gainetdinov, I., Colpan, C., Cecchini, K., Arif, A., Jouravleva, K., Albosta, P., Vega-Badillo, J., Lee, Y., Ozata, D.M., and Zamore, P.D. (2021). Terminal modification, sequence, length, and PIWI-protein identity determine piRNA stability. Mol Cell 81, 4826-4842 e4828. 10.1016/j.molcel.2021.09.012.
Ozata, D.M., Gainetdinov, I., Zoch, A., O'Carroll, D., and Zamore, P.D. (2019). PIWI-interacting RNAs: small RNAs with big functions. Nature Reviews Genetics 20, 89-108. 10.1038/s41576-018-0073-3.
Keywords: piRNA, C elegans, RNA decay
114. Codon and tRNA coevolution in Lactobaccilales impact on translation speed and accuracy
Chathuri Pathirage (Department of Chemistry, University of Michigan), Christopher D. Katanski (Department of Biochemistry and Molecular Biology, University of Chicago), Tyler J. Smith (Department of Chemistry, University of Michigan), Yichen Hou (Department of Biochemistry and Molecular Biology, University of Chicago), Kristin S. Koutmou (Department of Chemistry, University of Michigan), Tao Pan (Department of Biochemistry and Molecular Biology, University of Chicago)
Abstract:
Codon usage bias is widely observed in the genomes of different species, families and at the gene level within genomes. Coevolution of tRNA abundances and corresponding codon usage is supported by their positive correlation in natural selection. Here, we investigated the evolutionary divergence of Leu codon usage in four families of Lactobacillales order in correlation to their tRNA abundances and modifications. The families Carnobacteriaceae (C), Enterococcaceae (EF), Lactobacillaceae (L), and Streptococcaceae (SP) contain A/T rich genomes (~65%) and display an overall preference for UUA codon, while also exhibiting family-specific codon selectivity. Concurrently, the families show variation in the sequences, abundances and modifications in tRNAs. tRNA-seq of the oral microbiome revealed that tRNAGAGLeu, which recognizes CUC codon in EF is modified to Inosine (I) at wobble 34 in Streptococcus pyogenes of SP family. Using an E. coli reconstituted in vitro translation system, we demonstrate that I34 expands the Leu decoding ability to less-abundant codons, CUC and CUU in SP. Despite the overall high representation of UUA, certain genes in SP family such as those involved in glycolysis pathways have selectivity towards CUU and UUG. tRNA-seq of both oral and stool microbiomes showed an overall increase in ratio for tRNALeuCAA/UAA in S. pyogenes compared to Enterococcus faecalis, suggesting codon usage expansion to UUG in SP could be due to differences in corresponding tRNA abundances. Also, changes in variable loop sequences as observed between EF and SP tRNAUAALeu and tRNACAALeu may affect the overall tRNA stability and interactions. In vitro translation assays show that the changes in the variable loop of tRNALeu isodecoders lead to differences in translational efficiency, with SP tRNAUAALeu displaying a 10-fold decrease in rate compared to EF. This further supports the codon expansion to UUG, in SP. Overall, this work demonstrates how specific properties of tRNAs influence codon divergence leading to translation efficiency and optimality within families in Lactobacillales order.
Keywords: codon usage bias, tRNA coevolution, tRNA modifications
115. Title not available online - please see the booklet.
Brendan R. Pew (Department of Biological Chemistry and Pharmacology at The Ohio State University), Ian F. Price (Department of Biological Chemistry and Pharmacology at The Ohio State University), Wen Tang (Department of Biological Chemistry and Pharmacology at The Ohio State University)
Abstract not available online - please check the booklet.
116. Translation at the ER: Developing a smART-ER method
Ben Pockrass (Department of Biological Chemistry, University of Michigan Medical School), Rachel O. Niederer (Department of Biological Chemistry, University of Michigan Medical School)
Abstract:
It is generally understood that mRNAs encoding membrane/secreted proteins are targeted to the endoplasmic reticulum (ER) during early elongation via the signal recognition particle pathway. In this model, translation initiation occurs in the cytoplasm, and mRNA is only targeted to the ER upon emergence of an N-terminal signal sequence during early elongation. However, subsequent rounds of translation initiation on an already ER-localized mRNA would take place near the ER. Additionally, there is evidence that ribosomes remain in stable association with the ER following translation termination1 and that these ER-bound ribosomes are capable of direct translation initiation, including mRNAs encoding cytoplasmic proteins which do not encode a signal sequence2. Despite these findings, the nature of translation initiation at the ER and its regulation are largely unexplored. Because mRNA 5’ untranslated regions (5’ UTRs) are the site of translation initiation, and translation initiation is highly regulated, we hypothesize that 5’ UTR features regulate translation initiation at the ER. One prediction of this hypothesis is that 5’ UTRs from mRNAs encoding membrane proteins would have distinct features, as they uniquely require translation at the ER. A bioinformatic analysis of 5' UTR features from mRNAs encoding membrane proteins compared to those encoding non-membrane proteins revealed subtle but significant differences in their sequence composition, 5’-terminal nucleotide identity, and presence of RNA binding protein motifs. To systematically identify mRNA 5’ UTR features associated with enhanced/repressed translation initiation at the ER, we are developing a high-throughput method to measure translation initiation rates on ER-bound ribosomes for thousands of unique mRNAs. Discovery of these 5’ UTR features will guide further studies into the factors regulating translation initiation at the ER.
References:
1. Potter, M. D. & Nicchitta, C. V. Regulation of Ribosome Detachment from the Mammalian Endoplasmic Reticulum Membrane. Journal of Biological Chemistry 275, 33828–33835 (2000).
2. Jagannathan, S., Reid, D. W., Cox, A. H. & Nicchitta, C. V. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum. RNA 20, 1489–1498 (2014).
Keywords: translation initiation, ER, UTR
117. AAV-delivered Splice Switching Oligonucleotides: Restoring p53 function through MDM2 splicing modulation
Rafia Rahat (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University), Matias Montes (Department of Chemical and Systems Biology, Stanford University), Akila S Venkataramany (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University), Ravi Dhital (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics, The Ohio State University), Kevin Cassady (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University), Dawn Chandler (Center for Childhood Cancer Research, Nationwide Childrens Hospital and Department of Pediatrics and the Center for RNA Biology, The Ohio State University)
Abstract:
MDM2 negatively regulates the tumor suppressor p53, but its alternatively spliced isoform MDM2-ALT1 counteracts this by inhibiting full-length MDM2 (MDM2-FL). MDM2 is upregulated and drives oncogenesis in multiple sarcomas. Switching splicing from MDM2-FL to MDM2-ALT1 can restore p53 function and impede cancer progression. Our lab has shown that Splice Switching Oligonucleotides (SSOs) induce MDM2-ALT1 expression and reactivate p53 function. For in vivo target specific delivery, we expressed our SSO sequence as an antisense-RNA from a lentiviral plasmid and packaged it inside extracellular vesicles (EV). These SSO/antisense-RNA packaged in EVs successfully switched splicing and produced MDM2-ALT1. However, this effect was transient, likely due to degradation of the EV encapsulated antisense-RNA. To enhance stability, we plan to attach the SSO sequence to modified U7 snRNA and expressed it as a transgene from adeno-associated viral vectors (AAVs). We hypothesize that these engineered AAVs will facilitate in vivo delivery of SSOs to p53 wild-type tumor cells, induce MDM2-ALT1 expression, and restore p53 activity to reverse the cancer phenotype. We incorporated our SSO sequence in an AAV viral vector to be expressed as a transgene and validated its ability to induce MDM2-ALT1 splicing in vitro using RT-PCR. Next, we will construct viruses to express this transgene for in vivo delivery in xenograft tumor models. Additionally, we plan to package SSO/antisense-RNA attached to U7 snRNA in EVs to validate potentially increase in stability and target-specific delivery. The transgene expressed by the AAV viral vector successfully induced MDM2-ALT1 expression and stem-loop RT-qPCR analysis confirmed the packaging of the SSO/antisense-RNA within EVs collected from AAV vector transfected RH30 cells. We believe AAV or EV-mediated delivery of SSOs to p53 wild-type tumor cells will induce MDM2-ALT1 expression, enhancing p53 activity and potentially reversing the cancer phenotype.
References:
1. Comiskey, Daniel F Jr et al. “SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway.” Molecular cancer research : MCR vol. 18,2 (2020): 194-203. doi:10.1158/1541-7786.MCR-19-0541
2. Uchikawa, Hideki et al. “U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites.” Journal of human genetics vol. 52,11 (2007): 891-897. doi:10.1007/s10038-007-0192-8
3. Gadgil, Ankur, and Katarzyna Dorota Raczyńska. “U7 snRNA: A tool for gene therapy.” The journal of gene medicine vol. 23,4 (2021): e3321. doi:10.1002/jgm.3321
Keywords: Alternative splicing, Splice Switching Oligonucleotides, AAV mediated SSO delivery
118. Nucleoside modification mapping by genome-independent universal mass exclusion list of unmodified oligonucleotides during LC-MS/MS
Asif Rayhan (Chemistry, University of Cincinnati), Balasubrahmanyam Addepall (Chemistry, University of Cincinnati), Patrick A. Limbach (Chemistry, University of Cincinnati)
Abstract:
To evaluate the effectiveness of the UMEL for mapping RNA modifications in a single organism, RNase T1 digestion products from different organisms tRNA were analyzed separately and the number of modified digestion products detected using standard DDA and UMEL were compared. However, modifications containing only pseudouridine or one dihydrouridine were not considered in these experiments as they have the same mass as uridine.
A standard DDA based LC-MS/MS analysis of RNase T1 digestion products from E.coli total tRNAs resulted in detection of 62 post-transcriptionally modified digestion products. When the
UMEL approach was used, total 72 post-transcriptionally modified digestion products were detected. Thus, the use of an exclusion list increased the number of detected modified digestion products by 16%. To demonstrate the applicability of the universal exclusion list for RNA modification mapping, S.pombe was chosen which is previously uncharacterized. The modified nucleosides in S.pombe have not been reported previously. That’s why a census of modification was obtained from LC-MS/MS analysis of a nucleoside digest. Seventeen unique modified nucleosides were identified from S.pombe tRNAs. The RNase T1 digestion products from S.pombe total tRNA were analyzed both the standard DDA and UMEL approaches. The UMEL approach identified 12% more post-transcriptionally modified digestion products than the standard DDA analysis. Notably, we observed improved data acquisition for modified oligonucleotides, as the instrument prioritizes time for modified species by excluding unmodified ones. We will extend our analysis to include Thrmus Themophillus and Citrobacter koseri.
Keywords: tRNA , LCMS
119. Covalent Ligand Discovery for Selectively Targeting Homologous RNA Binding Protiens: hnRNP H and F
JOHANN ROQUE (Department of Chemistry and Biochemistry University of Notre Dame), Julia Haas (Department of Chemistry and Biochemistry University of Notre Dame), Ariel Thelander (Department of Chemistry and Biochemistry University of Notre Dame), Dr. Blanton Tolbert (Department of Biochemistry and Biophysics, Unviersity of Pennsylvania ), Dr. Brittany S. Morgan (Department of Chemistry and Biochemistry University of Notre Dame)
Abstract:
Dynamic and/or disordered proteins have historically been difficult to target by conventional, non-covalent small molecule methods. This is largely due to their lack of classical binding pockets and lack of protein structural information for rational design. One class of unligandable, dynamic and/or disordered proteins is RNA-binding proteins (RBPs). RBPs are master regulators of RNA biology and are the protein family with the most disease associated mutations. Despite their therapeutic potential, RBPs lack selective, potent small molecule probes, hindering RBP structure, function, and therapeutic studies. This is due in part to their structural conservation, where the sites of greatest diversity are in unligandable dynamic loops and linkers. Utilizing heterogeneous nuclear RiboNucleoProtein (hnRNP H/F) as a model system, we hypothesized that covalent ligands could selectively target loop structures and differential loop sequence and dynamics could be exploited for ligand selectivity. To begin, lead ligands were identified through literature reported cell lysate screenings. The ligands were re-screened in vitro, which identified F4 as two-fold selective for hnRNP H. By commercial and synthetic structure-activity relationships (SAR), an analog has been discovered with ten-fold selectivity for hnRNP H. Furthermore, the SAR revealed: i) the reaction mechanism of the covalent warhead significantly alters selectivity; ii) elimination of a single amide bond leads to a dramatic decrease in potency; and iii) isosteres of adamantane lead to higher selectivity. This work corroborates the potential of covalent ligands to selectively target dynamic and/or disordered proteins, particularly in systems with high sequence and structural conservation. Further, it has the potential to revolutionize our understanding of RBPs, leading to potent and selective probes for these “unligandable” proteins.
Keywords: Proteins, Covalent , Dynamic
120. Investigation of a unique vertebrate tRNA modification by Trmt10b in zebrafish
Olivia Roumaya (Ohio State Biochemistry Program, Ohio State University), Ben Jepson (Molecular, Cellular and Developmental Biology, Ohio State University), Thomas Gallagher (Department of Molecular Genetics, Ohio State University), Sharon Amacher (Department of Molecular Genetics, Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry, Ohio State University)
Abstract:
The tRNA methyltransferase 10 (Trm10) family methylates the N-1 atom of select purines at position 9 (m1R9) in the tRNA core using S-adenosyl methionine (SAM) and is ubiquitously expressed in Eukarya and Archaea. While most archaeal and single-celled eukaryotic species contain only one homolog of Trm10, vertebrates contain two cytoplasmic (Trmt10a and Trmt10b) and one mitochondrial (Trmt10c) enzyme. We and others have established that human and zebrafish (Danio rerio) Trmt10a enzymes perform m1G9-modification on a number of tRNAs, while Trmt10b has a non-redundant role in catalyzing m1A9-modification solely on tRNAAsp in human cells and in zebrafish embryos. Intriguingly, while the biological modification pattern catalyzed by human Trmt10b catalyzes m1A9 on tRNAAsp can be recapitulated in vitro with purified enzyme, recombinantly expressed and purified zebrafish trmt10b cannot catalyze the correct modification when tested in vitro. To investigate whether Trmt10b activity can be reconstituted in the context of another eukaryote, human and zebrafish Trmt10b were expressed in Saccharomyces cerevisiae trm10Δ strains. We demonstrated that human Trmt10b activity does not modify S. cerevisiae tRNAAsp, but exhibits the correct m1A9 modification activity on both human and zebrafish tRNAAsp species when they are also introduced into the yeast strain. However, zebrafish Trmt10b is not able to catalyze any detectable modification on either yeast, human or zebrafish tRNAAsp in this heterologous system, indicating that something unique to the zebrafish cellular environment may be required for m1A9 catalysis. To study the significance of these paralogs in a vertebrate organism, our lab generated homozygous mutant trmt10a and trmt10b zebrafish. To further probe the mechanism of zebrafish Trmt10b, we will use a proximity labeling assay to determine potential interactions necessary for catalysis in zebrafish embryos. To identify features of tRNAAsp important for catalysis, a mutant tRNAAsp library will be created to identify specific residues required for Trmt10b recognition. Altogether, this work will provide a further understanding of the mechanism and biological impact of the highly conserved vertebrate Trmt10a and Trmt10b enzymes.
Keywords: tRNA modification, Trm10, m1R9
121. PUS7 cytoplasmic localization directs a pseudouridine-mediated cellular stress response
Minli Ruan (University of Michigan, Department of Biological Chemistry, Ann Arbor, MI 48109), Sean M. Engels, Matthew R. Burroughs, Lydia M. Contreras (University of Texas, McKetta Department of Chemical Engineering, Austin, TX 78712), Dylan Bloch, Oleksandra Fanari, Stuart Akeson, Sara Rouhanifard, Miten Jain (Northeastern University, Department of Bioengineering, Boston, MA 02120), Daniel E. Eyler, Xiaoyan Li (University of Michigan, Department of Chemistry, Ann Arbor, MI 48109), Chase A. Weidmann (University of Michigan, Department of Biological Chemistry, Ann Arbor, MI 48109), Kristin S. Koutmou (University of Michigan, Department of Chemistry, Ann Arbor, MI 48109)
Abstract:
Pseudouridine (Y) is an abundant post-transcriptional modification found across all classes of RNA. Since its discovery in mRNAs, it has been widely speculated that Y might provide an avenue for cells to control post-transcriptional gene expression. Here we demonstrate that one of the principal mRNA pseudouridylating enzymes, pseudouridine synthase 7 (PUS7), exhibits a stress induced accumulation in the cytoplasm of yeast and human epithelial lung cells. The cytoplasmic localization of PUS7 promotes Y-incorporation into hundreds of mRNA sequences and increases cellular fitness under ROS and divalent metal ion stress. Quantitative proteomics reveal a reshaping of the proteome upon PUS7 relocalization under stress, with proteins that bind metal being particularly sensitive. Collectively, our data demonstrate that the post-transcriptional inclusion of Y into mRNA impacts protein production in cells. Furthermore, they suggest a conserved mechanism for a Y-mediated cellular stress response, whereby stressors relocalize PUS7 and modulate protein production from stress response mRNAs.
References:
1. Eyler, D. E. et al. Pseudouridinylation of mRNA coding sequences alters translation. Proc Natl Acad Sci USA 116, 23068–23074 (2019).
2. Carlile, T. M. et al. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515, 143–146 (2014).
3. Schwartz, S. et al. Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159, 148–162 (2014).
Keywords: Pseudouridine, PUS7, Gene regulation
122. Role of the lncRNA MEG9 in Vascular Endothelial Cells in Response to Stress
Sydney Rudolph (Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI), Miguel Nieto-Hernandez (Center for Molecular Medicine and Genetics,Wayne State University, Detroit, MI), Chayan Bhattacharya (Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI), Sudarshan Anand (Knight Cancer Institute, Oregon Health and Science University, Portland, OR), Cristina Espinosa-Diez (Center for Molecular Medicine and Genetics,Wayne State University, Detroit, MI)
Abstract:
Cellular stress of vascular endothelial cells can lead to a dysregulation of normal cellular functions, including a disruption in angiogenesis and vascular remodeling. Long non-coding RNAs (lncRNAs) are key regulators of vascular function that control gene expression through different mechanisms, including chromatin remodeling, gene splicing, and translation control, enabling endothelial cells to respond to environmental changes. The lncRNA, Maternally Expressed Gene 9 (MEG9), is encoded in the DLK1-DIO3 non-coding RNA cluster on Chromosome 14 and shows an increased expression in endothelial cells under stress. Our preliminary data illustrate that genotoxic stressors such as radiation etoposide, increase MEG9 expression, while exposure to vascular growth factors inhibits it. We hypothesize that the MEG9 could be part of vascular endothelial cells' adaptive stress-induced protective mechanism. We further tested MEG9 responses to oxidative stress by treating Human umbilical Vein Endothelial Cells (HUVECs) with Hydrogen Peroxide (H2O2) and Doxorubicin. We validated that MEG9 expression was upregulated in both instances compared to the non-treated control. To better understand the role of MEG9 in endothelial cells, we performed MEG9 loss of function using antisense oligonucleotide inhibitors (GapmeRs), specifically designed to knock down MEG9 expression and validate by RT-qPCR. We studied angiogenesis by performing tube formation assay, apoptosis with live/dead staining, and DNA damage by analyzing H2AX foci formation with and without exposure to stressors. Cells with MEG9 deficiency showed higher rates of cell death, and H2AX foci count, further enhanced by stress. Furthermore, loss of MEG9 decreased angiogenesis. In conclusion, our data indicate that loss of MEG9 impaired endothelial function and the ability to withstand cellular stressors. Therefore, MEG9 expression potentially is a compensatory adaptive protective mechanism in HUVECs in response to acute stress. In future studies, we will test the protective role of MEG9 in vitro and in vivo, together with its downstream regulators.
References:
DNA damage-induced lncRNA MEG9 impacts angiogenesis
Eugenia Fraile-Bethencourt, Sokchea Khou, RaeAnna Wilson, Adrian Baris, Rebecca Ruhl, Cristina Espinosa-Diez, Sudarshan Anand
bioRxiv 2022.12.07.519382; doi: https://doi.org/10.1101/2022.12.07.519382
Keywords: LncRNAs, Vascular Biology, Apoptosis
123. PYM1 limits non-canonical binding of exon junction complex and maintains homeostatic expression of mRNAs localized to endoplasmic reticulum
Manu Sanjeev (Department of Molecular Genetics, OSU), Lauren Woodward (Department of Molecular Genetics, OSU), Michael Schiff, Robert Patton, Sean Myers (Department of Physics, OSU), Bayley Carter (Department of Molecular Genetics, OSU), Ralf Bundschuh (Department of Physics, OSU), Guramrit Singh (Department of Molecular Genetics, OSU)
Abstract not available online - please check the booklet.
124. Identifying and characterizing charged and tautomeric G•U wobbles in RNA
Md Sharear Saon (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University), Catherine A. Douds (Center for RNA Molecular Biology, Department of Biochemistry and Molecular Biology, Pennsylvania State University), Andrew J. Veenis (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University), Neela Yennawar (Huck Institutes of the Life Sciences, Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry, Center for RNA Molecular Biology, Department of Biochemistry and Molecular Biology, Pennsylvania State University)
Abstract:
RNA can be an enzyme (ribozyme), small molecule sensor (riboswitch), and a vaccine. It was also critical to the origin of life in the RNA world scenario. This is astonishing given that the intrinsic molecular diversity of RNA is quite limited. To date, covalent modifications of nucleotides have received widespread attention, but much less is known about any non-covalent changes of the nucleotides. The latter can be classified into novel charged and tautomeric forms of the bases, which involve changes in protonation state and proton location, respectively. Like covalent modifications of RNA, non-covalent modifications may provide additional stability and functionality to the RNA. We used a cheminformatic approach to identify non-covalent changes to RNA and propose two novel orientations of G•U wobble base pairs: one in which U is negatively charged and one in which either of the bases is tautomeric. We provide widespread structural support of these proton-altered G•U wobbles within RNA 3D structures in the Protein Data Bank. Cheminformatics pipelines like this one can be designed with other non-Watson-Crick-Franklin pairs to identify further novel protonation states of RNA.
Keywords: anionic RNA, Rare wobble base pairs, RNA modifications
125. The 3' end of MALAT1 forms a partial triple helix in the presence of mascRNA
Mika Schievelbein (Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 45665), Nikhil Vijai (Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556), Jake M. Peterson (Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011), Walter N. Moss (Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556)
Abstract:
MALAT1 is a long noncoding RNA whose 3' end is processed via RNase P due to the presence of a tRNA-like structure known as mascRNA. The resulting 3' end of mature MALAT1 terminates in a triple helix formed between a U-rich internal loop and A-rich tract. In the premature transcript, the 3' end of mascRNA engages with a portion of the A-rich tract to form a pre-mascRNA structure that is essential for efficient RNase P cleavage and precludes mature triple helix formation. Yet, the 3'-end structure of the premature MALAT1 is generally unknown. Using UV thermal melts and the MALAT1 triple helix-binding protein METTL16, our results support the formation of a partial triple helix in an RNA containing the regions for the triple helix and pre-mascRNA. Furthermore, our de novo structural model posits that a short stretch of A residues in the A-rich tract engage with U residues in the lower region of the U-rich internal loop. Our preliminary in vitro exonucleolytic degradation assays suggest the partial triple helix is not a stability element in the premature RNA, although METTL16 binding may protect the premature RNA from degradation. Altogether, these findings suggest MALAT1 triple helix formation is a dynamic process and may offer insight into the structural details of MALAT1 maturation.
Keywords: RNA Triple Helix, MALAT1, mascRNA
126. Understanding Connections between PYM1 Function in Exon Junction Complex Binding and Gene Architecture
Michael Schiff (Department of Physics, The Ohio State University), Manu Sanjeev (Department of Molecular Genetics, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, The Ohio State University)
Abstract:
The exon junction complex (EJC) is a protein complex deposited near the junction of two exons during pre-mRNA splicing. The EJC is a hallmark of spliced RNAs and plays many important roles in RNA processes including mRNA surveillance. It has been previously suggested that PYM1 acts as an EJC disassembly factor through a direct interaction with the EJC [1]. However, recent experiments from our group suggest no role for PYM1 in translation dependent EJC disassembly. To better understand PYM1's function and effect on EJC occupancy and/or stability, we have generated and analyzed RNA-seq and EJC-footprinting data under conditions that limit EJC-PYM1 interaction. An exploratory analysis of these data has suggested a link between gene architecture and altered gene expression and EJC occupancy upon loss of EJC-PYM1 interaction. We thus set out to use a machine learning based, unbiased approach to identify which specific aspects of gene architecture are most relevant for the observed differences. RNA-seq and EJC-footprint foldchanges between samples from different conditions were predicted using gene architecture features as the predictors. The importance of each feature in the learned models was subsequently measured. A feature appearing important to the model thus could shed light on the role of PYM1 in EJC occupancy. This method was tested using synthetic data to verify that known predictors are indeed identified by this approach. Current progress on experimentally obtained data thus far has pointed to a connection between PYM1 effect on EJC occupancy and the GC content, which previously has been linked with gene architecture [2]. Thus, PYM1 function in regulating EJC's RNA binding is likely dependent on gene architectural features such as GC content.
References:
[1] Gehring NH, Lamprinaki S, Kulozik AE, Hentze MW. Disassembly of exon junction complexes by PYM. Cell. 2009 May;137(3):536-548. DOI: 10.1016/j.cell.2009.02.042. PMID: 19410547
[2] Mordstein, C., Savisaar, R., Young, R. S., Bazile, J., Talmane, L., Luft, J., Liss, M., Taylor, M. S., Hurst, L. D., & Kudla, G. (2020). Codon usage and splicing jointly influence mrna localization. Cell Systems, 10(4). https://doi.org/10.1016/j.cels.2020.03.001
Keywords: Exon Junction Complex (EJC), PYM1, Machine Learning
127. Exploring the roles of UPF3 functional domains within Nonsense-Mediated mRNA Decay
Savanna P. Schutte (Department of Molecular Genetics, The Ohio State University), Rabab Abu Alhasan (Department of Molecular Genetics, The Ohio State University), Rene M. Arvola (Department of Molecular Genetics, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University)
Abstract:
Nonsense-mediated decay (NMD) is a crucial post-transcriptional mechanism that regulates both quality and quantity of eukaryotic mRNAs. This pathway targets mRNAs containing premature termination codons (PTCs) for degradation to prevent the production of C-terminal truncated protein products. NMD also regulates gene expression by targeting a percentage of all endogenous mRNAs where termination is apparently aberrant. Prematurely terminating ribosomes are first recognized by the core NMD factors UPF1, UPF2, and UPF3, and the NMD pathway is activated upon UPF1 phosphorylation. While the paralogous UPF3 factors, UPF3A and UPF3B, are known NMD enhancers, their precise molecular function remains unclear as they are non-essential in the mammalian NMD pathway. Canonical NMD targets bear exon-junction complexes (EJCs) downstream of PTCs which associate with mRNA exon-exon junctions post-splicing. UPF3 has been proposed to enhance NMD by linking the premature termination complex with the EJC. Previous work in our lab, however, has shown that UPF3 still acts as an NMD enhancer without its EJC binding activity. In exploring the other roles of UPF3 in the premature termination complex, we found that the knockout of UPF3B in a human colorectal cancer cell line (HCT116) increases UPF1 and UPF2 association with ribosomal complexes. Our ongoing work suggests that UPF3 has distinct roles in composition and activity of the premature termination complex. Our data supports the hypothesis that UPF3B influences the arrangement of ribosome-associated premature termination complexes likely via its UPF2-binding activity rather than through bridging with the EJC. We also found that disrupting UPF2 binding has little effect on the decay of UPF3B-dependent NMD targets while decay is strongly inhibited when the loss of UPF2 binding is coupled with partial deletion of UPF3B’s mid domain. Thus, functional domains of UPF3B could act semi-redundantly to facilitate NMD enhancement.
References:
Yi, Z., Arvola, R. M., Myers, S., Dilsavor, C. N., Abu Alhasan, R., Carter, B. N., Patton, R. D., Bundschuh, R., & Singh, G. (2022). Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding. The EMBO Journal, e109202
Keywords: nonsense-mediated mRNA decay, UPF3, translation termination
128. Use of a small molecule microarray screen to identify inhibitors of the catalytic RNA subunit of Methanobrevibacter smithii RNase P
Vaishnavi Sidharthan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Christopher D. Sibley, Kara Dunne-Dombrink, Mo Yang (Chemical Biology Laboratory, National Cancer Institute), Walter J. Zahurancik (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Christian Heryakusuma, Biswarup Mukhopadhyay (Department of Biochemistry, Virginia Tech University), Damien B. Wilburn (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), John S. Schneekloth Jr, Venkat Gopalan (Chemical Biology Laboratory, National Cancer Institute; Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University)
Abstract:
There is great interest in leveraging binding pockets in structured long non-coding (lnc) RNAs to develop therapeutics that allow metabolic fine-tuning. For example, regulating methanogens in intestinal tracts may offer a modality to mitigate methane emission from livestock and to treat type 2 diabetes mellitus in human. In this context, drug targets in Methanobrevibacter smithii (Msm) warrant consideration as Msm and close relatives constitute a major part of cattle rumen and human intestine methanogen population. To this end, we have used a small molecule microarray (SMM) screen to find inhibitors of a lncRNA ribozyme: the Msm RNase P RNA (Msm RPR, ~300 nt). The ribonucleoprotein form of RNase P, which catalyzes the 5'-maturation of precursor tRNAs, is a suitable drug target as it is essential, structurally diverse across life domains, and present in low copy. From an SMM screen of 7,300 compounds followed by selectivity profiling, we identified 48 hits that bound specifically to the Msm RPR—the catalytic subunit in Msm (archaeal) RNase P. When we tested these hits in precursor-tRNA cleavage assays, we discovered that the drug-like M1, a diaryl-piperidine, inhibits Msm RPR (KI, 17 ± 1 µM) but not a structurally-related archaeal RPR, and binds to Msm RPR with a KD(app) of 8 ± 3 µM. Structure-activity relationship analyses performed with synthesized analogs pinpointed groups in M1 that are important for its ability to inhibit Msm RPR. Moreover, M1 retards the growth of Msm in culture. Overall, the SMM method offers prospects for advancing RNA druggability by identifying new privileged scaffolds/chemotypes that bind lncRNAs.
References:
1.Gopalan et al. (2018) RNA 24, 1–5
2.Connelly et al. (2017) Methods Mol Biol 1518, 157−175
3.Danielson et al. (2017) Front Microbiol 8, 226
Keywords: RNase P, drug discovery, small molecule microarray, Methanobrevibacter smithii
129. Quantitative mapping of modified RNA nucleosides in tRNAs
Kaley M. Simcox (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA), Joshua D. Jones (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA), Robert T. Kennedy (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA), Kristin S. Koutmou (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA)
Abstract:
Transfer RNAs are the most abundantly modified RNA species with up to 20% of sites containing a modified nucleoside. RNA modifications influence all aspects of the RNA lifecycle including tRNA structure, stability, and tRNA:mRNA interactions during translation. The location of a modification on a tRNA is an important determinant of function. Therefore, rigorous methods are needed to confidently identify nucleosides and sequence tRNAs. Using a multiplexed methodology like liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) nucleoside discovery and sequencing are possible from total tRNA mixtures. Using two orthogonal enzymatic digestions, we were able to achieve high sequence coverage for E. coli total tRNA by exploiting tRNA secondary structure with single-stranded specific ribonucleases. Additionally, we have developed an untargeted LC-MS/MS method for nucleoside discovery from E. coli total tRNA. The combination of these two methodologies will be essential to elucidate the RNA modification landscape and give further insight to the role of RNA modifications.
Keywords: RNA modifications, LC-MSMS
130. Dual-Action Circular ADAR-Recruiting RNAs: A Novel Approach for Reversible Gene Modulation through RNA Editing and Antisense Effects
Shashi S. Singh (Department of Biochemistry and Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Joseph M. Luna (Department of Biochemistry and Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)
Abstract:
RNA editing offers a promising approach for reversible gene modulation, addressing the limitations of permanent genome editing. We present a novel strategy using circular ADAR-recruiting guide RNAs (circ-arRNAs) to harness endogenous Adenosine Deaminase Acting on RNA (ADAR) activity for precise adenosine-to-inosine editing. These self-cleaving, self-ligating circ-arRNAs bind specific transcripts, recruiting ADAR to induce targeted base edits. We demonstrate circ-arRNA efficacy in two key applications: (1) stop codon removal in a fluorescent reporter system, and (2) exon skipping in KRAS through intron-exon boundary editing. Direct RNA delivery of circ-arRNAs showed superior editing kinetics compared to plasmid-based methods. Unexpectedly, we observed antisense oligonucleotide (ASO)-like activity of circ-arRNAs, with splice site switching occurring even in ADAR knockout cells, indicating a mechanism independent of RNA editing. This dual functionality of circ-arRNAs - combining ADAR-mediated editing and ASO-like effects - presents a versatile tool for manipulating gene expression. We are developing a bichromatic reporter system to assess circ-arRNA-mediated effects in various cancer cell lines. This approach holds significant potential for therapeutic applications, particularly in scenarios where transient modulation of gene expression is preferable to permanent genetic alterations.
Keywords: RNA Editing, Circular RNAs, Splicing modifier
131. Free tRNA introns act as novel noncoding regulatory RNAs
Paolo L. Sinopoli (Department of Molecular Genetics & Center for RNA Biology, The Ohio State University), Regina T. Nostramo (Department of Molecular Genetics & Center for RNA Biology, The Ohio State University), Alicia Bao (Department of Molecular Genetics & Center for RNA Biology, The Ohio State University), Sara Metcalf (Department of Molecular Genetics & Center for RNA Biology, The Ohio State University), Anita K. Hopper (Department of Molecular Genetics & Center for RNA Biology, The Ohio State University)
Abstract:
tRNAs are created through transcription of tRNA genes, a subset of which contain introns that are excised during tRNA maturation. tRNA intron sequences are highly conserved between closely related species within Drosopholid and Saccharomycetaceae families, yet there is no known sequence-dependent role for tRNA introns. We report that the sequences of tRNA introns in Saccharomyces cerevisiae have remarkable and statistically improbable regions of complementarity to coding open reading frames (ORFs) of mRNAs. Contrastingly, tRNA exons have none or minimal complementarity to ORFs. The data lead us to propose that tRNA introns might interact with mRNA sequences similarly to regulatory microRNAs that are found in other organisms. To test this hypothesis, we generated a strain that lacks the tRNAIle introns from both tRNAIle genes (IleiΔ). In the IleiΔ strain, there are significant increases in levels of mRNAs with complementarity to the tRNAIle intron, but not for mRNAs lacking complementarity. Congruently, inducible overexpression of the tRNAIle intron results in a reduction of mRNAs with complementarity to the intron relative to mRNAs lacking complementarity. Further, mutations of yeast genes that accumulate tRNA introns also display reductions in the levels of mRNAs with complementarity to tRNA introns. Together, the data indicate that tRNA introns possess complementarity-dependent inhibitory effects on mRNA levels. This inhibitory effect is dependent on nuclear and cytoplasmic ribonucleases, indicating direct action of tRNA introns on mRNA stability. Levels of mRNAs with tRNA intron complementarity in their UTRs are unaffected in our experiments, differentiating regulation by tRNA introns from microRNAs. This is consistent with S. cerevisiae lacking the argonaute proteins integral to microRNA function, indicating that mRNA regulation by tRNA introns occurs through an alternative, currently unknown, mechanism. Our findings lead us to propose that free introns of tRNAs act as a new class of small regulatory RNAs.
References:
Nostramo RT, Sinopoli PL, Bao A, Metcalf S, Peltier LM, Hopper AK. FitRNA for duty: Free introns of tRNAs as novel complementarity-dependent regulators of gene expression. In revision, Molecular Cell.
Keywords: tRNA, tRNA introns, regulatory RNA
132. Identifying the role of 3’UTRs in transcriptional regulation of the calmodulin gene family
Emily Slobodenyuk (Cellular and Molecular Biology Program, University of Michigan), Brittany Bowman (Department of Biological Chemistry, University of Michigan), Chase Weidmann (Department of Biological Chemistry, University of Michigan)
Abstract:
Calmodulin (CaM) is an essential, highly conserved, ubiquitously expressed calcium-binding protein, and interacts with many proteins to regulate a myriad of cellular functions, including the cell cycle, cell motility, and the regulation of ion channels. Due to calmodulin’s importance, mutations in the calmodulin gene or misregulation of the protein contributes to cancer, heart disease, and neurological disorders. Interestingly, in mammals, three CaM genes at three separate autosomal loci express divergent mRNA (CALM1, CALM2, CALM3) that each encode an identical calmodulin protein. Expression of CaM varies in different tissues, variants localize in unique patterns, and mutations in each CaM gene yield distinct phenotypes. The idea of unique transcript roles is further supported by evolutionary conservation, as there is a higher degree of conservation of CaM variants between species than between the CaM variants within each species, especially in their 3′UTRs. Therefore, we hypothesize that sequences within the 3′UTRs govern mRNA localization, with local translation of the transcripts determining the subcellular localization of CaM protein, and thus, unique signaling responses. We will the determine the contribution of each transcript to the total protein and determine if there are transcript-specific protein compartments using RNA FISH and immunofluorescent tagging of each protein. We will determine the cis-regulatory elements necessary for the localization of the transcripts by chemical probing and genetic screening/ We hope to better understand the transcript-level regulation of CaM genes, with the resulting knowledge being broadly applicable to other RNAs.
References:
Kahl et al., Endocr Rev., 2003
Villalobo, et al. Int J Mol Sci, 2020
Wu et al., Int J Mol Sci, 2021
Beghi et al. , Int J Mol Sci, 2022
O’Day et al., Int J Mol Sci, 2020
Gnegy et al., Brain Res, 1991
Munk et al., Cell Calcium, 2022
Toutenhoofd, et al., Cell Calcium, 2000
Palfi et al., J Histochem Cytochem, 1999
Palfi et al., Life Sci., 2002
Zhang et al., Neurosci., 1993
Bogdanov et al., Biophys J., 2022
Bogdanov et al., Biophys J., 2024
Fischer et al., J Biol Chem., 1988
Keywords: 3UTRs, mRNA localization
133. RNase H1-S233A mutant fails to suppress R loop associated genomic instability
Niraja Soman (Masonic cancer center, Department of Pharmacology, University of Minnesota), Hai Dang Nguyen (Masonic cancer center, Department of Pharmacology, University of Minnesota)
Abstract:
R loops are three stranded RNA:DNA hybrid structures having multiple biological applications in natural cellular processes. They play important roles in gene expression, DNA replication and repair. However, how the formation of unscheduled R loops causes genomic instability and its association with cancer is unknown. To avoid detrimental effects of R loops, they need to be tightly regulated. RNaseH1 is an enzyme that degrades the RNA strand in the RNA:DNA hybrid to resolve R loops. Yet, how RNaseH1 is regulated is not fully understood. Here, we found that RNaseH1 is phosphorylated at Ser76 and Ser233 in cells by phospho-proteomics. In this project, we focused on how RNase H1 phosphorylation at Ser233 plays a role in R-loop regulation. The Ser233 is located on the DNA binding channel in the catalytic domain (CAT) at a basic protrusion in a phosphate binding pocket, which is unique to human RNaseH1 protein (Nowotny et al., Mol Cell 2007). To investigate RNaseH1 function in cells, we generated doxycycline inducible GFP-tagged phospho-mutant (S233A) and phospho-mimetic (S233E) in U2OS cell lines. To assess R-loop-associated genomic instability, we treated cells with a PARP1/2 specific inhibitor, olaparib (PARPi), and measured DNA damage levels by immunofluorescence staining using anti-γH2AX. In contrast to RNaseH1WT-GFP overexpressing cells, neither individual RNaseH1S76A-GFP nor RNaseH1S233A-GFP mutants were able to suppress PARPi-induced DNA damage. Future studies will be conducted to investigate the molecular function of Ser233 phosphorylation on RNaseH1 activity.
References:
Yang, W., Lee, J. Y., & Nowotny, M. (2006). Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity. Molecular cell, 22(1), 5-13.
Keywords: RNase H1, Genomic instability, R loops
134. SARS-CoV-2 Infection Alters CPSF73 Expression, Modulating Immune Response via Alternative Polyadenylation
Ankita Srivastava (Tufts University), Salwa Mohd Mustafa, Wendy Puryear, Jonathan Runstadler, (Tufts University), Luyang Wang, Bin Tian (The Wistar Institute), Claire Moore (Tufts University)
Abstract:
The emergence of SARS-CoV-2 in 2019 sparked a global pandemic, leading to extensive research into its impact on the host immune system, key to understanding viral replication and disease severity. mRNA maturation requires 3' end cleavage and poly(A) (pA) tail addition, which stabilize mRNA and promote translation. Approximately 70% of human genes undergo alternative polyadenylation (APA), generating diverse mRNA isoforms. Previous studies have shown that viral infections, such as Influenza and HIV-1, modify APA to enhance immune responses or support viral replication. We hypothesize that SARS-CoV-2 modulates APA to facilitate its infection.
To test this, we analyzed 3' end processing of ACTB and MYC mRNAs in SARS-CoV-2-infected samples using RT-qPCR. We observed a significant increase in unprocessed ACTB (1.8-fold) and MYC (2.5-fold), suggesting disrupted mRNA processing. Next, we conducted 3' mRNA sequencing to assess pA site usage and found notable activation of intronic polyadenylation (IPA) (834 instances) versus 116 3' UTR modifications. Genes undergoing IPA were enriched in pathways related to DNA damage response and RNA metabolism. Gene Set Enrichment Analysis (GSEA) further highlighted APA alterations in TNF-alpha signaling pathways.
To understand why mRNA 3' end processing is altered, we quantified cleavage/polyadenylation (C/P) proteins post-infection and found reduced levels of CPSF73 (the pA site nuclease) and CPSF6 (an RNA-binding protein in the C/P complex). RNA-seq analysis from CPSF73 knockdown and SARS-CoV-2 infection showed a 40-fold increase in read-through at the EEF1A2 pA site, suggesting impaired processing. Additionally, we noted a 2.1-fold increase in UTR lengthening of CPSF6 mRNA in both conditions.
Ectopic expression of SARS-CoV-2 nonstructural protein NSP12 disrupted 3' end processing of MYC and ACTB and reduced CPSF73 levels via the ubiquitin-proteasome pathway. Restoring CPSF73 levels reversed the processing defects. Our findings suggest that SARS-CoV-2 targets the C/P complex, inducing APA and influencing viral pathogenesis, which could offer new therapeutic targets.
Keywords: cleavage, polyadenylation, APA, CPSF73, mRNA, SARS-Cov2
135. Probing functional differences in pri-miR-20a binding by the 2 RRMs of hnRNPA2B1
Sydney M. Steele (Department of Chemistry, University of Michigan), Cade T. Harkner (Department of Chemistry, University of Michigan), Yaping Liu (Biophysics Program, University of Michigan), Sarah C. Keane (Department of Chemistry, University of Michigan and Biophysics Program, University of Michigan)
Abstract:
MicroRNAs are short (~22 nt) RNAs which function to regulate gene expression. Primary microRNA-20a (pri-miR-20a) is an oncogenic miRNA whose enzymatic processing is post-transcriptionally regulated by RNA-binding proteins (RBPs). The apical loop of pri-miR-20a is known to be a critical binding site for RBPs, including heterogeneous nuclear ribonucleoproteins (hnRNPs). Our laboratory has shown that hnRNPA2/B1 binds pri-miR-20a and forms a 1:1 complex. However, the molecular details of the intermolecular interaction remain unknown. For example, it is not clear whether one or both of the tandem RNA recognition motifs (RRMs) preferentially binds pri-miR-20a or if the RRMs have functionally distinct or redundant roles. Isothermal titration calorimetry (ITC) data revealed that each isolated RRM bound pri-miR-20a, with similar affinity. Interestingly, the binding of each isolated RRM was similar to that measured for the intact RNA-binding domain (RBD), which contains both RRMs. To elucidate potential functional differences between the RRMs, we used site-directed mutagenesis to mutate residues in each RRM predicted to be important for RNA binding. We are currently investigating how these mutations, in the isolated RRMs and in the full-length RBD, impact the binding of pri-miR-20a using ITC. Our studies will uncover important functional differences between the RRMs and reveal the mechanism by which hnRNPA2/B1 binds pri-miR-20a.
Keywords: microRNA, Protein-RNA interaction, hnRNP
136. Engineered Suppressor tRNAs Rescues Nonsense Mutations In TP53
Alejandro Tapia (Microbiology & Immunology), Jennifer Flora (Biochemistry & Molecular Biology), Olabode Dawodu (Biochemistry & Molecular Biology), Caitlin Specht (Biochemistry & Molecular Biology), Roland W. Herzog (Microbiology & Immunology), Jeffery Tharp (Biochemistry & Molecular Biology)
Abstract:
Background:
Over 10% of genetic diseases are caused by mutations that introduce stop codons into protein coding genes, known as premature termination codons (PTCs). mRNA transcripts harboring PTCs result in premature polypeptide cleavage, leading to loss of protein function. One promising approach for PTC-readthrough is the development of engineered suppressor transfer RNAs (sup-tRNAs), which are modified to recognize one of the 3 stop codons. Sup-tRNAs can incorporate amino acids (AAs) at PTCs, outcompeting endogenous factors that terminate translation, inducing stop codon readthrough. The application of sup-tRNAs for cancer therapeutics has not been explored. Tumor suppressor protein p53 (encoded by TP53) is one of the most mutated proteins in cancer—11% of cancer-associated mutations on TP53 are PTCs. We hypothesize that sup-tRNAs can induce translational readthrough of PTCs in the TP53 gene to restore p53 protein levels, rescue p53 function, and inhibit cancer cell proliferation.
Methods:
We developed a novel class of sup-tRNAs that efficiently suppress all 3 stop codons. We tested the ability of these sup-tRNAs to elicit translational readthrough of PTCs in cancer cells with homozygous TP53 mutations. p53 protein levels were quantified by immunoblotting. p53 activity was quantified with a combination of luciferase-based transcriptional assays and qRT-PCR.
Results:
Sup-tRNA transfection resulted in increased levels of p53 protein levels and function. Moreover, sup-tRNA-treated cells showed increased expression of several downstream genes that are transactivated by p53.
Conclusions:
Sup-tRNAs can restore functionally active p53 protein in cancer cells carrying PTC mutations in TP53. Sup-tRNAs are a potential approach to help drive tumor suppression. The use of sup-tRNAs for gene therapy can overcome current hurdles of vector transgene capacity. Further validating this therapy in animal models will help solidify its potential as a clinical therapeutic.
References:
Mort, M., et al. (2008). Human Mutation 29(8): 1037-1047.
Floquet, C., et al. (2010). Nucleic Acids Research 39(8): 3350-3362.
Albers, S., et al. (2023). Nature 618(7966): 842-848.
Biorender
Keywords: p53, sup-tRNA, PTC-readthrough
137. Fused in Sarcoma protein recognizes and binds to mRNA methylation
Chamali T. Mudiyanselage (Chemistry and Biochemistry, Kent State University), Sudeshi M. Abedeera (Chemistry and Biochemistry, Kent State University), Aftab Mollah (Chemistry and Biochemistry, Kent State University), Sanjaya Abeysirigunawadena (Chemistry and Biochemistry, Kent State University)
Abstract:
Epitranscriptomics is the post-transcriptional gene expression regulation through RNA modifications and editing. Various RNA modifications, including N1-methyladenosine (m1A), pseudouridine (Ψ), 5-methylcytidine (m5C), and N6-methyladenosine (m6A), which is the most prevalent nucleotide modification in eukaryotic mRNA are observed in epitranscriptomic gene regulation. The m6A modification exerts its influence by recruiting m6A-binding proteins known as m6A readers, such as YTH-domain-containing proteins like YTHDF1-3 and YTHDC1-2. These m6A reader proteins, independently or in conjunction with accessory proteins, play specific roles in various RNA metabolic processes, including mRNA splicing, stabilization, and decay. Reader proteins also influence translation. We have discovered several proteins that bind to methylations, specifically using an RNA pulldown assay. FUS protein was identified as a potential methyl reader that specifically recognizes m6A methylated RNA during these pulldown assays. FUS (Fused in Sarcoma) is a nuclear RNA binding protein, a multidomain protein composed of RRM and ZnF domains known to bind to RNAs. This study identified a sequence close to the ZnF motif that can likely bind to m6A methylations. FUS protein can form membraneless biological condensates with other proteins and nucleic acids under normal cellular conditions. The abnormal aggregation of FUS is linked to several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and essential tremor. Our findings will give insights into the role of m6A methylation in RNA on various diseases related to FUS protein.
References:
Miller LG, Demny M, Tamamis P, Contreras LM. Characterization of epitranscriptome reader proteins experimentally and in silico: Current knowledge and future perspectives beyond the YTH domain. Comput Struct Biotechnol J. (2023) Jun 30;21:3541-3556.
Deng, H., Gao, K. & Jankovic, J. The role of FUS gene variants in neurodegenerative diseases. Nat Rev Neurol 10, 337–348 (2014).
Jiang, X., Liu, B., Nie, Z. et al. The role of m6A modification in the biological functions and diseases. Sig Transduct Target Ther 6, 74 (2021)
Keywords: FUS, m6A, ALS
138. Investigating the role of bS21 paralogs in ribosome heterogeneity in Agrobacterium fabrum
Annie Urban (Ohio State University), Fawwaz Naeem (Ohio State University), Kurt Fredrick (Ohio State University)
Abstract:
The ribosome is the machine responsible for translation of mRNAs into proteins needed for cellular functions. In bacteria, ribosomes are made up of two subunits, small 30S and large 50S. The 30S subunit contains the 16S rRNA which has the anti-Shine Dalgarno (ASD) sequence located on its 3`end. Typically, during translation initiation, the ASD base pairs with the Shine-Dalgarno (SD) sequence located upstream of the start codon on mRNAs and this SD-ASD interaction positions the start codon in the P-site. Located near the ASD is the ribosomal protein bS21 which help stabilize the ASD-SD binding. bS21 is essential in some bacteria while completely absent from others, and some bacteria have multiple copies. Recently, it has been shown that bS21 is involved in regulation of its own synthesis in Flavobacterium johnsoniae. Furthermore, bS21 is also a common ribosomal protein encoded by phages. It has been shown to get incorporated into the ribosome during late-stage infection and may explain how viruses replicate using host machinery.
In Agrobacterium fabrum, there are three different rpsU (bS21) genes: ATU4064, ATU3123, and ATU3637, denoted as rpsU-A, rpsU-B, and rpsU-C respectively. The genes are identical except in the C-terminal, suggesting duplications or redundancy functions; however, preliminary data suggests these homologs can act as global regulators of translation. RNA-seq studies show differential expression of the three genes in different media. In rich media, rpsU-A was more highly expressed; however, in minimal media, rpsU-B, and rpsU-C are more expressed. This suggests that there could be heterogenous populations of ribosomes within the cell as a funcntion of which bS21 homolog is incorporated into the ribosomes, which could serve as a stress response to changing nutrient conditions. We have generated multiple deletion strains to investigate the role of each of the three native A. fabrum genes along with phage mutants that only contain phage encoded S21 proteins from three different phages. These phage genes are identical to the native A. fabrum genes except for a variable and extended C-terminal region. Growth assays from these strains show a surprising phenotype where the rpsU-C deletion grows faster in minimal media, while the rpsU-B deletion is slower in rich media. Strains in which phage genes were inserted into A. fabrum genome show a defect in growth. Currently, we are performing Ribo-Seq on those various strains to elucidate the role of bS21 paralogs in ribosome heterogeneity and investigate the possibility of bS21 phage genes being used to redirect translation of the phage machinery in the host.
Keywords: RNA, Ribosome heterogeneity, Phages
139. Investigating the role of ribosomal RNA pseudouridylation in regulating translation
Leidy Vanegas-Cano (Department of Biological Chemistry, University of Michigan), Rachel O. Niederer (Department of Biological Chemistry, University of Michigan)
Abstract:
Pseudouridine is the most ubiquitous modification across eukaryotic ribosomes. Dyskerin (DKC1), the primary pseudouridine synthase responsible for rRNA pseudouridylation, utilizes small nucleolar RNA as a guide to direct the specific positioning of uridine at its active site, where the enzyme subsequently modifies the rRNA[1,2]. In mammalian cells with DKC1 deficiency also show compromised translation initiation and fidelity[2,3]. Although these studies indicate that rRNA pseudouridylation regulates translation by the ribosome, there is still limited understanding of how these modifications are regulated and their potential effects on selective mRNA translation. To gain insights into how rRNA pseudouridylation regulates ribosome function, we started by investigating whether DKC1 expression levels affect ribosome pseudouridylation and mRNA translation. First, we used the Genotype-Tissue Expression (GTEx) Portal to select three cell lines from tissues with differential expression of DKC1, ranging from low to high abundance (SKOV3, A549, and Hep G2). To validate the differential expression of DKC1 among these three cell lines, we performed western blots monitoring DKC1. Our preliminary results show lower DKC1 expression in SKOV3 compared to A549 and Hep G2, which is in agreement with the expression data from the GTEx Portal. Additionally, to evaluate the overall translation activity of these cell lines, we performed in vitro translation assays using cells extracts derived from each cell line. The results showed that SKOV3 extracts exhibited significantly higher translation activity than Hep G2 and A549 suggesting that high levels of DKC1 might impact translation possibly through ribosome pseudouridylation. Future experiments using direct RNA sequencing using Nanopore will elucidate the rRNA pseudouridylation level in these cell lines as well as any cell-specific Ψ sites. This work constitutes the first step in characterizing the effect of natural variation in DKC1 expression levels on rRNA pseudouridylation in different cell types and will allow us to investigate the impact of rRNA pseudouridylation in mRNA recruitment by the ribosome.
References:
[1]Keszthelyi, T. M.; Tory, K. The Importance of Pseudouridylation: Human Disorders Related to the Fifth Nucleoside. Biologia Futura 2023, 74, 3–15. https://doi.org/10.1007/s42977-023-00158-3.
[2]Borchardt, E. K.; Martinez, N. M.; Gilbert, W. V. Regulation and Function of RNA Pseudouridylation in Human Cells. Annual Review of Genetics 2020, 54 (1), 309–336. https://doi.org/10.1146/annurev-genet-112618-043830.
[3]Yoon, A.; Peng, G.; Brandenburg, Y.; Zollo, O.; Xu, W.; Rego, E.; Ruggero, D. Impaired Control of IRES-Mediated Translation in X-LinkedDyskeratosis Congenita. Science 2006, 312 (5775), 902–906. https://doi.org/10.1126/science.112383.
Keywords: DKC1, cell lines, in-vitro translation
140. Elucidating the structure of the 3′ end of premature MALAT1 using SHAPE
Nikhil Vijai (Department of Chemistry and Biochemistry, University of Notre Dame), Mika J. Schievelbein (Department of Chemistry and Biochemistry, University of Notre Dame), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)
Abstract:
Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a long non-coding RNA that contains an RNA triple helix at its 3′ end. Additionally, MALAT1 has been associated with cancer progression and is upregulated in many forms of cancer. MALAT1 undergoes a unique 3′ end processing mechanism, whereby it is cleaved by ribonucleases P (RNase P) and Z to form mature MALAT1 and a tRNA-like structure known as MALAT1-associated cytoplasmic RNA (mascRNA). The RNase P cleavage event enables the formation of the triple helix. However, the structure of MALAT1 before it is cleaved by RNase P is unknown. UV thermal denaturation and METTL16-binding assays indicate that a partial triple helix forms at the 3′ end of premature MALAT1, but the extent to which one forms is unclear. To provide insight into the putative triple helix structure of premature MALAT1, we employed selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). First, we generated an RNA in which the 3′ end of the premature MALAT1 sequence is inserted in a SHAPE cassette with a 3′ reverse transcriptase primer-binding site. Next, we optimized concentrations of RNA and primer for primer binding, reaction conditions for reverse transcription, ddNTP concentrations for sequencing, and NMIA concentrations for acylation. We then submitted some initial reactions for Sanger sequencing and analyzed the results using the QuSHAPE software. By elucidating the structure of premature MALAT1, we hope to better understand the life of the MALAT1 triple helix. Additionally, the structure of premature MALAT1 could represent a novel anti-cancer therapeutic target.
Keywords: MALAT1, SHAPE, Triple Helix
141. Mutation of C. albicans snR10c leads to translational inhibitor sensitivity
Rheanna E. Walther (Biology Department, Ball State University ), Douglas A. Bernstein (Biology Department, Ball State University )
Abstract:
Candida albicans is a dimorphic opportunistic fungal pathogen that causes infections ranging in severity from superficial skin and mucosal surface infections to organ and blood infections. Due to its prevalence as a pathogen, it is important to understand what distinguishes C. albicans from mammals at a molecular level. Pseudouridine is found in all species and understanding pseudouridine synthesis in C. albicans may help us better understand the process in other species and/or potentially serve as a therapeutic target. Some pseudouridine synthases are guided to their substrates by H/ACA box snoRNAs. snR3a, snR10b, and snR10c are three C. albicans H/ACA snoRNAs predicted to target rRNA residue 2919 of the large subunit. We modified the targeting region of C. albicans snR3a, snR10b, and snR10c using CRISPR-Cas9 genome editing so that they could no longer target rRNA. We find that the alteration of snR3a at its binding site eliminates modification of uridine 2919 of cells grown in nutrient-rich media (YPD) at 30°C. Conversely, we find that alteration of snR10b and snR10c has no impact on pseudouridylation at residue 2919. This suggests that snR10b and snR10c are nonessential for pseudouridylation under our tested conditions. We next tested if our modified snoRNA strains displayed altered growth. We find 2 mutant isolates of snR10c that have additional insertion and point mutations downstream of the annotated snoRNA gene result in sensitivity to translation inhibitors. RNA seq data suggests these downstream regions are transcribed. These findings suggest that snR10c is longer than annotated and these extended regions are important for growth and filamentation under translational stress.
Keywords: snoRNA, Candida albicans, pseudouridine
142. Computational modelling of RNA-protein binding interactions under an external force
Danielle Wampler (Department of Physics, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University)
Abstract:
RNA binding proteins play a crucial role in post-transcriptional gene regulation by controlling the transport, processing, and translation of their target RNAs. Post-transcriptional gene regulation leads to the differential expression of genetic material and loss of regulation or overregulation relates to a large range of cancers and diseases – many of which have directly been associated with RNA binding proteins and their target RNAs. To understand RNA, RNA binding proteins, and how they function in gene expression, it is essential to characterize how RNA binding proteins interact with their target RNAs. The current project aims to assess the potential for single molecule force spectroscopy experiments to be used in the characterization of RNA-protein binding by investigating to what extent a change of extension due to RNA-protein binding is experimentally measurable and what aspects of the interaction can be deduced from such measurements. We predict the effect of protein binding on RNA force extension measurements via the open-source ViennaRNA package, which we have modified to simultaneously consider an external force, protein binding, and RNA secondary structure. From this work, we see protein concentration–dependent responses to external forces with discernable differences in predicted extensions around biologically relevant concentrations for several RNA binding proteins.
Keywords: RNA-protein binding, RNA secondary structure, external force
143. Investigating AT-hook Proteins and Their Role in PATC-Mediated Gene Regulation in C. elegans
Chin Wang (Department of Biological Chemistry and Pharmacology, Molecular, Cellular and Developmental Biology Program), Yi-Hui Wang (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, Ohio State Biochemistry Program), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology), Wen Tang (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, Ohio State Biochemistry Program, Molecular, Cellular and Developmental Biology Program)
Abstract:
The animal germ line produces gametes that transmit genetic and epigenetic information from generation to generation. Organisms have evolved an array of specialized pathways to ensure proper gene expression. One such regulatory element in C. elegans is the periodic An/Tn-cluster (PATC), which features a 10-base pair periodicity and has been implicated in promoting the expression of both transgenes and endogenous genes (Fire et al., 2006, Aljohani et al., 2020, Seroussi et al., 2022). However, the mechanisms how PATCs regulate gene expression remain poorly understood. Our unpublished data suggest that AT-hook proteins, known to bind to AT-rich motifs, may play a role. We hypothesize that AT-hook proteins interact with PATC sequences to promote gene expression. To test this, we conducted a RNAi based screen targeting individual members of the AT-hook family in C. elegans strains expressing a GFP reporter with PATCs (Frøkjær-Jensen et al., 2016). Among the nine AT-hook genes tested, knockdown of two resulted in GFP silencing specifically in the germ line. Our ongoing experiments, including ChIP-seq and transcriptome profiling, aim to elucidate the mechanisms by which AT-hook proteins interact with PATCs and regulate gene expression in the germ line.
References:
1. Fire, A., Alcazar, R., and Tan, F. (2006). Unusual DNA structures associated with germline genetic activity in Caenorhabditis elegans. Genetics 173, 1259–1273.
2. Aljohani, M.D., El Mouridi, S., Priyadarshini, M., et al. (2020). Engineering rules that minimize germline silencing of transgenes in simple extrachromosomal arrays in C. elegans. Nat. Commun. 11, 6300.
3. Seroussi, U., Li, C., Sundby, A.E., Lee, T.L., Claycomb, J.M., and Saltzman, A.L. (2022). Mechanisms of epigenetic regulation by C. elegans nuclear RNA interference pathways. Semin. Cell Dev. Biol. 127, 142–154.
4. Frøkjær-Jensen, C., Jain, N., Hansen, L., Davis, M.W., Li, Y., Zhao, D., Rebora, K., Millet, J.R.M., Liu, X., Kim, S.K., Dupuy, D., Jorgensen, E.M., and Fire, A.Z. (2016). An abundant class of non-coding DNA can prevent stochastic gene silencing in the C. elegans germline. Cell 166, 343–357.
Keywords: AT-hook, PATCs (Periodic AnTn Clusters), Gene expression regulation
144. An AT-hook transcription factor promotes transcription of histone, spliced-leader, and piRNA clusters
Yi-Hui Wang (Department of Biological Chemistry and Pharmacology; The Center for RNA Biology), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology; The Center for RNA Biology), Benjamin Pastore (Department of Biological Chemistry and Pharmacology; The Center for RNA Biology), Wen Tang (Department of Biological Chemistry and Pharmacology; The Center for RNA Biology)
Abstract:
In all three domains of life, genes with related functions can be organized into specific genomic regions known as gene clusters. In eukaryotes, histone, piRNA, and rDNA (ribosomal DNA) clusters are among the most notable clusters which play fundamental roles in chromatin formation, genome integrity, and translation, respectively. These clusters have long been thought to be regulated by distinct transcriptional mechanisms. In this study, using C. elegans as a model system we identify ATTF-6, a member of the AT-hook family, as a key factor for the expression of histone, piRNA, and 5S rDNA-SL1 (spliced leader 1) clusters. ATTF-6 forms distinct nuclear foci at both piRNA and 5S rDNA-SL1 clusters. Loss of ATTF-6 leads to a depletion of histone mRNAs and SL1 transcripts. Additionally, we demonstrate that ATTF-6 is required for the recruitment of USTC (Upstream Sequence Transcription Complex) to piRNA clusters, which is necessary for piRNA production. Collectively, our findings reveal a unifying role for an AT-hook transcription factor in promoting the expression of fundamental gene clusters.
Keywords: piRNA biogenesis, AT-hook transcription factor
145. Contribution of an alternative 16S rRNA helix to biogenesis of the 30S subunit of the ribosome
Benjamin Warner (Department of Microbiology, The Ohio State University), Kurt Fredrick (Department of Microbiology, The Ohio State University)
Abstract:
30S subunits become inactive upon exposure to low Mg2+ concentration, because of a reversible conformational change that entails nucleotides (nt) in the neck helix (h28) and 3′ tail of 16S rRNA. This active-to-inactive transition involves partial unwinding of h28 and repairing of nt 921–923 with nt 1532–1534, which requires flipping of the 3′ tail by ∼180°. Growing evidence suggests that immature 30S particles adopt the inactive conformation in the cell, and transition to the active state occurs at a late stage of maturation. Here, we target nucleotides that form the alternative helix (hALT) of the inactive state. Using an orthogonal ribosome system, we find that disruption of hALT decreases translation activity in the cell modestly, by approximately twofold, without compromising ribosome fidelity. Ribosomes carrying substitutions at positions 1532–1533 support the growth of Escherichia coli strain Δ7 prrn (which carries a single rRNA operon), albeit at rates 10%–20% slower than wild-type ribosomes. These mutant Δ7 prrn strains accumulate free 30S particles and precursor 17S rRNA, indicative of biogenesis defects. Analysis of purified control and mutant subunits suggests that hALT stabilizes the inactive state by 1.2 kcal/mol with little-to-no impact on the active state or the transition state of conversion.
Keywords: ribosome assembly, rRNA folding, translation
146. Investigating structural features of pre-miR-122 that affect Dicer processing
Megan N. Westwood (Biophysics, University of Michigan), Sarah C. Keane (Biophysics, University of Michigan)
Abstract:
Proper cellular function depends on both protein coding and non-coding (nc) RNAs. Mature microRNAs (miRNAs) play a critical role in post-transcriptional regulation of gene expression, regulating more than half of human messenger RNAs. MiRNAs are transcribed from DNA into primary (pri-) miRNAs which are processed in the nucleus to make precursor (pre-) miRNAs. Subsequent cytoplasmic processing of pre-miRNAs involves Dicer and transactivation response element RNA-binding protein (TRBP) to produce mature miRNAs. Pre-miR-122 is a tissue-specific miRNA that is highly expressed in the liver and aberrant expression of miR-122 is a hallmark of various tumors and cancers including hepatocellular carcinoma. Pre-miR-122 has no known RNA-binding proteins that modulate its biogenesis post-transcriptionally. Therefore, pre-miR-122 may possess intrinsic structural properties that regulate its processing. To gain atomic-level insight into the structure(s) of pre-miR-122, I will use nuclear magnetic resonance (NMR) spectroscopy. Interestingly, secondary structure predictions and NOEs observed near the apical loop suggest that pre-miR-122 may adopt alternative conformations. Comparative processing assays with Dicer:TRBP between the wildtype and mutant RNAs, designed to stabilize specific alternative structures, will be used to assess which structural features regulate its biogenesis. Future work will include using NMR dynamics experiments to quantify the populations and rate constants of conformational exchange. Together, this work will provide unprecedented insight into the structural and dynamic features of pre-miR-122 to understand how its biogenesis is regulated post-transcriptionally.
Keywords: microRNA
147. The AUGCs: Learning the chemistry of RNA through a mass-spectrometry screen of electrophiles
Adam B. Wier (University of Notre Dame Department of Chemistry & Biochemistry), Marla Gravino (University of Notre Dame Department of Chemistry & Biochemistry), Adam Albritton (University of Notre Dame Department of Chemistry & Biochemistry), Brittany S. Morgan, Ph.D. (University of Notre Dame Department of Chemistry & Biochemistry)
Abstract:
RNA possesses multiple nucleophiles that can react with small-molecule electrophiles, such as 2’-hydroxyls that are acylated by SHAPE reagents.1 These nucleophile-electrophile reactions anchor methods of learning about the higher-order structures and small-molecule binding sites of the transcriptome, providing a readout that persists through RNA’s dynamic nature.2,3 Despite their applicability, only a narrow range of RNA-reactive electrophiles have been demonstrated compared to those that are known to react with nucleophilic amino acids. Furthermore, there are few validated nucleophile-specific electrophiles for RNA, whereas amino acid residue-specific electrophiles such as acrylamides for cysteine or hydroxysuccinimides for lysine are well known.4 To address this gap in knowledge, I have developed a liquid chromatography-mass spectrometry (LC-MS) platform for screening reactions between electrophilic fragments and 5’-monophosphate ribonucleotides. Electrophiles are sorted into those that produce a mass-spectrometry identifiable product and those that are unreactive for each canonical nucleotide. In the future, reactive electrophiles will be quantified for differences in percentage of covalent bond formation using high-performance liquid-chromatography coupled with UV-absorbance (HPLC-UV). Products will be identified using 1H and 13C NMR. This work is the first systematic census of electrophile reactivity with ribonucleotides, revealing the myriad chemical transformations RNA can experience. I expect the results of this screen to provide greater context for existing electrophilic chemistries used with RNA and to broaden the available tools for researchers looking to develop covalent strategies for studying the transcriptome.
References:
(1): Merino, E.J.; Wilkinson, K.A.; Coughlan, J.L.; Weeks, K.M. RNA Structure Analysis at Single Nucleotide Resolution by Selective 2‘-Hydroxyl Acylation and Primer Extension (SHAPE). JACS 2005, 127, 12, 4223-4231
(2): Chan, D.; Feng, C.; Spitale, R.C. Measuring RNA structure transcriptome-wide with icSHAPE. Methods 2017, 120, 85-90.
(3): Fang, L.; Velema, W.A.; Lee, Y.; Xiao, L.; Mohsen, M.G.; Kietrys, A.M.; Kool, E.T. Pervasive transcriptome interactions of protein-targeted drugs. Nature Chemistry 2023, 15, 1374-1383.
(4): Gehringer, M. and Laufer, S.A. Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology. J. Med. Chem. 2019, 62, 12, 5673-5724.
Keywords: Electrophile, Ribonucleotide, LCMS
148. Elongation Factor P is required for antibiotic resistance in β-lactam resistant Escherichia coli
Kelsey Shaffer (Ohio University Heritage College of Osteopathic Medicine), Tiffany Kelley (Ohio University Heritage College of Osteopathic Medicine), Annie Witzky (Department of Biological and Environmental Sciences, Capital University)
Abstract:
The rise of antibiotic resistant bacteria has been identified as a global health crisis. The CDC has specifically identified carbapenem resistant and extended-spectrum beta-lactamase producing Enterobacterales as urgent and serious threats respectively. Combatting these threats will rely on better understanding the mechanisms that Enterobacterales use to maintain resistance. Elongation factor P (EF-P) is a translation factor that influences antibiotic sensitivity in many bacterial species. EF-P assists the ribosome in translation of polyproline motifs by entering the ribosome through the E-site and stimulating peptide bond formation. Though the functional role of EF-P in translation has been established, the significance it may have in antibiotic resistant bacteria has not been characterized. Here, we aimed to assess the role of EF-P in β-lactam resistant Escherichia coli. Initial disc diffusion results confirmed that loss of EF-P activity in E. coli resulted in increased susceptibility to ampicillin, penicillin, meropenem, and carbapenem. When this experiment was repeated in E. coli that harbored plasmids conferring resistance, loss of EF-P resulted in hypersensitivity to meropenem and carbapenem, but not ampicillin or amoxicillin. To assess the genetic basis for the role of EF-P in β-lactam resistance, a suppressor screen was used to identify Δefp mutants that had suppressed antibiotic hypersensitivity. Initial screening resulted in identification of an Δefp suppressor mutant with restored resistance to multiple antibiotics. Together, these results indicate that EF-P is required for antibiotic resistance in β-lactam resistant E. coli, and this requirement can be by-passed through a suppressor mutation. Future work will center on identification and characterization of the suppressor mutation as well as further screening for additional mutants.
Keywords: Elongation Factor P, Antibiotic Resistance, Translation
149. Characterizing Structural and Thermodynamic Properties of a Human Homolog of TRMT1
Matthew Yacoub (Department of Chemistry, University of Michigan), Catherine Wilhelm (Department of Chemistry, University of Michigan), Shayna Brotzman (Department of Chemistry, University of Michigan), Daniel Corey (Department of Chemistry, University of Michigan), Markos Koutmos (Department of Chemistry, University of Michigan)
Abstract:
Post-transcriptional modifications of transfer RNA (tRNA) affect the stability, three-dimensional structure, and folding of these dynamic biomacromolecules. Such modifications exert downstream effects on critical biological processes such as translation and aminoacylation. TRMT1, a tRNA methyltransferase, modifies guanosine at position 26 of its tRNA substrates to form both monomethylated (m2G26) and dimethylated (m22G26) products. Mutations in human TRMT1 (hTRMT1) have been linked to numerous diseases, including autosomal-recessive intellectual disability, mitochondrial encephalomyopathies, and cancer. Homologs of this enzyme found in Saccharomyces cerevisiae, Pyrococcus horikoshii, and Aquifex aeolicus have previously been kinetically, thermodynamically, and structurally characterized; however, the same characterization has yet to be performed for hTRMT1. This is likely due to the complexity of the human homolog, as it is 300 amino acids longer than its non-human counterparts, conferring instability and intrinsically disordered regions (as predicted by AlphaFold) that make it more difficult to express, purify, and handle. We successfully purified hTRMT1 using affinity and size exclusion chromatography. Next, we aim to determine the structural and thermodynamic properties of the resulting protein samples by setting up crystallization screening trays and negative stains for characterization by X-ray crystallography and cryo-EM, respectively. We further plan to conduct binding and catalytic activity assays in order to characterize the enzyme’s affinity for and ability to methylate its tRNA substrates. These studies will lay the groundwork for future investigations into the mechanisms of pathogenesis caused by patient mutations in hTRMT1.
References:
Machnicka et al. RNA Biol. (2014) 11(12), 1619-1629.
Zhang et al. Hum. Mutat. (2020) 41(3), 600-607.
Ihsanawati et al. J. Mol. Biol. (2008) 383, 871-884.
Awai et al. J. Biol. Chem. (2011) 286(40), 35236-35246.
Keywords: hTRMT1, Modification, Methyltransferase
150. Experimental determination of pKa values of canonical and analogue nucleosides
Andrew A. Yaldo (Chemistry, Wayne State University ), Sun Jeong Im (Chemistry, Wayne State University ), Christine S. Chow (Chemistry, Wayne State University )
Abstract:
Nucleosides play critical roles in biological systems. Modified nucleoside analogues such as fleximers (synthetic variants of purine bases in which the bicyclic ring is split into two linked heterocycles) have shown potential as novel antiviral and cancer drugs. A key characteristic that plays a role in a molecule’s binding mode with a biological target is the pKa value. The pKa value is a measure of protonated or deprotonated states at specific locations on the molecule at a given pH value. The pKa values of nucleoside analogues such as fleximers have not been well studied. Depending on the protonation state, the compounds could have very different properties and participate in different interactions with a target molecule. In this study, the pKa values of nucleosides were determined experimentally using two methods: 1) carbon-13 NMR and chemical shift changes at varying pH conditions, and 2) UV-visible spectroscopy by measuring the absorbance values at various pH conditions. The pros and cons of both methods will be discussed alongside the experimental results. The ability to determine pKa values of analogue nucleosides will be beneficial for the development of novel drugs.
References:
Seley, K. L.; Zhang, L.; Hagos, A.; Quirk, S. “Fleximers”: design and synthesis of a new
class of novel shape-modified nucleosides. J Org Chem 2022 67(10), 3365-3373.
Thapa, B.; Raghavachari, K. Accurate pKa evaluations for complex bio-organic molecules
in aqueous media. J Chem Theory Comput 2019, 15(11), 6025-6035.
Jones, E. L.; Mlotkowski, A. J.; Hebert, S. P.; Schlegel, H. B.; Chow, C. S. Calculations of
pKa values for a series of naturally occurring modified nucleobases. J Phys Chem A 2022,
126(9), 1518-1529.
Vidal-Salgado, L. E.; Vargas-Hernández, C. “Spectrophotometric Determination of the pKa, Isosbestic Point and Equation of Absorbance vs. pH for a Universal pH Indicator”. American Journal of Analytical Chemistry, 2014, v. 5(17), p. 1290-1301. DOI: 10.4236/ajac.2014.517135
Keywords: Fleximer, pKa
151. Gathering insights into differences between substrate recognition by ADR-1 and ADR-2 in vivo
Boyoon Yang (Biochemistry Graduate Program, Indiana University Bloomington, IN ), Ryan J. Andrews, Brenda L. Bass (2Department of Biochemistry, University of Utah, UT), Douglas B. Rusch (Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN), Heather A. Hundley (Department of Biology, Indiana University Bloomington, IN )
Abstract:
All animals generate transcriptomic diversity by modifying genetic information through RNA editing. A-to-I RNA editing is mediated by Adenosine deaminases that act on RNA (ADARs). ADARs convert specific adenosines into inosines in double-stranded RNA (dsRNA). ADARs have a high potential as therapeutic means to correct specific G-to-A mutations at the RNA level without modifying the genome. Therefore, it is crucial to understand how ADARs bind specific transcripts and select an individual adenosine for editing in vivo.
In Caenorhabditis elegans, RNA editing is mediated by ADR-2, whereas ADR-1 is an editing-deficient ADAR family member which positively or negatively regulates editing by ADR-2. While in vitro studies have shown that ADARs bind dsRNA in a structure-dependent manner, how ADARs recognize specific dsRNA substrates in vivo is poorly understood. To investigate how ADR-1 and ADR-2 bind specific targets, we performed chimeric crosslinking immunoprecipitation (CLIP) coupled to high-throughput sequencing. ADR-1/ADR-2 eCLIP revealed that the majority of ADR-1 binding occurs at 3’ UTRs, while most ADR-2 binding occurs at intronic regions, indicating ADR-1 and ADR-2 may recognize substrates differently. Elucidating the mechanism of how ADR-1 and ADR-2 co-occupy target mRNAs will provide new insights into how A-to-I editing is mediated in vivo.
Keywords: RNA editing, ADAR, Substrate specificity
152. Oligomerization-mediated activation of a short prokaryotic Argonaute
Xiaoyuan Yang (Department of Biological Chemistry and Pharmacology, The Ohio State University ), Zhangfei Shen (Department of Biological Chemistry and Pharmacology, The Ohio State University), Shiyu Xia (Divison of Biology and Biological Engineering, California Institute of Technology), Wei Huang, Derek J. Taylor (Department of Biochemistry, Case Western Reserve University School of Medicine), Kotaro Nakanishi (Department of Chemistry and Biochemistry, The Ohio State University), Tianmin Fu (Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract:
Argonautes (Agos) are highly conserved proteins that widely exist in all domains of life, and they play a crucial role in post-transcriptional gene regulation and immune surveillance against foreign invaders. While eukaryotic Agos (eAgos) and long prokaryotic Agos (pAgos) employ short nucleotide guides to cleave complementary target nucleic acids, short pAgos lack such cleavage activity and are usually associated with distinct accessory proteins for function. However, compared to the well-studied eAgos and long pAgos, the short pAgos, which constitute the majority of pAgos, remain underexplored. Here, we present a hierarchical activation pathway of a short pAgo system named SPARTA (Short Prokaryotic Argonautes and associated TIR-APAZ proteins). SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA-DNA-bound state, two dimeric RNA-DNA-bound states, and a tetrameric RNA-DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. Apo SPARTA is inactive, its RNA-DNA-binding channel occupied an auto-inhibitory motif in TIR-APAZ. Upon RNA-DNA binding, SPARTA transitions from a monomer to a symmetric and then an asymmetric dimer, in which two TIR domains interact via charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolyzing NAD(P)+. Additionally, we observed unique features of SPARTA-RNA-DNA interactions, including competition between the DNA 3’ end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA-DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgo systems and offer insights into repurposing SPARTA as valuable tools for cancer diagnostics and therapy.
References:
1. Koopal, B., Potocnik, A., Mutte, S. K., Aparicio-Maldonado, C., Lindhoud, S., Vervoort, J. J. M., Brouns, S. J. J., & Swarts, D. C. (2022). Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA. Cell, 185(9), 1471–1486.e19. https://doi.org/10.1016/j.cell.2022.03.012
2. Shen, Z., Yang, XY., Xia, S. et al. Oligomerization-mediated activation of a short prokaryotic Argonaute. Nature 621, 154–161 (2023). https://doi.org/10.1038/s41586-023-06456-z
Keywords: Short Prokaryotic Argonautes, Oligomerization-mediated activation
153. BR-bodies switch from mRNA decay to mRNA storage in stationary phase
Hadi Yassine (Biological Sciences, Wayne State University), Luis Ortiz-Rodriguez (Chemistry, University of Michigan Ann Arbor), Nadra Al-Husini (Biological Sciences, Wayne State University), Vidhyadhar Nandana (Biological Sciences, Wayne State University), Julie Biteen (Chemistry, University of Michigan Ann Arbor), Jared Schrader (Biological Sciences, Wayne State University)
Abstract:
Bacterial Ribonucleoprotein bodies (BR-bodies) are phase-separated condensates found in Caulobacter crescentus (Ccr) that help organize mRNA decay machinery during exponential growth phase. During exponential growth, BR-bodies quickly assemble to promote mRNA decay on the 1-4 minute timescale, and upon mRNA decay, BR bodies rapidly disassemble. Interestingly, mRNA decay rates drop in stationary phase of many bacteria, yet it has remained unclear what role BR-bodies play in the deceleration of mRNA decay in stationary phase. Time-lapse microscopy and FRAP revealed that BR-bodies become dynamically arrested upon entry into stationary phase, resembling a solid-like state. We observe that as BR-body dynamics become gradually arrested, there is a corresponding reduction in mRNA decay rates. However, upon nutrient replenishment, solid-like BR-bodies melt back into a dynamic liquid-like state, which occurs prior to when the cells resume growth. Translation rates assayed by amino acid incorporation (BONCAT) demonstrated that upon the addition of fresh media to stationary phase cells, we find an anti-correlation of translation activity and BR-body assembly, suggesting that solid-like BR-bodies may be sequestering poorly translated mRNAs. RNase E together with RNA drive BR-bodies phase separation, where its intrinsically disordered C-terminal domain (CTD) is necessary and sufficient to drive phase separation. To investigate the physiological roles of BR-bodies in stationary phase, we generated an RNEΔCTD strain that cannot form BR-bodies. When comparing its growth rate to the wild-type, RNEΔCTD has only subtle growth defects in log phase. However, when stationary phase cells are provided with fresh media, RNEΔCTD tends to have an extended lag-phase phenotype compared to wild-type. This suggested that BR-bodies promote faster entry into log-growth upon nutrient replenishment. Overall, we propose that BR-bodies shift their function from mRNA decay to mRNA storage when cells enter the stationary phase, and upon nutrient availability, BR-bodies dissolve and release the stored mRNA to promote fast regrowth.
References:
1- Al-Husini N, Tomares DT, Pfaffenberger ZJ, Muthunayake NS, Samad MA, Zuo T, Bitar O, Aretakis JR, Bharmal MM, Gega A, Biteen JS, Childers WS, Schrader JM. BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates. Mol Cell. 2020 May 21;78(4):670-682.e8. doi: 10.1016/j.molcel.2020.04.001.
2- Al-Husini N, Tomares DT, Bitar O, Childers WS, Schrader JM. α-Proteobacterial RNA Degradosomes Assemble Liquid-Liquid Phase-Separated RNP Bodies. Mol Cell. 2018 Sep 20;71(6):1027-1039.e14. doi: 10.1016/j.molcel.2018.08.003.
Keywords: BR-bodies, Material properties, mRNA storage
154. Translational output of CFTR is regulated by multiple elements in 5’UTR
Alice M. Youle (Biological Chemistry, University of Michigan), Rachel O. Niederer (Biological Chemistry, University of Michigan)
Abstract:
Cystic fibrosis is a monogenic disorder primarily caused by mutations to the chloride ion channel protein CFTR. Over 2000 causative genomic variants have been identified, which result in a wide range of molecular phenotypes, including low expression of CFTR, or expression of CFTR with reduced or abolished function1. The amount of functional CFTR is tightly connected to the severity of disease, which has focused therapeutic efforts on increasing expression of functional CFTR. The 5’ untranslated region (UTR) of an RNA is an important feature in regulating translation of an RNA into protein. CFTR has several elements in its 5’UTR which are commonly inhibitory for translation, including secondary structure and an upstream start codon (uAUG). However, the interplay of these regulatory elements within 5’UTR, and their combined impact on translational output requires further characterization. We identified that secondary structure within the 5’UTR suppresses translation of CFTR in vitro particularly when located at the 5’end of the mRNA, and that shifting the structure downstream by only 4 nucleotides is sufficient to promote translation. This is consistent with previous work suggesting the highly structured 5’ end of CFTR RNA inhibits translation initiation2. Surprisingly, we also find that the uAUG is not inhibitory for translation of CFTR as expected, but rather enhances translational output. We propose that the uAUG may enhance translation by promoting translational re-initiation. These findings establish the CFTR 5’UTR as an interesting model for uncovering mechanistic details of RNA translation regulation, as well as a potential therapeutic target to increase CFTR expression in CF patients.
References:
1) Cystic Fibrosis Mutation Database (2011). Available online at: http://www.genet.sickkids.on.ca
2) Sasaki, S., Sun, R., Bui, H.-H., Crosby, J. R., Monia, B. P., & Guo, S. (2019). Steric Inhibition of 5’ UTR Regulatory Elements Results in Upregulation of Human CFTR. Molecular Therapy: The Journal of the American Society of Gene Therapy, 27(10), 1749–1757. https://doi.org/10.1016/j.ymthe.2019.06.016
Keywords: 5UTR, secondary structure, translation reinitiation
155. Liquid Chromatography-Mass Spectrometry Analysis of Total tRNA using RNase 4
Hina Zain (Department of Chemistry, University of Cincinnati, OH), Bibek Hamal (Department of Chemistry, University of Cincinnati, OH), Asif Rayhan (Department of Chemistry, University of Cincinnati, OH), Patrick A. Limbach (Department of Chemistry, University of Cincinnati, OH)
Abstract:
Tandem liquid chromatography-mass spectrometry (LC-MS/MS) is one of the best tools for the analysis of the RNA sequence without amplification and converting it into cDNA. Identifying the RNA sequence and locating the specific ribonucleoside modifications requires the digestion of RNA into oligonucleotides that can then be analyzed by LC-MS/MS. Various ribonucleases with different cleavage specificity can be used to generate these oligonucleotides. Recently, human RNase 4, which cleaves at the sequence motif Up(A/G), became available commercially. Past work has focused on the use of this enzyme for the digestion of messenger RNA (mRNA). However, transfer RNAs (tRNAs) contain far more modifications than are found in mRNAs. In the present study, different incubation periods and enzyme concentrations for human RNase 4 were optimized with a 78-nt tRNA mimic. Once the conditions were optimized, the reaction was applied to yeast total tRNA. We found that RNase 4 cleavage motif can include the modified uridines, pseudouridine and dihydrouridine. Additionally, a Cp(A/G) cleavage motif was also found. Optimization of reaction conditions for human RNase 4 digestion of complex tRNA mixtures will enable improved tRNA modification mapping by LC-MS/MS
Keywords: Human RNase 4, tRNA, LC-MSMS
156. Incorporating common ASO modifications into mRNAs impacts translation speed
Maddy Zamecnik (University of Michigan, Department of Chemistry), Dan OReilly (University of Massachusetts Chan Medical School, RNA Therapeutics Institute), Anastasia Khvorova (University of Massachusetts Chan Medical School, RNA Therapeutics Institute), Kristin Koutmou (University of Michigan, Department of Chemistry)
Abstract:
RNA therapeutics require chemical modifications to their RNAs to extend their lifespans in the cell. Nucleobase modifications like N1-methylpseudouridine can increase the effectiveness of mRNA therapeutics, but a different suite of modifications has been developed for decades to solve a similar problem in other types of RNA therapeutics, including antisense oligonucleotides (ASOs). Specifically, modifications including 2’-O-methoxyethyl RNA (MOE), locked nucleic acids (LNA), and unlocked nucleic acids (UNA) have long been explored in ASOs for their abilities to increase binding affinities, decrease immunogenicity, and slow RNA degradation. However, these modifications have not been explored for their potential applicability to mRNA therapeutics. This lack of research is particularly surprising given that MOE is already being used in approved therapeutics, they are all compatible with existing ASO delivery techniques, and each has had a method of enzymatic synthesis already developed for it. Given this knowledge gap, I have employed an in vitro, reconstituted, E. coli translation system to assess how each of these modifications influences the kinetics of translation elongation when they are inserted into all 3 positions of a codon. Our findings indicate that the effects of the modifications on peptide bond formation are context dependent, varying depending on the position of the modification in the codon. While some modified codons produced only mild (2-fold) or nonsignificant changes in peptide bond formation relative to an unmodified codon, others stopped translation entirely. These results suggest that the suite of RNA modifications already developed for ASOs may provide a viable avenue to expand the chemical toolbox for mRNA therapeutics development.
Keywords: therapeutics, rna modifications, translation kinetics
157. Expanding the function and activity of a unique deadenylase involved in non-coding RNA processing and regulation
Lizette Zavala (Biochemistry and Molecular Biology, University of Chicago), Magadalena Sobien (Biochemistry and Molecular Biology, University of Chicago), Cassandra Hayne (Biochemistry and Molecular Biology, University of Chicago)
Abstract:
Target of the Egr1 (TOE1) is a unique 3'-to-5' Cajal-associated deadenylase involved in RNA processing of small non-coding RNAs (ncRNAs). It’s essential for trimming poly(A) tails for the proper maturation of small nuclear RNAs, and while it functions like other canonical deadenylases, TOE1 also has a unique 3'-exonuclease activity. TOE1 is an essential protein that is indispensable for early embryonic development of chordates, however, minimal biochemical studies characterization limits our understanding of the activity, stability, and regulation of this fascinating deadenylase1. Additionally, TOE1 has been linked to a neurological disorder—pontocerebellar hypoplasia type 7 (PCH7) due to the abundant accumulation of snRNA precursors upon TOE1 depletion in cells and the presence of specific point mutations in TOE12.
Here, we share our work to decipher how PCH7 linked TOE1 variants affect TOE1’s stability and activity by utilizing in vitro assays and interpret these findings in the context of AlphaFold generated models to provide mechanistic explanations for how these PCH7 linked mutations may cause disease. Using a recombinant system, we purified many TOE1 variants. Preliminary deadenylation assays and differential scanning fluorimetry (DSF) indicates distinct differences between wild-type TOE1 and PCH7-linked TOE1 variants. Our findings indicate that some variants impact TOE1’s thermal stability while others impact TOE1’s cleavage activity and possibly RNA recognition. We determined select mutations appear to have mild impacts on TOE1’s thermal stability from the DSF results, altered activity based on the deadenylation assays, and used AlphaFold models to decipher other ways mutations may impact TOE1 function or regulation. As an essential protein involved in the maturation of snRNAs, it is vital to discern the actual impact PCH7 TOE1 variants have, and to also expand our current understanding of how deadenylase can recognize and target their specific RNA substrates, deadenylate them, and aid in both protecting and moving the RNA substrates to their next stage in processing.
References:
1. Huynh, T. N., & Parker, R. (2023). The PARN, TOE1, and USB1 RNA deadenylases and their roles in non-coding RNA regulation. The Journal of biological chemistry, 299(9), 105139. https://doi.org/10.1016/j.jbc.2023.105139
2. Lardelli, R. M., et al. (2017). Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing. Nature genetics, 49(3), 457–464. https://doi.org/10.1038/ng.3762Skeparnias, I., Αnastasakis, D., Shaukat, A. N., Grafanaki, K., & Stathopoulos, C. (2017). Expanding the repertoire of deadenylases. RNA biology, 14(10), 1320–1325. https://doi.org/10.1080/15476286.2017.1300222
Keywords: RNA processing, Non-coding RNAs, deadenylase
158. Gene silencing by cityRNAs
Huaqun Zhang (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210, USA), GeunYoung Sim (Molecular, Cellular and Developmental Biology, Center for RNA Biology, The Ohio State University, Columbus, Ohio, 43210, USA), Audrey C. Kehling, Vishal Annasaheb Adhav (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210, USA), Andrew Savidge (Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, 43210, USA), Benjamin Pastore, Wen Tang (Center for RNA Biology, Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, 43210, USA), Kotaro Nakanishi (Department of Chemistry and Biochemistry, Molecular, Cellular and Developmental Biology, Center for RNA Biology, Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, 43210, USA)
Abstract:
Huaqun Zhang, GeunYoung Sim, and Audrey C. Kehling contributed equally.
Argonaute proteins (AGOs) load 20-23 nucleotide (nt) microRNAs (miRNAs) to form the RNA-induced silencing complex (RISC), the effector of RNA interference. Among the four human AGO paralogs, only AGO2 and AGO3 cleave target RNAs that are fully complementary to the guide RNA1. Recently, we discovered that AGO3 maximizes its endoribonuclease activity by loading specific guide RNAs of approximately 14 nt in length. These newly identified small noncoding RNAs have been termed cleavage-inducing tiny guide RNAs (cityRNAs)2. In this study, our in vitro assays demonstrated that the upstream sequence of the tiny RNA binding site (UTy) on the target RNA is crucial for efficient cleavage by AGO3, with a similar trend observed in AGO23. We further evaluated the potential of cityRNAs as a gene-silencing tool. To this end, we developed two auxiliary RNA systems, referred to as Booster, which facilitate the loading of any desired cityRNAs into endogenous AGO proteins. Using Dual-Luciferase Reporter assays, we demonstrated that cityRNAs efficiently induced gene silencing in HEK293T, HCT116, A549, and HeLa cells, especially for targets with specific UTy sequences. Unexpectedly, our data suggest that cityRNA-mediated silencing is highly dependent on target RNA cleavage. This could potentially reduce the off-target effect that designed small interfering RNAs bind to undesired target RNAs and induce silencing via translational repression.
References:
1. Park, M.S., Phan, H.D., Busch, F., Hinckley, S.H., Brackbill, J.A., Wysocki, V.H., and Nakanishi, K. (2017). Human Argonaute3 has slicer activity. Nucleic Acids Res 45, 11867-11877. 10.1093/nar/gkx916.
2. Park, M.S., Sim, G., Kehling, A.C., and Nakanishi, K. (2020). Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA. Proc Natl Acad Sci U S A 117, 28576-28578. 10.1073/pnas.2015026117.
3. Zhang, H., Sim, G.Y., Kehling, A.C., Adhav, V.A., Savidge, A., Pastore, B., Tang, W., Nakanishi, K. Cell Rep. accepted
Keywords: Argonaute, cityRNA, gene silencing
159. Abrogation of intronic polyadenylation: CRISPR/Cas9-based gene-editing to circumvent acquired resistance to DNA Topoisomerase II-targeted anticancer agents
Xinyu Zhao (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Xinyi Wang (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Sarah E. Eaton (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Terry S. Elton (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University), Jack C. Yalowich (Division of Pharmaceutics & Pharmacology, College of Pharmacy, The Ohio State University)
Abstract:
DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is a nuclear enzyme that plays a key role in chromosomal segregation at mitosis by catalyzing transient DNA double-stranded breaks, allowing replicated DNA duplexes to pass through one another. The clinical efficacy of TOP2α-targeted anticancer agents is often limited due to acquired drug resistance which is predominantly associated with decreased TOP2α/170 levels, attenuated drug-induced stabilization of TOP2α-DNA complexes, and reduced DNA damage/cytotoxicity.
We previously demonstrated decreased expression of TOP2α/170 protein in an acquired etoposide-resistant human leukemia K562 cell line (K/VP.5) due to the production of a novel C-terminal truncated 90 kDa isoform (TOP2α/90), the result of intronic polyadenylation (IPA) within Intron 19 (I19) of TOP2α mRNA. Circumvention of resistance was accomplished by gene-editing of the E19/I19 5'-splice site.
Truncated TOP2α mRNA transcript and protein have also been reported within a mitoxantrone-resistant subline (HL60/MX2) derived from human HL-60 leukemia cells as a result of I33 IPA, yielding a C-terminal truncated TOP2α/160 and reduced TOP2α/170 mRNA/protein. TOP2α/160 is predominantly distributed in the cytoplasm due to loss of a nuclear localization signal (NLS).
Utilizing 3'-Rapid Amplification of cDNA Ends (3'-RACE) and Sanger sequencing, IPA at I33 in HL60/MX2 cells was confirmed. In addition, use of THZ531, a CDK12 inhibitor, demonstrated a 30-fold increase in TOP2α/160 mRNA in HL60/MX2 cells compared to the parental HL60 cell line, further validating IPA in this resistant cell line.
CRISPR/Cas9 with homology-directed repair was used to successfully gene edit/mutate/enhance the intrinsically weak exon 33/intron 33 (E33/I33) splice-site in HL60/MX2 cells. Studies are underway to demonstrate abrogation of IPA, restoration of wild-type TOP2α/170 mRNA/protein level, and circumvention of anticancer drug sensitivity in these gene-edited cells.
Keywords: Intronic polyadenylation, DNA topoisomerase II
160. The polyadenylation factor CPSF30 is important for root development and plant response to salt stress
Lichun Zhou (Department of Plant and Soil Sciences, University of Kentucky, Lexington KY 40546), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky, Lexington KY 40546)
Abstract:
Message RNA polyadenylation is an essential step for eukaryotic mRNA transport, translation, and turnover. Alternative polyadenylation (APA) serves an important role in regulation of gene expression. The Arabidopsis thaliana ortholog of the 30-kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (CPSF30) gene encodes two proteins, CPSF30S and CPSF30L. Prior research has shown CPSF30S is a calmodulin-regulated RNA-binding protein, regulates APA, and links with cellular signaling and stress response. To better understand how CPSF30 contributes to stress responses and connects to environmental signals, we subjected a set of mutant and complemented Arabidopsis lines to salt stress. The set of lines included a mutant (oxt6) that does not express CPSF30 as well as lines that express either CPSF30S or CPSF30L in the oxt6 background. Seedlings were used to count lateral root numbers and collected to perform Poly (A) Tag Sequencing (PATSeq). The results show both CPSF30S and CPSF30L are functional in lateral root development. However, CPSF30S and CPSF30L have different functions in response to stress treatment. Specifically, CPSF30L is required for APA in salt stress. More generally, after stress treatment, genes associated with stress responses tended to use proximal poly(A) sites. Taken together, these results provide new insights into the connections between mRNA polyadenylation and environment signaling.
Keywords: Plant Polyadenylation, CPSF30
161. Cancer-associated snaR-A noncoding RNA interacts with core splicing machinery and disrupts processing of mRNA subpopulations
Sihang Zhou (Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign), Simon Lizarazo (Department of Molecular and Integrative Physiology, University of Illinois Urbana-Champaign), Leela Mouli,Ruiying Cheng (School of Molecular and Cellular Biology, University of Illinois Urbana-Champaign), Sandip Chorghade,Auinash Kalsotra (Department of Biochemistry, University of Illinois Urbana-Champaign), Rajendra K C (Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign), Kevin Van Bortle (Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign)
Abstract:
RNA polymerase III (Pol III) activity in cancer is linked to the production of small noncoding (nc)RNAs that are otherwise silent in most tissues. snaR-A (small NF90-associated RNA isoform A) - a hominid-specific ncRNA shown to enhance cell proliferation, migration, and invasion - is a cancer-emergent Pol III product that remains largely uncharacterized despite promoting growth phenotypes. Here, we applied a combination of genomic and biochemical approaches to study the biogenesis and subsequent protein interactions of snaR-A and to better understand its role as a putative driver of cancer progression. By profiling the chromatin landscapes across a multitude of primary tumor types, we show that predicted snaR-A upregulation is broadly linked with unfavorable outcomes among cancer patients. At the molecular level, we unexpectedly discover widespread interactions between snaR-A and mRNA splicing factors, including SF3B2 - a core component of the U2 small nuclear ribonucleoprotein (snRNP). We find that SF3B2 levels are sensitive to high snaR-A abundance and that depletion of snaR-A alone is sufficient to decrease intron retention levels across subpopulations of mRNA enriched for U2 snRNP occupancy. snaR-A sensitive genes are characterized by high GC content, close spatial proximity to nuclear bodies concentrated in pre-mRNA splicing factors, and functional enrichment for proteins involved in deacetylation and autophagy. We highlight examples of splicing misregulation and increased protein levels following snaR-A depletion for a wide-ranging set of factors, suggesting snaR-A-driven splicing defects may have far-reaching effects that reshape the cellular proteome. These findings clarify the molecular activities and consequences of snaR-A in cancer, and altogether establish a novel mechanism through which Pol III overactivity may promote tumorigenesis.
Keywords: ncRNA, Cancer, Splicing
162. Genome-wide application of a tRNA assay to uncover proteins involved in the tRNA nuclear retrograde pathway
Ernesto J. Roldan-Bonet (Department of Molecular Genetics, OSU), Paolo L. Sinopoli (Department of Molcular Genetics, OSU), Bianca Lederfeind (Department of Biochemistry and Molecular Biology, UF), Anita K. Hopper (Department of Molecular Genetics, OSU)
Abstract:
Transfer RNAs(tRNAs) are initially transcribed in the nucleus and subsequently transported to the cytoplasm where they function as essential adaptor molecules in translation. Given their cytoplasmic function, tRNA subcellular trafficking was thought to be unidirectional – from the nucleus to the cytoplasm. It is now well documented that after primary nuclear export, tRNAs are shuttled back into the nucleus in a process called retrograde tRNA nuclear import before re-localizing to the cytoplasm (tRNA nuclear re-export). These latter trafficking steps have been implicated in several important functions, yet our understanding of the proteins involved is incomplete. Here, we developed a genome-wide assay in Saccharomyces cerevisiae to identify proteins involved in the tRNA nuclear retrograde pathway via the detection of the retrograde pathway-dependent, post-transcriptional tRNA modification: wybutosine (yW). Over 15% of the annotated genome has been queried, and we have identified three proteins involved in yW biosynthesis – validating our unbiased approach. Additionally, we queried all the members of the karyopherin-beta family, which comprise several nuclear exportins, importins and biportins, representing the largest known family of tRNA nuclear transporters. Taken together, our studies aim to identify the missing proteins involved in the widely conserved tRNA nuclear retrograde pathway.
References:
Nostramo, R. T., & Hopper, A. K. (2020). A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae. Nucleic acids research, 48(20),11577–11588. https://doi.org/10.1093/nar/gkaa879
Shaheen, H. H., & Hopper, A. K. (2005). Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 102(32), 11290–11295. https://doi.org/10.1073/pnas.0503836102
Takano, A., Kajita, T., Mochizuki, M., Endo, T., & Yoshihisa, T. (2015). Cytosolic Hsp70 and co-chaperones constitute a novel system for tRNA import into the nucleus. eLife, 4, e04659. https://doi.org/10.7554/eLife.04659
Keywords: tRNA retrograde pathway, subcellular trafficking, wybutosine