Poster abstracts
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1. PufA regulates R-loop formation, DNA damage repair and chemotherapy resistance
chenyu Lin (The James Comprehensive Cancer Center, The Ohio State University), Jian Ouyang (Massachusetts General Hospital Cancer Center, Harvard Medical School), Eshan Khan (The James Comprehensive Cancer Center, The Ohio State University), Hannah Hylton (The James Comprehensive Cancer Center, The Ohio State University), Lee Zou (Massachusetts General Hospital Cancer Center, Harvard Medical School), Wayne Miles (The James Comprehensive Cancer Center, The Ohio State University)
Abstract:
Double-strand DNA breaks (DSBs) promote the formation of RNA/DNA hybrids (R-loops), as an active mechanism to ensure accurate repair. However, how these RNA/DNA hybrids are formed and protected remains poorly understood. In this study, we identified PufA as a novel regulator of R-loops that are important for R-loops sites that form during transcriptional termination. We find that PufA restricts DNA damage induced by aberrant transcription and also modulates the accumulation of RNA/DNA hybrids at DSBs sites that are required for homologous recombination (HR)-mediated repair. Our data shows that PufA plays an important role in HR-DNA repair and R-loop activity. We find that overexpression of PufA accelerates HR-mediated repair. The loss of PufA leads to the sensitivity of tumor cells to cisplatin, offering new therapeutic opportunities in PufA-deficient tumors.
References:
1. Qiu, C.; McCann, K. L.; Wine, R. N.; Baserga, S. J.; Hall, T. M. T. A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization. Proceedings of the National Academy of Sciences 2014, 111 (52), 18554.
2. Chang, H.-Y.; Fan, C.-C.; Chu, P.-C.; Hong, B.-E.; Lee, H. J.; Chang, M.-S. hPuf-A/KIAA0020 modulates PARP-1 cleavage upon genotoxic stress. Cancer research 2011, 71 (3), 1126.
3. Peng, G.; Lin, S.-Y. Exploiting the homologous recombination DNA repair network for targeted cancer therapy. World journal of clinical oncology 2011, 2 (2), 73.
4. Ouyang, J.; Yadav, T.; Zhang, J.-M.; Yang, H.; Rheinbay, E.; Guo, H.; Haber, D. A.; Lan, L.; Zou, L. RNA transcripts stimulate homologous recombination by forming DR-loops. Nature 2021, 594 (7862), 283.
Keywords: PufA, R-loop , DNA damage
2. Peptides to inhibit the T-Box Riboswitch
Emily Fairchild (Ohio University), Danushika Hearth (Ohio University), Jennifer Hines (Ohio University)
Abstract:
T-Box riboswitches are found in gram positive bacteria and control systems related to amino acid sensing, making it a crucial target for antibiotic design. There are currently no known predictive docking protocols for targeting the T-Box riboswitch. In order to develop a computationally predictive method we designed a tetra- peptide library to target the T-box riboswitch antiterminator RNA element. The library was composed of all combinations and permutations of natural amino acids, giving way to 160,000 peptides built. This library was then docked to the NMR-derived structure of the antiterminator of the T-Box riboswitch using iteratively filtered docking methods. From this optimized docking workflow, two peptides, RDCH and YQQW, were tested for affinity and inhibition of the T-Box riboswitch. The peptides showed preliminary antagonist and agonist activity, respectively.
Keywords: T-Box Riboswitch, Computational Docking, Peptides
3. Robust discovery and quantification of transcript isoforms from error-prone long-read RNA sequencing data
Yuan Gao, Feng Wang (Center for Computational and Genomic Medicine, The Childrens Hospital of Philadelphia), Robert Wang (Center for Computational and Genomic Medicine, The Childrens Hospital of Philadelphia), Eric Kutschera, Yang Xu, Stephan Xie (Center for Computational and Genomic Medicine, The Childrens Hospital of Philadelphia), Yuanyuan Wang, Kathryn E. Kadash-Edmondson (Center for Computational and Genomic Medicine, The Childrens Hospital of Philadelphia), Lan Lin (Department of Pathology and Laboratory Medicine, University of Pennsylvania), Yi Xing (Center for Computational and Genomic Medicine, The Childrens Hospital of Philadelphia)
Abstract:
Long-read RNA sequencing (RNA-seq) holds great potential for characterizing transcriptome variation and full-length transcript isoforms, but the relatively high error rate of current long-read sequencing platforms poses a major challenge. We present ESPRESSO, a computational tool for robust discovery and quantification of transcript isoforms from error-prone long reads. ESPRESSO jointly considers alignments of all long reads aligned to a gene and uses error profiles of individual reads to improve the identification of splice junctions and the discovery of their corresponding transcript isoforms. On both a synthetic spike-in RNA sample and human RNA samples, ESPRESSO outperforms multiple contemporary tools in not only transcript isoform discovery but also transcript isoform quantification. In total, we generated and analyzed ~1.1 billion nanopore RNA-seq reads covering 30 human tissue samples and three human cell lines. ESPRESSO and its companion dataset provide a useful resource for studying the RNA repertoire of eukaryotic transcriptomes.
Keywords: long-read RNA sequencing, transcript isoform variation, alternative splicing
4. A congenital hydrocephalus causing mutation in Trim71 results in stem-cell and neural-differentiation defects through inhibiting Lsd1 mRNA translation
Kailey Worner (Department of Biochemistry and Molecular Biology, Mayo Clinic), Qiuying Liu (Department of Biochemistry and Molecular Biology, Mayo Clinic), Mariah K. Novak (Department of Biochemistry and Molecular Biology, Mayo Clinic), Rachel M. Pepin, Katharine R. Maschhoff (Department of Biochemistry and Molecular Biology, Mayo Clinic), Xiaoli Chen, Shaojie Zhang (Department of Computer Science, University of Central Florida), Wenqian Hu (Department of Biochemistry and Molecular Biology, Mayo Clinic)
Abstract not available online - please check the booklet.
5. The dynamic, motile and deformative properties of RNA nanoparticles facilitate the third milestone of drug development
Abhjeet S. Bhullar (Biophysics, The Ohio State Univeristy), Peixuan Guo (Pharmacy, Ohio State University), Daniel Binzel (Pharmacy, Ohio State University)
Abstract:
Besides mRNA, rRNA, and tRNA, cells contain many other noncoding RNA that display critical functions in the regulation of cellular functions. Human genome sequencing revealed that the majority of non-protein-coding DNA actually codes for non-coding RNAs. The dynamic nature of RNA results in its motile and deformative behavior. These conformational transitions such as the change of base-pairing, breathing within complemented strands, and pseudoknot formation at a 2D level as well as the induced-fit and conformation capture at the 3D level are important for their biological functions including regulation, translation, and catalysis. The dynamic and motile and catalytic activity has led to a belief that RNA is the origin of life. We have recently reported that the deformative property of RNA nanoparticles enhances their penetration the leaky blood vessel of cancers which leads to high-efficiency tumor accumulation. This special deformative property also enables RNA nanoparticles to pass the glomerulus, overcoming the filtration size limit, resulting in fast renal excretion and rapid body clearance, thus low or no toxicity. The biodistribution of RNA nanoparticles can be further improved by the incorporation of ligands for cancer targeting. In addition to the favorable biodistribution profiles, RNA nanoparticles possess other properties including self-assembly, negative charge, programmability, and multivalency; making it a great material for pharmaceutical applications. The intrinsic negative charge of RNA nanoparticles decreases the toxicity of drugs by preventing nonspecific binding to the negative charge cell membrane and enhancing the solubility of hydrophobic drugs. The polyvalent property of RNA nanoparticles allows the multi-functionalization which can apply to the approach to overcome drug resistance. This review focuses on the summary of these unique properly of RNA nanoparticles, describes the mechanism of RNA dynamic, motile and deformative properties, and elucidates and prepares to welcome the RNA therapeutics as the third milestone in pharmaceutical drug development.
Keywords: RNA
6. The DEAD-box helicase Drs1 plays a role in compaction of 25S rRNA domain III during nucleolar stages of large subunit assembly
Fiona Fitzgerald (Department of Biological Sciences, Carnegie Mellon University), Collin Bachert (Department of Biological Sciences, Carnegie Mellon University), Stephanie Biedka (Department of Biological Sciences, Carnegie Mellon University), Yungyang Zhang (State Key Laboratory of Membrane Biology, Peking University), Ning Gao (State Key Laboratory of Membrane Biology, Peking University), John L. Woolford (Department of Biological Sciences, Carnegie Mellon University)
Abstract:
The nucleolar stages of large subunit (LSU) assembly are characterized by compaction of individual domains of 25S ribosomal RNA (rRNA) around the root helices, beginning with compaction of domains I, II, and VI, followed by domain III, and ultimately domains IV and V. The compaction of these rRNA domains is important for formation of ribosome functional centers, e.g., the nascent polypeptide exit tunnel (NPET) and the peptidyl-transferase center (PTC). We also believe that before these rRNA domains are compacted, they function to form a network of interactions between pre-ribosomes to create the nucleolar condensate.
Some DEAD-box helicases (DBHs) involved in ribosome biogenesis use their ATPase activity to coordinate the folding of rRNA through removal of structural snoRNAs or assembly factors (AFs), and rearrangement of rRNA structure. However, the precise functions of most DBHs involved in ribosome assembly remain unclear.
The DBH Drs1 functions in early stages of LSU assembly. Depletion of Drs1 leads to accumulation of 27SA2 and 27SA3 pre-rRNAs. In contrast, the drs1-1 mutation near a conserved ATPase motif results in cold-sensitive growth and accumulation of 27SB pre-rRNA. We seek to understand the role of the ATPase activity of Drs1 in late-nucleolar stages of LSU assembly.
Previous studies using protein-protein crosslinking and co-transcriptional rRNA association indicate that Drs1 might bind in or near domain III of assembling pre-ribosomes. Further examination of the drs1-1 missense mutant, using a combination of proteomics, cryo-EM, and genetics, indicates that Drs1 may play an important role in compaction of rRNA domain III.
Keywords: rRNA, ribosome, helicase
7. Computationally determined pKa values of modified nucleobases: more than just a series of numbers
Alan J. Mlotkowski (Department of Chemistry, Wayne State University), Evan L. Jones (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:
Modifications play a role in fine tuning structure and function of RNA. Both natural and nonnatural modifications have been studied for their impact on RNA biology, but also used as chemical tools and building blocks for therapeutics. Despite a large amount of information about modifications being available, some details regarding their physical properties are still lacking. Nucleobases and their modifications contain one or more sites of protonation/deprotonation with unique pKa values. Experimentally, these values can be challenging to determine, particularly for cases in which multi-step syntheses of the modified nucleosides are required. In this study, we performed theoretical calculations to obtain the pKa values of a series of natural and nonnatural modified nucleobases. The calculated pKa values of modified nucleobases were compared to their unmodified counterparts to determine the impact of the added chemical functionalities. Differences in the pKa values between modified and unmodified nucleobases range from 0.1 to 9.1, with an average of 1.8. These nucleobase pKa values can provide insight into the structural and energetic impacts of modifications within a wide range of RNAs.
References:
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, 1518–1529.
Keywords: Nucleobase, Modification, Structure
8. Investigating Natural Variation in SAM-I Riboswitch Structure and Function
Ian Hall (Department of Chemistry, University of Michigan), Kate Zablock (Program in Biophysics, University of Michigan), Raeleen Sobetski (Program in Biophysics, University of Michigan), Sarah Keane (Department of Chemistry and Program in Biophysiscs, University of Michigan)
Abstract:
Methionine and its metabolites are essential for the growth and development of all organisms. Mechanisms for the recognition and regulation of methionine levels are diverse among the domains of life. In many bacteria, the metabolite S-adenosyl methionine (SAM) is recognized by SAM-I riboswitches which control the transcription of methionine biosynthesis and transport genes in a SAM-dependent manner. SAM-I riboswitches are non-coding (nc)RNAs found in the 5 ′ untranslated region (UTR) of some bacterial messenger (m)RNAs. SAM-I riboswitches bind SAM within an aptamer domain which directs the co-transcriptional folding of an expression platform. When SAM is bound, a terminator hairpin forms, causing premature termination of transcription. Without SAM, the expression platform folds into an anti-terminator hairpin that permits transcription of downstream genes.
Previous work in Bacillus subtilis discovered natural variation in the ability of SAM-I riboswitches to bind SAM and terminate transcription.1 Seven candidate SAM-I riboswitches were identified in Listeria monocytogenes and named SAM riboswitch elements A-G (SreA-G).2 These candidate riboswitches have yet to be functionally validated but are predicted to fold into the conserved SAM-I riboswitch secondary structure. The current work is a robust biochemical and biophysical characterization of the candidate riboswitches. The ligand affinity and specificity of the riboswitches were determined by isothermal titration calorimetry (ITC). The ability and efficiency of the riboswitches to terminate transcription were evaluated by a radiolabeled transcription termination assay. The secondary and tertiary structures of the riboswitches were investigated by chemical probing and small-angle X-ray scattering (SAXS), respectively. The goal of this work is to functionally validate SreA-G as SAM-I riboswitches and to understand how natural variance in the riboswitches' ability to bind SAM and terminate transcription is related to riboswitch structure and genomic location.
References:
1. Tomšič et al. Natural Variability in S-Adenosylmethionine (SAM)-Dependent Riboswitches: S-Box Elements in Bacillus subtilis exhibit Differential Sensitivity to SAM In Vivo and In Vitro. Journal of Bacteriology. 2008.
2. Toledo-Arana et al. The transcriptional landscape of Listeria monocytogenes: switch from saprophytism to virulence. Nature. 2009.
Keywords: Riboswitch, RNA Structure, Molecular Recognition
9. Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit
Aparna R. Biswas (Biology, Saint Louis University), Yu-Fong Peng, Poonam Roshan (Biology, Saint Louis University), Jonah Elliff (Department of Immunology, The University of Iowa, Department of Biological Sciences, Marquette University), Sahiti Kuppa, Nicola Pozzi, Edwin Antony (Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine ), Brian Bothner (Department of Chemistry and Biochemistry, Montana State University), Sofia Origanti (Biology, Saint Louis University)
Abstract:
Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its regulation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6, we have uncovered the critical interface entailing 8 key residues in the C-tail of RPL23 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provides a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to RPL23 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting a key residue in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Further, we are establishing a system to study the domain dynamics of SBDS, an eIF6 release factor, by using non-canonical amino acids and bio-orthogonal chemistry to site-specifically label individual domains. Determining the mechanism of eIF6 release from 60S and establishing the key interfaces that regulate eIF6 will provide a therapeutic framework for targeting eIF6 in cancers and SDS.
Keywords: eIF6-uL14 interaction, eIF6 somatic mutations, Shwachman-Diamond Syndrome
10. Pre-mRNA 3D structural scaffold as a contributor to the mammalian splicing code
Kaushik Saha (Department of Chemistry and Biochemistry, University of California San Diego), Mike M. Fernandez (Department of Chemistry and Biochemistry, University of California San Diego), Whitney England (Department of Pharmaceutical Sciences, University of California Irvine), Robert C. Spitale (Department of Pharmaceutical Sciences, University of California Irvine), Gourisankar Ghosh (Department of Chemistry and Biochemistry, University of California San Diego)
Abstract:
The specific recognition of splice signals at or near exon-intron junctions by the early spliceosomal components is not explained by their weak sequence conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We show that the early spliceosomal components recognize a pre-mRNA 3D structural scaffold beyond the short splice signal sequences. Interestingly, structural remodeling of the pre-mRNA 3D scaffold by the splicing regulatory proteins (SRps) appeared to be critical for its specific recruitment by the early spliceosomal components. We also find that exonic loops immediately upstream of the intron regulate the functional recruitment of SRps and consequent pre-mRNA structural remodeling. These exonic segments are more hybridized upstream of the retained introns compared to the spliced introns across the transcriptome.
Additional investigations into the mechanism of recognition of the pre-mRNA 3D structural scaffold by the early spliceosomal components suggest that the SRps bind the pre-mRNAs cooperatively, generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65, and U2AF35.
Overall, the central hypothesis supported by this work is that the early spliceosomal components recognize a regulatable 3D structural scaffold in addition to the short splice signal sequences. This effectively constitutes the newest element of the mammalian splicing code in addition to the splice signal sequences, the splicing regulatory elements, and the pre-mRNA secondary structure.
References:
1. Saha, K., England, W., Fernandez, M.M., Biswas ,T., Spitale, R.C., Ghosh, G. (2020). Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing. Nucleic Acids Res 48, 6294-6309. DOI:10.1093/nar/gkaa358
2. Saha, K., Fernandez, M.M., Biswas, T., Joseph, S. and Ghosh, G. (2021) Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code. Nucleic Acids Res, 49, 7103-7121. DOI:10.1093/nar/gkab533
3. Saha, K. and Ghosh, G. (2022) Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly. Nucleic Acids Res. Ahead of print. DOI:10.1093/nar/gkac636
Keywords: Pre-mRNA 3D structure, Mammalian splice site recognition, SR proteins
11. Title not available online - please see the booklet.
Feng Jiang (Department of Biochemistry & Biophysics, University of Rochester School of Medicine & Dentistry, Rochester, New York 14586), Omar Hedaya (Department of Biochemistry & Biophysics, University of Rochester School of Medicine & Dentistry, Rochester, New York 14586), EngSoon Khor (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, New York 14586), Matthew Auguste (Department of Biological Sciences Molecular Genetics, University of Rochester, Rochester, New York 14642), Peng Yao (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, New York 14586)
Abstract not available online - please check the booklet.
12. Analysis of tRNA methyltransferase homologs from zebrafish reveals similarities and differences to human Trm10 enzymes
Ben Jepson (Department of Chemistry and Biochemistry, OSU), Jane Jackman (Department of Chemistry and Biochemistry, OSU)
Abstract:
The tRNA m1R9 methyltransferase (Trm10) family enzymes methylate the N-1 atom of purine residues at the ninth position of tRNAs. Higher eukaryotes encoding up to three homologs of Trm10. Human TRMT10A catalyzes m1G9 formation on multiple tRNAs, whereas TRMT10B forms m1A9 specifically on human tRNAAsp. Familial TRMT10A mutations are associated with human disease, further supporting the non-redundant function of the two homologs in vivo. We sought to develop a vertebrate model system to further probe the significance of these distinct activities using Danio rerio and have created mutant lines for the cytosolic homologs. Like human TRMT10A, expression of zebrafish Trmt10a rescues the trm10∆ growth phenotype in S. cerevisiae and methylates yeast tRNAs in vivo. Conversely, neither zebrafish Trmt10b nor human TRMT10B are capable of rescuing the phenotype or methylating yeast substrates in vivo. However, zebrafish Trmt10a and Trmt10b do not show the same pattern of in vitro substrate specificities as the human homologs. Since the modification status of zebrafish tRNAs had not been previously determined, we carried out a genome-wide study to map tRNA modification in zebrafish using mim-tRNAseq on wild-type embryos. We have now identified and validated multiple tRNA modifications in zebrafish, providing the first global map of tRNA modification in this organism, including target tRNAs for m1R9 modification. Analysis of R9 modification in the trmt10a and trmt10b mutant fish show that although each homolog acts on unique tRNA substrates similar to modification in humans, other substrates are shared between homologs, consistent with our in vitro experiments with purified enzymes. Overall, our data reveals that while the zebrafish enzymes share important similarities with their human counterparts there are distinct differences in some patterns of activity, deeper investigation of which could help uncover important insights into complex substrate specificities of Trm10 homologs.
Keywords: tRNA modification, Trm10, m1R9
13. Manganese-dependent microRNA trimming by 3′→5′ exonucleases generates 14-nucleotide or shorter tiny RNAs.
GeunYoung Sim (Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA, Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH 43210, USA), Mi Seul Park (Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA), Audrey C. Kehling, Jackson Secor, Huaqun Zhang (Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Nipun Malhotra, Divyaa Bhagdikar, Ekram A. El-Wahaband (Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Cameron Divoky (Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA), Kotaro Nakanishi (Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA)
Abstract not available online - please check the booklet.
14. Role of Modified Nucleotides in Mediating Structure and Oxidative Damage of U2 snRNA
Sun Jeong Im (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)
Abstract:
Reactive oxygen species (ROS) are free radicals that are necessary for cellular processes. However, imbalanced levels of ROS cause oxidative stress in biological systems, leading to undesired side effects such as damage to biomolecules. A number of laboratories have carried out research to understand how ROS reacts with DNA and proteins, but the relationship between ROS and RNA is much less understood. For this project, the impact of ROS on structure and chemical stability of variants of the highly modified U2 small nuclear RNA (snRNA) is being determined. U2 snRNA is a key component of the spliceosome. This RNA contains two different modified nucleotides in the stem I region. Disruption of the U2 snRNA structure due to ROS could lead to dysfunction of pre-mRNA splicing. We hypothesize that modified nucleotides will mediate the damage responses. Chemically synthesized and in vitro transcribed stem I U2 snRNA variants with and without modifications are being analyzed by circular dichroism and 1D 1H NMR spectroscopy to assess their global and secondary structures, respectively. In addition, the U2 snRNA responses towards hydroxyl radicals (•OH) are being studied to elucidate which nucleotide modifications at specific sites will impact radical reactivity. The outcomes of this project will contribute to a better understanding of oxidative damage to RNA molecules and the relationship to modification status, structure, and human diseases.
Keywords: Oxidative damage, U2 snRNA
15. Telomerase RNA biogenesis requires direct interactions of a core group of nucleolar family proteins in the parasitic protist, Trypanosoma brucei
Justin A. Davis (Department of Biological Sciences, University of North Carolina at Charlotte ), Nitika, Andrew W. Truman, Kausik Chakrabarti (Department of Biological Sciences, University of North Carolina at Charlotte ), Andres V. Reyes, Shou-Ling Xu (Department of Plant Biology and Carnegie Mass Spectrometry Facility, Carnegie Institution for Science), Arpita Saha, Bibo Li (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Sciences, College of Arts Sciences, Cleveland State University,), Donald J. Wolfgeher (Department of Molecular Genetics and Cell Biology, The University of Chicago)
Abstract:
Telomeres are the nucleoprotein structures found at the ends of eukaryotic linear chromosomes. Telomeric dysfunction and instability poses a significant threat to the genome integrity of eukaryotes. Telomeres are maintained by the actions of the ribonucleoprotein enzyme telomerase. Unlike human somatic cells, telomerase is constantly active in parasitic protozoa, which makes these cells proliferate rapidly to establish chronic, long-term infections. Significantly shortened telomeres in the protozoan parasite Trypanosoma brucei (T. brucei) have been shown to affect antigenic variation, which facilitates the parasite’s ability to evade its host’s immune responses. We and others have identified and characterized the telomerase RNA (TR) and the reverse transcriptase protein component (TERT) of T. brucei telomerase. Our recent in vivo probing of T. brucei telomerase RNA (TbTR), revealed developmental specific TR structural changes, which are intimately linked to parasite development, telomerase activity and cell proliferation. Interestingly, we found that the telomerase catalytic subunit TERT does not partake in the TR folding process for telomere synthesis in the human infective form (bloodstream form) of T. brucei. To gain a global view of the proteome that might be responsible for biogenesis and assembly of T. brucei telomerase, we have now identified the global interactors of T. brucei telomerase reverse transcriptase (TbTERT) using an affinity-purification based mass spectrometry approach. This approach identified >100 interactors, including known orthologs from yeast, human and ciliate species that can bind TERT and the TR. Most surprisingly, we identified an array of snoRNPs that can carry out 2’-O-ribose methylation, which is required for proper folding, stability and RNA-protein interactions. Deletion of the C/D box domain of the Telomerase RNA impairs binding of snoRNP NOP58, suggesting its potential role in the maturation and structural changes in this RNA during the telomerase reaction cycle. Therefore, we hypothesize that these proteins are required for TbTR biogenesis and have the potential to act as chaperones for RNA folding in T. brucei telomerase
Keywords: Telomerase , Telomerase RNA , T brucei
16. isoCirc catalogs full-length circular RNA isoforms in human transcriptomes
Ruijiao Xin, Yan Gao, Yuan Gao, Robert Wang, Kathryn E. Kadash-Edmondson (The Childrens Hospital of Philadelphia), Lan Lin (The Childrens Hospital of Philadelphia), Yi Xing (The Childrens Hospital of Philadelphia)
Abstract:
Circular RNAs (circRNAs) have emerged as an important class of functional RNA molecules. Short-read RNA sequencing (RNA-seq) is a widely used strategy to identify circRNAs. However, an inherent limitation of short-read RNA-seq is that it does not experimentally determine the full-length sequences and exact exonic compositions of circRNAs. Here, we report isoCirc, a strategy for sequencing full-length circRNA isoforms, using rolling circle amplification followed by nanopore long-read sequencing. We describe an integrated computational pipeline to reliably characterize full-length circRNA isoforms using isoCirc data. Using isoCirc, we generate a comprehensive catalog of 107,147 full-length circRNA isoforms across 12 human tissues and one human cell line (HEK293), including 40,628 isoforms ≥500 nt in length. We identify widespread alternative splicing events within the internal part of circRNAs, including 720 retained intron events corresponding to a class of exon-intron circRNAs (EIciRNAs). Collectively, isoCirc and the companion dataset provide a useful strategy and resource for studying circRNAs in human transcriptomes.
References:
Nat Commun. 2021 Jan 12;12(1):266. doi: 10.1038/s41467-020-20459-8.
Keywords: Circular RNA, RNA-seq, nanopore
17. Title not available online - please see the booklet.
Ian F. Price (Department of Biological Chemistry and Pharmacology, Ohio State University), Jillian Wagner (Department of Biological Chemistry and Pharmacology, Ohio State University), Wen Tang (Department of Biological Chemistry and Pharmacology, Ohio State University)
Abstract not available online - please check the booklet.
18. Massive upregulation of COII mRNA editing correlates with alterations in RESC protein and RNA interactions during T. brucei differentiation
Joseph T. Smith Jr. (Department of Microbiology and Immunology, University at Buffalo), Arunasalam Naguleswaran (Institute of Cell Biology, University of Bern), Brianna L. Tylec (Department of Microbiology and Immunology, University at Buffalo), Isabel Roditi (Institute of Cell Biology, University of Bern), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo)
Abstract not available online - please check the booklet.
19. Substrate recognition by two 3′ to 5′ RNA polymerases in Dictyostelium discoideum
Grace Johnecheck (Department of Chemistry and Biochemistry and OSBP, The Ohio State University), Yicheng Long (Department of Chemistry and Biochemistry and OSBP, The Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry and OSBP, The Ohio State University)
Abstract not available online - please check the booklet.
20. LC-MS/MS-based detection of 5-methyl cytidine in E. coli tRNAs arising from oxidative stress
Satenik Valesyan (University of Cincinnati), Patrick A. Limbach (University of Cincinnati), Balasubrahmanyam Addepalli (University of Cincinnati)
Abstract:
Oxidative stress caused by endogenous and exogenous sources has been associated with a number of human diseases. One exogenous source is ultraviolet radiation (UVR). Our lab earlier found that post-transcriptional modifications in transfer ribonucleic acids (tRNAs) are damaged by the reactive oxygen species (ROS) generated by UVA. Those studies also revealed that some photoproducts were more readily identified during RNA modification mapping by liquid chromatography tandem mass spectrometry (LC-MS/MS) rather than relying solely on their detection during nucleoside analysis. Another approach for generating ROS is the Fenton reaction, whereby Fe2+ catalyzes the generation of hydroxyl radicals (HO.) from hydrogen peroxide (H2O2). Here we have been exposing Escherichia coli (E. coli) cells to ROS conditions generated by the Fenton reaction. Surprisingly, our preliminary nucleoside analysis results reveal that 5-methylcytidine (m5C) is found at significantly higher levels in tRNAs upon exposure to Fenton conditions. Because m5C has not been reported in any published E. coli tRNA sequences, we have focused our RNA modification mapping analyses on discovery possible sequence locations in specific tRNAs where this modification may be occurring. At present, Tyr-QUA appears to be the most likely tRNA containing this additional modification, which appears induced by the Fenton reaction. Results from our analyses along with a discussion into the possible causes for this change in modification status will be presented.
References:
1. Liguori et al. (2018) Clin Interv Aging. 13: 757–772.
2. Bhattacharyya et al. (2014) Physiol Rev. 94(2): 329–354
3. Sun et al. (2018) ACS Chem Biol., 13(3): 567–572.
4. Zhao et al. (2019) Aging Med (Milton). 2(2): 82–87.
5. Dedon et al. (2010) PLoS Genet., 6(12): e1001247.
6. Sun et al. (2020) Biomolecules. 8;10(11):1527
7. Yu et al. (2017) ACS. 89, 20, 10744–10752
8. Lobue et al. (2019) Methods. 156:128-138
Keywords: Fenton reaction, tRNA, 5-methylcytidine (m5C)
21. Title not available online - please see the booklet.
Madeline M. Glennon (Chemistry and Biochemistry, University of Notre Dame), Krishna M. Shivakumar (Chemistry and Biochemistry, University of Notre Dame ), Martina Zafferani, Anita Donlic (Chemistry, Duke University ), Charlotte N. Kunkler (Chemistry and Biochemistry, University of Notre Dame), Amanda E. Hargrove (Chemistry, Duke University ), Jessica A. Brown (Chemistry and Biochemistry, University of Notre Dame)
Abstract not available online - please check the booklet.
22. RNA Tert-Seq - a biochemical pipeline to discover novel functional RNAs with tertiary structure
Nishanti Sudhakar (Department of Biochemistry andMolecular Biology and Center for RNA Molecular Biology, Penn State University), Dr. Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, and Center for RNA Molecular Biology, Penn State University)
Abstract not available online - please check the booklet.
23. Investigating transcriptional program of C. elegans piRNA expression
Yi-Hui Wang (Department of Biological Chemistry and Pharmacology), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology), 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:
PIWI-interacting RNAs (piRNAs) are a class of germline small non-coding RNA that are required for silencing the transposable elements. The piRNAs biogenesis involves processing of long transcript precursors into mature piRNAs that are 21-35 nucleotides (nts) in length. In C. elegans, piRNA precursors are capped small RNAs (csRNAs) (Gu et al., 2012). After 5’ and 3’ processing, the mature piRNAs uniformly start with 5’ Uracil and are 21 nts in length, thus referred to as 21U-RNAs. Most 21U-RNAs are produced from the two large piRNA clusters in chromosome IV, which encode over 15,000 unique piRNA sequences. The upstream of these piRNAs gene contains a conserve 8 nts motif, named Ruby motif, which is associated with transcription factors, including TOFU-5, TOFU-4, SNPC-4, and PRDE-1 (Ruby et al.,2006; Weng et al., 2019). To comprehensively define the transcriptional program of piRNA genes, we used a proximity-based labeling approach in combination with mass spectrometry to identify the proteins associated with piRNA clusters (Hertz et al., 2022). Our proteomics data revealed several promising candidates for piRNA biogenesis. We show that a novel transcription factor colocalizes with the piRNA cluster and its depletion causes the diffusion of PRDE-1 from the piRNA cluster, suggesting it acts upstream or co-dependently of PRDE-1. Moreover, upon loss of the transcription factor, the expression levels of 21U-RNA and csRNAs are significantly reduced. Our ongoing research will elucidate the function of this novel transcription factor and provide insights into transcription and processing of piRNAs.
References:
1. Gu, W., Lee, H. C., Chaves, D., Youngman, E. M., Pazour, G. J., Conte Jr, D., & Mello, C. C. (2012). CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors. Cell, 151(7), 1488-1500
2. Ruby, J. G., Jan, C., Player, C., Axtell, M. J., Lee, W., Nusbaum, C., ... & Bartel, D. P. (2006). Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell, 127(6), 1193-1207.
3. Weng, C., Kosalka, J., Berkyurek, A. C., Stempor, P., Feng, X., Mao, H., ... & Miska, E. A. (2019). The USTC co-opts an ancient machinery to drive piRNA transcription in C. elegans. Genes & development, 33(1-2), 90-102.
4. Hertz, H. L., Price, I. F., & Tang, W. (2022). Visualization and Purification of Caenorhabditis elegans Germ Granule Proteins Using Proximity Labeling. Bio-protocol, 12(8), e4386-e4386.
Keywords: piRNA biogenesis, Transcription factor, Ruby motif
24. Quantifying cleavage of known and novel twister ribozymes in vivo in rice
Reuben G. Kern (Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University), Lauren N. McKinley (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University), Sarah M. Assman (Department of Biology, Center for RNA Molecular Biology, Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry, Department of Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University)
Abstract not available online - please check the booklet.
25. Elucidating the role of RNA modifications in acetate-grown and methanol-grown Methanosarcina acetivorans
Anwesha Ghosh (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802), Allison M. Williams (Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802), Michel Geovanni Santiago-Martinez (Department of Molecular and Cellular Biology, University of Connecticut, Stamford, CT 06901 ), Ashley Shay (Metabolomics Core Facility, Pennsylvania State University, University Park, PA 16802), James G. Ferry (Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802)
Abstract not available online - please check the booklet.
26. Nucleic Acid Protective and Rescuing Properties of Early Earth Coacervates
Haiyun Liu (Department of Biochemistry and Molecular Biology, Pennsylvania State University, , University Park, PA 16802), Christine D. Keating (Department of Chemistry, Pennsylvania State University, University Park, PA 16802), Philip C. Bevilacqua (Department of Chemistry, Pennsylvania State University, University Park, PA 16802)
Abstract:
It is known that RNA, as one form of genetic information carrier, can also act as catalyst, which makes it important in the emergence of life. In addition, DNA is also known to catalyze chemical reactions. Therefore, both nucleic acids could be vital molecules in prebiotic evolution.
However, it was not easy for nucleic acids to maintain their catalytic activities and keep their genetic information while evolving toward the first cell. The early Earth surface was under strong UV radiation and rich in reactive chemicals, therefore nucleic acids were facing UV and chemical damage; also, proofreading systems were less likely to exist for the earliest RNA or DNA replicase. All these factors make it challenging for nucleic acids to maintain their function and information. We propose that coacervates, which are a plausible form of encapsulation of the earliest cell, protected and rescued the nucleic acids from the damages and mutations. To test this hypothesis, we are artificially damaging nucleic acids with UV, high pH, as well as introducing mutations and modifications, and attempting to rescue them with coacervates.
Here I present results in damaging the 10-23 DNAzyme and some variants of the DNAzyme with 254 nm UV and broad-band UV light and rescuing them with Asp10 - PDAC53 coacervates. Variants with more Ts showed more sensitivity to UV damage; concentrations of the DNAzyme had different effects on the sensitivity to UV damage in different ion conditions. Coacervates were able to improve the catalytic activity of both damage and undamaged DNAzymes, however there was not a significant difference on the magnitude of the improvement.
Keywords: Complex coacervates, early Earth, UV damage
27. Does the MALAT1 Triple Helix Form in the Presence of mascRNA?
Mika Schievelbein (Department of Chemistry and Biochemistry, University of Notre Dame), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)
Abstract not available online - please check the booklet.
28. Functional implications of a cancer-associated mutation on LARP1 DM15 region
Jahree A. Sosa (University of Pittsburgh), Kevin C. Cassidy (University of Pittsburgh), Jacob D. Durrant (University of Pittsburgh), Andrea J. Berman (University of Pittsburgh)
Abstract not available online - please check the booklet.
29. Disruption of spliceosomal proteins reduces NMD efficiency
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:
Regulation of the RNA lifecycle is critical to ensure proper protein production. Pre-mRNA splicing is an early RNA processing step that is tied to other downstream regulatory steps. Most notably is nonsense mediated mRNA decay (NMD), a quality control pathway that monitors translation for premature termination. Using data from the ENCODE consortium we have analyzed changes in RNA following knockdown of proteins involved in the catalytic process of splicing. Knockdown of these proteins results in the overwhelming of the NMD pathway and stabilization of NMD targets. To examine the disruption of a spliceosomal protein in a disease context we examined EFTUD2, a GTPase in the U5 snRNP. A number of pathogenic mutations in EFTUD2 reduce interactions with other members of the U5 snRNP, preventing EFTUD2's role in spliceosome activation.
Keywords: Splicing, NMD, Quality Control
30. Bioconjugation of functionalized oligodeoxynucleotides to hydrophobic fluorescence reporters
Erwin Doe (Ball State University), Hannah Hayth (Ball State University), Emil Khisamutdinov (Ball State University)
Abstract:
In the field of oligonucleotide (ODN) therapeutics, there is an imperative need to improve the ODNs properties by either chemical modification of the oligonucleotides structure or covalently linking reporter or therapeutic moieties with biologically relevant properties. The chemical conjugation can thus significantly improve the intrinsic property not only of ODNs but also reporter/therapeutic molecules. Bioconjugation of nucleic acids to small molecules also serve as nano-delivery facilities to transport drugs to specific targets. This study deployed azide–alkyne cycloaddition, a click reaction, to successfully conjugate a cyanine 3 alkyne moiety to an azide functionalized oligodeoxynucleotide 12-mer, as well as 3–azido–7–hydroxy–chromen–2–one, a coumarin azide to an alkyne functionalized ODN 27-mer. The application of Huisgen 1,3–dipolar cycloaddition of azides to terminal alkynes via a Cu(I)–catalyzed reaction pathway has been well established to produce high yield and stable 1,5–disubstituted 1,2,3–triazoles. Denaturing polyacrylamide gel electrophoresis confirmed the successful formation of the clicked products. Although cyanine 3 alkyne and coumarin azide are inherently fluorescent compounds, post-conjugation investigation of the clicked products demonstrated a drastic amplitude of fluorescent intensity by multiple fold. This amplification of fluorescence is attributable to the successful formation of the clicked ODNs which are characterized by the formation of the 1,2,3-triazole linkers.
References:
Bock VD, Hiemstra H, Maarseveen JH-V. CuI-Catalyzed alkyne-azide “click” cycloadditions from a mechanistic and synthetic perspective. Eur. J. Org. Chem 2006:51–68.
Chassaing S, Kumarraja M, Sido AS-S, Pale P, Sommer J. Click chemistry in CuI-zeolites: the huisgen [3 + 2]-cycloaddition. Org. Lett 2007;9:883–886. [PubMed: 17286410]
Hein, C.D., Liu, X-M & Wang, D. (2008). Click Chemistry, a powerful tool for pharmaceutical sciences. National Institute of Health, 25 (10), 2216 - 2230.
Huisgen R. 1,3-Dipolar cycloadditions. Angew. Chem 1963;75:604–637.
Rodriguez, D. F., Moglie, F., Ramirez-Sarmiento, C. A., Singh, S. K., Dua, K. and Zacconi, F. C. (2022). Bio-click chemistry: a bridge between biocatalysis and click chemistry. Royal Society of Chemistry, 1932 - 1949.
Keywords: Bioconjugation, Fluorescence, Oligodeoxynucleotide
31. Title not available online - please see the booklet.
Gowthami Mahendran (Department of Chemistry and Biochemistry), Kurtis Breger (Department of Chemistry and Biochemistry), Philip J. McCown, (Department of Chemistry and Biochemistry), Jacob P. Hulewicz (Department of Chemistry and Biochemistry), Jessica A. Brown (Department of Chemistry and Biochemistry)
Abstract not available online - please check the booklet.
32. Dissecting cross-talks between intron turnover pathway and nonsense mediated decay via the interactions of RNA debranching enzyme, DBR1
Tiffany D. Barwell (Department of Biological Sciences, University of North Carolina at Charlotte), Kausik Chakrabarti, PhD (Department of Biological Sciences, University of North Carolina at Charlotte)
Abstract not available online - please check the booklet.
33. Comparative analysis of piRNA sequences, targets and functions in nematodes
Hannah L. Hertz (Biology, Chemistry, and Pharmacology; The Ohio State University), Benjamin Pastore (Biology, Chemistry, and Pharmacology; Center for RNA Biology; Ohio State Biochemistry Program; The Ohio State University), Wen Tang (Biology, Chemistry, and Pharmacology; Center for RNA Biology; The Ohio State University)
Abstract:
Piwi proteins and Piwi-interacting RNAs (piRNAs) are best known for their roles in suppressing transposons and promoting fertility. Yet piRNA biogenesis and its mechanisms of action differ widely between distantly related species. To better understand the evolution of piRNAs, we characterized the piRNA pathway in C. briggsae, a sibling species of the model organism C. elegans. Our analyses define 25,883 piRNA producing-loci in C. briggsae. piRNA sequences in C. briggsae are extremely divergent from their counterparts in C. elegans, yet both species adopt similar genomic organization and transcription programs that drive piRNA expression. By examining production of Piwi-dependent secondary small RNAs, we identified a set of protein-coding genes that are evolutionarily conserved piRNA targets. In contrast to C. elegans, small RNAs targeting ribosomal RNAs or histone transcripts are not hyper-accumulated in C. briggsae Piwi mutants. Instead, we found that transcripts with few introns are prone to small RNA overamplification. Together our work highlights evolutionary conservation and divergence of the nematode piRNA pathway and provides insights into its role in endogenous gene regulation.
Keywords: piwi-interacting small RNAs, C elegans, evolution
34. MLSnitch: Towards a Machine Learning Method for RiboSNitch Prediction
Kobie J. Kirven (Pennsylvania State University, Graduate Program in Bioinformatics and Genomics), Philip C. Bevilacqua (Pennsylvania State University, Department of Biochemistry and Molecular Biology, Department of Chemistry), Sarah M. Assmann (Pennsylvania State University, Department of Biology), Vasant Honavar (Pennsylvania State University, College of Information Sciences and Technology)
Abstract not available online - please check the booklet.
35. Title not available online - please see the booklet.
Krishna M. Shivakumar (Department of Chemistry and Biochemistry, University of Notre Dame), Madeline M. Glennon (Department of Chemistry and Biochemistry, University of Notre Dame), Charlotte N. Kunkler (Department of Chemistry and Biochemistry, University of Notre Dame), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)
Abstract not available online - please check the booklet.
36. Polyadenylation site choice correlates with epitranscriptomic mark, N6-methyladenosine (m6A), in Plasmodium falciparum
Cassandra Catacalos (Department of Biological Sciences, University of North Carolina, Charlotte, NC, USA), Manohar Chakrabarti, Arthur Hunt (Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA), David Zhu, Kate Meyer (Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA), Robert Reid (Department of Bioinformatics, University of North Carolina, Charlotte, NC, USA), Kausik Chakrabarti (Department of Biological Sciences, University of North Carolina, Charlotte, NC, USA)
Abstract:
The recent discoveries of post-transcriptional modifications of mRNA have opened the possibility to understand a previously underappreciated regulatory layer involved in biological processes. Among all RNA modifications, N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark and regulates numerous cellular processes. Functional roles of this mark are carried out by m6A binding proteins, “readers”, which bind m6A residues and contribute to various aspects of mRNA regulation. Although the function of m6A in mRNA stability and translation has been well established, the implication of m6A in the control of mRNA 3’ end processing, remains obscure. Polyadenylation is the post-transcriptional addition of multiple adenine (A) nucleotides to the tail of a mRNA transcript, which requires recognition of a sequence element, AAUAAA, for efficient cleavage and poly(A) tail addition. However, an unresolved question remains in eukaryotic biology is how polyadenylation machinery is able to distinguish ‘bona-fide’ polyadenylation signals from the multiple look-alikes interspersed along the 3' UTRs? This is particularly intriguing in the Apicomplexan family with exceedingly A/T-rich genomes, such as the malaria pathogen Plasmodium falciparum. This pathogen’s gene expression varies hourly during its 48 hour human intraerythrocytic developmental cycle (IDC). We found that its genome harbors a cleavage and polyadenylation specificity factor, CPSF30, which possesses a characteristic reader domain. The strategic locations of m6A marks in Plasmodium mRNAs, nearby poly(A) sites, lends itself to the hypothesis that m6A may be a determinant of 3’ end processing in P. falciparum. We will present data from our global identification of m6A sites in P. falciparum, transcriptome-wide characterization of 3’ end processing events and molecular analysis of Plasmodium polyadenylation machinery that links epitranscriptomics 3’-end signatures in Plasmodium falciparum.
Keywords: Plasmodium, m6A, polyadenylation
37. The N-Terminal Domain of Eukaryotic Translation Initiation Factor 4B promotes P body Levels in Yeast
Qian He (SUNY at Buffalo, Department of Biological Sciences, Buffalo, NY), Ansuman Sahoo, Xiaozhuo Liu and Sarah E. Walker (SUNY at Buffalo, Department of Biological Sciences, Buffalo, NY)
Abstract:
P bodies are membrane-less cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes for storage and degradation of mRNA. However, the mechanisms that mediate sorting of mRNAs into P bodies and other granules are not well understood. Eukaryotic initiation factor eIF4B, which accelerates the rate and stability of translation initiation complex assembly on mRNAs, has been shown to reside in P bodies in response to certain stresses in Saccharomyces cerevisiae. The NTD and 7-repeats of eIF4B promote translation of structured mRNAs and bind to the small subunit of the ribosome. We recently showed that the NTD also promotes resistance to various stressors by reprogramming translation of mRNAs known to aggregate in P bodies. Here we investigated whether P bodies and resulting mRNA turnover play a role in translation reprogramming mediated by eIF4B. First, we determined that eIF4B enhanced resting P bodies levels in yeast, by following localization of Edc3-GFP, a protein localized to these cytoplasmic mRNP granules. Deletion of the NTD of eIF4B decreased the number of P bodies per cell, but deletion of the RRM, which promotes RNA binding, or the 7-repeats domain, which like the NTD stimulates mRNA recruitment to the ribosome, but with a more deleterious phenotype, did not affect P body levels. Consistent with a specific role for the eIF4B NTD in promoting P body levels, we found that an eIF4A mutant compromised in eIF4B interaction showed reduced P body formation when eIF4B ∆ntd was overexpressed. Current work is exploring additional interactions of eIF4B and their roles in P body formation and stability. Together these data suggest that eIF4B plays a direct role in assembly or maintenance of P bodies, and that changes in translatome composition as a result of this activity allows resistance of yeast to various stressors. This work also highlights the impact of mRNA structure in the granule-mediated degradation of mRNAs through competition with translation.
Keywords: eIF4B, P bodies, Translation initiation
38. Small-angle scattering techniques as a structural tool to study RNA:RNA complexes
Aldrex Munsayac (Department of Chemistry, University of Michigan), Ian Hall (Department of Chemistry, University of Michigan), Sarah C. Keane (Department of Chemistry and Program in Biophysics, University of Michigan)
Abstract:
Non-coding RNAs (ncRNAs) play numerous roles to maintain cellular homeostasis, regulating gene expression at the levels of transcription, RNA processing, and translation.1 The functions of many ncRNAs depends on how their three-dimensional structure changes in response to changes in the cellular environment (concentration of metabolites and/or metal ions, temperature) and interactions with other biomolecules (proteins and other RNAs).2 In the foodborne pathogen Listeria monocytogenes, two regulatory RNA elements—the positive regulatory factor A (prfA) RNA thermosensor (RNAT) and S-Adenosyl methionine riboswitch element A (SreA)—work in tandem to regulate its virulence.3 However, the precise molecular mechanism governing this interaction remains unclear due to difficulties in elucidating RNA:RNA complex structures. Therefore, there is a need for new strategies to probe structural information of RNA:RNA complexes. Biomolecular small-angle X-ray/neutron scattering (SAXS/SANS) are powerful solution-based techniques that provides information on molecular masses, particle dimensions and interactions, and low-resolution conformations. SANS is particularly well-suited to study multicomponent systems; by systematically changing the deuteration level of either the macromolecule or buffer medium, specific parts within the molecule can be singled out.4 Using the well-characterized human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) kissing complex as a model complex, we report strides towards the development of SANS as a novel tool for structure determination of RNA-only complexes. We have acquired high-quality SAXS data of the HIV-1 DIS complex and are currently in the process of taking SANS measurements of the partially deuterated complex to establish the methodology, which will then be applied to the prfA RNAT:SreA complex to unveil the global rearrangements of the individual components. SANS, complemented with other biophysical and biochemical techniques, improves our understanding towards how RNAs can fold into higher order assemblies to carry out their functions.
References:
1. Cech, T. R.; Steitz, J. A. The Noncoding RNA Revolution—Trashing Old Rules to Forge New Ones. Cell 2014, 157 (1), 77–94.
2. Ganser, L. R.; Kelly, M. L.; Herschlag, D.; Al-Hashimi, H. M. The Roles of Structural Dynamics in the Cellular Functions of RNAs. Nat. Rev. Mol. Cell Biol.
3. Loh, E.; Dussurget, O.; Gripenland, J.; Vaitkevicius, K.; Tiensuu, T.; Mandin, P.; Repoila, F.; Buchrieser, C.; Cossart, P.; Johansson, J. A Trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria Monocytogenes. Cell 2009, 139 (4), 770–779.
4. Hoogerheide, D. P.; Forsyth, V. T.; Brown, K. A. Neutron Scattering for Structural Biology. Phys. Today 2020, 73 (6), 36–42.
Keywords: Small-angle scattering, RNA-RNA complexes, Integrative structural biology
39. Determining the role of the 3’-5’ reverse polymerase in Myxococcus xanthus
Brandon Iwaniec (Chemistry and Biochemistry, The Ohio State University), Ashanti Matlock (Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry )
Abstract not available online - please check the booklet.
40. Trans-spliced Leader Sequences Present a Novel Evolutionary Target of the DM15 Region of LARP1
Nicholas Arredondo (Department of Biological Sciences; University of Pittsburgh), Andrea Berman (Department of Biological Sciences; University of Pittsburgh)
Abstract:
The RNA-binding protein La-related protein 1 (LARP1) is present in most eukaryotes. In vertebrates, LARP1 regulates the translation of the mRNAs that encode the translation machinery. These mRNAs are characterized by a 5’ terminal oligopyrimidine (TOP) motif, which is directly bound by the LARP1 DM15 domain to repress their translation in response to mTOR kinase signaling inhibition. Interestingly, lower eukaryotes including the cnidarian Hydra also have LARP1 and TOR kinase signaling but lack TOP mRNAs. What is the target of the LARP1 DM15 region in organisms that lack TOP mRNAs?
The DM15 region is well conserved throughout evolution, especially along its RNA-binding surface. Because of this, we expect the LARP1 targets in Hydra have sequence and structural similarities to the TOP motif. Many Hydra mRNAs undergo a posttranscriptional processing called spliced leader (SL)-based trans-splicing. This phenomenon exists in many lower eukaryotes, in part, to post-transcriptionally resolve polycistronic pre-mRNAs into individual mRNAs. In this process, an SL sequence containing a m2,2,7G-trimethylated (TMG) cap is appended to the 5’ end of an mRNA. Similar to TOP motifs, SL sequences are pyrimidine rich. Therefore, we hypothesize that this TMG-capped SL sequence represents a potential motif for LARP1-directed translation regulation of these transcripts. Preliminary data demonstrates that the LARP1 DM15 region from cnidarian Styllophora pistallata directly and specifically binds an SL sequence, and likely associates with the TMG cap. Interestingly, in a chordate that undergoes SL-based trans-splicing, the translational regulation of SL-associated transcripts is controlled by TOR signaling (Danks, 2019). It has also been shown that in regenerative flatworms, 85% of their neoblast (stem cell) genes are trans-spliced. Neoblast proliferation and differentiation are responsible for the bulk of flatworm regeneration. Therefore, this study aims to elucidate the molecular mechanism by which these transcripts are regulated. Results from this study could also implicate LARP1’s differential binding ability in the regenerative capacities of lower eukaryotes.
Keywords: Trans-splicing, LARP, RBP
41. Structural basis for terminal loop recognition and stimulation of miR-20a processing by hnRNPA1 and hnRNPA2B1
Yaping Liu (University of Michigan), Sarah Keane (University of Michigan)
Abstract not available online - please check the booklet.
42. Structural and Functional Profiling of telomerase RNA domain deletion mutants in Trypanosoma brucei, a deep-branching parasitic agent of neuropathology in mammals
Kaitlin E. Klotz (Department of Biological Sciences-University of North Carolina at Charlotte), Abhishek Dey (Department of Biology-University of North Carolina at Chapel Hill), Arpita Saha (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Sciences, College of Sciences and Health Professions-Cleveland State University), Bibo Li (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Sciences, College of Sciences and Health Professions-Cleveland State University), Kausik Chakrabarti (Department of Biological Sciences-University of North Carolina at Charlotte)
Abstract:
The ribonucleoprotein (RNP) telomerase consists of a telomerase reverse transcriptase (TERT) protein to catalyze the addition of telomeric repeats to the ends of linear chromosomes and a telomerase RNA (TR) which provides the instructions for the addition of telomeric repeats. Recently, we demonstrated that the RNA component of active telomerase complex of the parasite Trypanosoma brucei, the causative agent of African sleeping sickness, adopts two different conformations in its two different life stages in mammals and insects, which might affect stage-specific parasite proliferation rate and telomerase. To better understand which domains of T. brucei RNA are necessary for proper folding of the RNA and telomerase activity, we have created eleven deletion mutants of T. brucei TR, determined the structural folds of six of these RNA mutants by in vivo RNA SHAPE analysis and performed telomerase activity assays to measure the effects of each domain deletion on the ability of telomerase to lengthen T. brucei telomeres. Here, we document how each domain is associated with the structure and function of telomerase and propose some ideas regarding how telomerase function may be modulated through induced mutations to telomerase RNA structural domains. Notably, deletion of an RNA helical structure, distal to the templating domain of the RNA for telomere synthesis and a unique C/D box snoRNA domain, significantly affects RNA biogenesis and activity of T. brucei telomerase. These results provide the first comprehensive nucleotide-resolution analysis of telomerase structure-function in an evolutionarily and clinically important parasitic protist.
References:
Abhishek Dey, Anais Monroy-Eklund, Kaitlin Klotz, Arpita Saha, Justin Davis, Bibo Li, Alain Laederach, Kausik Chakrabarti, In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei, Nucleic Acids Research, Volume 49, Issue 21, 2 December 2021, Pages 12445–12466, https://doi.org/10.1093/nar/gkab1042
Keywords: Telomerase RNA, Structure Dynamics, Trypanosoma brucei
43. Regulatory structural features of pre-miR-31 for Dicer processing
Sicong Ma (Department of Biophysics, University of Michigan), Anita Kotar (Slovenian NMR Centre, National Institute of Chemistry), Sarah C. Keane (Department of Biophysics and Department of Chemistry, University of Michigan)
Abstract:
microRNAs (miRs) are a class of non-coding RNA that post-transcriptionally regulates gene expression. Cleavage by Drosha converts primary microRNAs (pri-miRs) into precursor microRNAs (pre-miRs). Pre-miRNAs are subsequently cleaved by Dicer to generate mature miRs. Although many protein factors are known to regulate Dicer processing, how the intrinsic structure and stability of pre-miR affect Dicer processing are poorly understood, in part due to the limitation of high-resolution tertiary structures of this family of RNAs. Here, we determined the tertiary structure of pre-miR-31 using NMR spectroscopy. We show that an A∙C mismatch within the stem of pre-miR-31 stabilizes pre-miR-31 through the formation of an A+-C base pair near neutral pH. While mismatches within the stem of pre-miR-31 have little impact on Dicer processing, mismatches near the Dicer cleavage site can have significant impacts. We discovered that the presence of a single nucleotide mismatch structure near the cleavage site promotes Dicer processing and 5’ strand cleavage while the larger internal loop structures hinder this processing. Meanwhile, we observed that apical loop size could regulate Dicer processing efficiency bidirectionally, and that loop size needs to be optimized for efficient short hairpin RNA (shRNA) design. Furthermore, we reveal that the top three base pairs of pre-miR-31 are a finely-tuned intrinsic structural feature that helps promote optimal binding and processing by Dicer.
Keywords: pre-miR-31, regulatory structural feature, Dicer processing
44. Title not available online - please see the booklet.
Deepak Shrestha (Chemistry, Wayne State University ), Christine Chow (Chemistry, Wayne State University )
Abstract not available online - please check the booklet.
45. DecoyFinder: Identification of Decoy Sequences in Sets of Homologous RNA Sequences
Mingyi Zhu (Center for RNA Biology, University of Rochester Medical Center; Department of Biochemistry and Biophysics, University of Rochester Medical Center), Jeffrey Zuber (Center for RNA Biology, University of Rochester Medical Center; Department of Biochemistry and Biophysics, University of Rochester Medical Center), Zhen Tan (Center for RNA Biology, University of Rochester Medical Center;Department of Biochemistry and Biophysics, University of Rochester Medical Center), Gaurav Sharma (Department of Electrical and Computer Engineering, University of Rochester;Department of Computer Science,University of Rochester), David H. Mathews (Center for RNA Biology, University of Rochester Medical Center; Department of Biochemistry and Biophysics, University of Rochester Medical Center;Department of Biostatistics and Computational Biology)
Abstract:
Abstract
Motivation: RNA structure plays essential roles for its functions. Using multiple homologous sequences, which share structure and function, secondary structure can be predicted with much higher accuracy than with a single sequence. It can be difficult, however, to establish a set of homologous sequences. We developed a method to identify sequences in a set of putative homologs that are in fact non-homologs.
Results: Previously, we developed TurboFold, to estimate the conserved structure using multiple, unaligned input homologs. Here, we report that TurboFold’s predication accuracy reduces due to the presence of contamination, but the reduction is significant. We developed a method called DecoyFinder, which applies machine learning trained with the feature data from TurboFold, to detect sequences that are not homologous with the other sequences in the set. DecoyFinder can identify approximately 45% of non-homologous sequences, at a rate of 5% misidentification of true homologous sequences.
Availability: DecoyFinder and TurboFold are incorporated in RNAstructure, which is provided free and open source under the GPL V2 license. It can be downloaded at http://rna.urmc.rochester.edu/RNAstructure.html
References:
Mathews, D. H. (2004). Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization. RNA, 10(8), 1178-1190. https://doi.org/10.1261/rna.7650904
Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., & Turner, D. H. (2004). Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A, 101(19), 7287-7292. https://doi.org/10.1073/pnas.0401799101
Tan, Z., Fu, Y., Sharma, G., & Mathews, D. H. (2017). TurboFold II: RNA structural alignment and secondary structure prediction informed by multiple homologs. Nucleic Acids Res, 45(20), 11570-11581. https://doi.org/10.1093/nar/gkx815
Keywords: RNA structure predication , Machine learning, RNA secondary structure
46. Investigating PYM1 function in cellular and flaviviral gene expression
Bayley Carter (Department of Molecular Genetics and Center for RNA Biology, OSU), Manu Sanjeev (Department of Molecular Genetics and Center for RNA Biology, OSU), Lauren Wooodward (Department of Molecular Genetics and Center for RNA Biology, OSU), Robert Patton, Sean Myers (Department of Physics, OSU), Ralf Bundschuh (Department of Physics, OSU and Department of Chemistry and Biochemistry, Division of Hematology), Guramrit Singh (Department of Molecular Genetics and Center for RNA Biology, OSU)
Abstract:
Post-transcriptional regulation of RNAs in the cell is critical to proper expression of the genome. The Exon Junction Complex (EJC) is an RNA binding protein complex that has important role in post-transcriptional regulation of RNAs. An EJC is deposited on an mRNA by the spliceosome ~24 nucleotides upstream of exon-exon junctions (canonical position) to serve as an architectural marker of intron positions. Presence of an EJC on mRNA can enhance its export and its translation. Further, presence of an EJC after a stop codon can mark an mRNA for degradation through a process called nonsense mediated decay (NMD). NMD is an essential pathway in eukaryotic cells that degrades premature termination codon (PTC)-containing mRNAs and also regulates expression of many normal mRNAs. Our unpublished work suggests that PYM1, a ribosome associated protein, may regulate EJC binding at positions away from exon-exon junctions (non-canonical). Loss of EJC-PYM1 interaction leads to a decrease of EJC deposition at -24 nucleotide canonical EJC sites and increased EJC deposition away from exon junctions at non-canonical sites on spliced mRNAs and lncRNAs. Upon loss of PYM1-EJC interaction, non-canonical EJC binding is also greatly increased on intronless RNAs, which do not normally have bound EJCs. This has potential implications for the understanding of gene expression of flaviviruses (e.g., Zika virus, West Nile virus), as PYM1 is reported to interact with flavivirial capsid proteins. This interaction may sequester PYM1 away from its role in regulating non-canonical EJCs, leading to EJC assembly on the intronless flaviviral RNAs and thereby influencing their translation and/or NMD. In this work, we are investigating the molecular basis of PYM1-flaviral capsid protein interaction, the effects of PYM1 depletion on nuclear functions of the EJC, as well as the potential translational effects of non-canonical EJC deposition on intronless RNAs. The proposed study will help us understand the role of the EJC in post-transcriptional regulation of RNAs and provide insights into the mechanism of flaviviral infection.
References:
1. Bono, et al. (2004), “Molecular Insights into the Interaction of PYM with Magoh-Y14 Core of the Exon Junction Complex”, EMBO Reports, vol. 5, No. 3, pp.304-310.
2. Gehring, et al. (2009), “Disassembly of the Exon Junction Complexes by PYM”, Cell 137, pp.536-548.
3. Minghua, et al. (2019), “Identification of antiviral roles for the exon–junction complex and nonsensemediated decay in flaviviral infection”, Nature Microbiology 4, pp.985–995.
4. Tan, et al. (2020), “Capsid protein structure in Zika virus reveals the flavivirus assembly process”. Nature Commun 11, 895.
Keywords: Exon Junction Complex , PYM1, Flavivirus
47. Structural and functional analysis of Crassostrea gigas OAZ-PK RNA
Rhiannon McCracken (Department of Chemistry and Biochemistry, Creighton University), Spencer Thompson (Department of Chemistry and Biochemistry, Creighton University), Siddharth Venkatraman (Department of Chemistry and Biochemistry, Creighton University), Garrett Soukup (Department of Biomedical Sciences, Creighton University), Juliane Soukup (Department of Chemistry and Biochemistry, Creighton University)
Abstract not available online - please check the booklet.
48. Development of a chemo-transcriptomic platform for identifying RNA-binding small molecules
Jose A. Reyes Franceschi (Program in Chemical Biology, University of Michigan ), Emilio L. Cardenas, PhD (Department of Medicinal Chemistry, University of Michigan ), Prof. Amanda L. Garner, PhD (Department of Medicinal Chemistry, University of Michigan )
Abstract:
Dysregulation of RNA functions has been implicated in multiple human pathologies, such as bacterial and viral infections, as well as playing a role in other diseases including cancers. Because of RNAs’ involvement in diseases, the transcriptome has become an area of interest in the field of drug discovery. Current efforts for targeting RNA with small molecules has yielded therapeutics that treat bacterial infections and cancer; however, these target a small number of RNA molecules meaning that this field is still vastly underexplored. One big reason for the lack of RNA-targeting therapeutics is the lack of technologies for accurately screening RNA-binding small molecules in cells. This is a major issue since RNA is a very dynamic biomolecule whose structure depends upon its surrounding environment. Therefore, it is important to develop new approaches that allow us to screen for RNA-binding small molecules in living cells. In this project, we explore the synthesis and biological characterization of novel chemical probes that can help elucidate small molecule-RNA interactions in cells. This probe will consist of a small molecule fragment of interest linked to an RNA-selective covalent warhead that will react with the RNA to enable its covalent modification, and subsequent identification via affinity purification and sequencing. Efforts toward the development of this approach will be discussed.
Keywords: RNA, Small Molecule, Chemical Probe
49. KREH1 RNA helicase activity promotes utilization of initiator gRNAs across multiple mRNAs in trypanosome RNA editing
ASHUTOSH P DUBEY (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Brianna L. Tylec (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Amartya Mishra (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Runpu Chen (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Yijun Sun (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Laurie K. Read (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA)
Abstract not available online - please check the booklet.
50. Aminoacyl-tRNA synthetase complex-interacting multifunctional protein 3 (AIMP3) is essential for physiological function of heart
Anindhya S. Das (Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus 43210, USA), Charles P. Rabolli (Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus 43210, USA), Colton R. Martens (Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus 43210, USA), Federica Accornero (Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University)
Abstract:
In eukaryotes, tRNA multi-synthetase complex (MSC) has been proposed to evolve to facilitate protein synthesis by efficiently providing charged tRNAs to the translating ribosome. The human MSC consists of 8 aminoacyl-tRNA synthetases (aaRS) and 3 non-enzymatic proteins known as aminoacyl-tRNA synthetase complex-interacting multifunctional proteins (AIMP1, 2, and 3). Aside from their role in translation, these multifunctional proteins are involved in other biological processes such as inflammation, apoptosis, and DNA damage response. However, their functions in specialized cells such as cardiomyocytes (CM) have not been studied. Here, we report that CM-specific conditional knockout (cKO) of AIMP3 in mice induces severe fibrosis and leads to immune cell infiltration concomitant with pathological cardiac remodeling. AIMP3 cKO mice show significant impairment of cardiac function, which ultimately progresses to develop heart failure within 1 month of deletion. The global protein synthesis is unperturbed in CMs from cKO hearts, suggesting that AIMP3 plays a critical translation-independent role in maintaining physiological cardiac functions and homeostasis. Currently, we are probing into the key cellular defects and their molecular mechanisms in cKO hearts that might help us to understand the CM-specific functions of AIMP3 with a therapeutic potential for the treatment of heart failure.
Keywords: tRNA multi-synthetase complex, AIMP3, Heart failure
51. Evaluation of isosteric substitutions of non-Watson-Crick basepairs in recurrent RNA 3D motifs
Emil F. Khisamutdinov (Department of Chemistry, Ball State University)
Abstract not available online - please check the booklet.
52. Zinc-dependent regulation of sod1 mRNA transcript levels.
Amdrew T. Weeks (Department of Human Nutrition,The Ohio State University), Derek M. Boehm (Department of Molecular Genetics, The Ohio State University), Amanda J. Bird (Department of Human Nutrition, Department of Molecular Genetics, The Ohio State University)
Abstract not available online - please check the booklet.
53. Super resolution nanoscopy of Trypanosoma brucei RNA binding protein 42 during acute infection in mice
Vasilios Orologas (Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers- New Jersey Medical School), Kezia Lizardo (Hackensack Meridian Health), Luke Fritzky (Rutgers- New Jersey Medical School ), Vivian Bellofatto (Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers- New Jersey Medical School)
Abstract:
RNA binding proteins (RBPs) are intricately involved in mRNA dynamics, including intracellular localization-based stability, and as reversible mediators of mRNA fate during cell stress. The complex life cycle, and polycistronic gene expression of Trypanosoma brucei, the African Trypanosome requires an extensive use of RBP-based regulatory machineries that aid parasite transition from insect vector to mammalian host. In the mammalian host, the parasite occupies and adapts to a variety of tissue microenvironments to generate peak parasitemia at day 6 post-infection. RBP42 is a polysome-associated, essential RNA binding protein, acting as a post-translational regulator of mRNA-encoding enzymes involved in T. brucei Central Carbon Metabolism. Our hypothesis is that the internal localization of RBP42 in T. brucei fluctuates to allow the parasites to derive nutrients from multiple host environments. To test this hypothesis, we are employing Leica Stellaris 8 tau-STED Super Resolution Microscopy, and experimental immunohistochemical staining to visualize the sub-cellular localization of RBP42 in parasites infecting lung, kidney, heart, pancreas, and adipose tissue in the mouse model. We will show Super Resolution images of parasites stained with anti-Variant Surface Glycoprotein antibodies (VSG), anti-RBP42 antibodies, and PicoGreen (nuclear and kinetoplast DNA stain) to reveal detailed images of parasites during acute infection.
Keywords: RNA binding proteins , STED microscopy , Trypanosoma brucei
54. Characterization of pug1/gap2 double knockout in the human fungal pathogen C. albicans
Evan Spell (Department of Biology, Ball State University), Dr. Douglas Bernstein (Department of Biology, Ball State University)
Abstract:
Pseudouridine is the most common RNA modification, however its function is not well understood. Psuedouridine is broken down in many organisms by pseudouridine glycosidases, yet in humans breakdown is absent. Pseudouridine glucosidases have not been characterized in any eukaryotes. To characterize the role of psuedouridine glycosidase (PUG1) it was knocked out in Candia albicans, a eukaryotic model organism and human fungal pathogen. When the gene coding for psuedouridine glycosidase was deleted, a number of genes were up and down regulated. One of these genes GAP2, an amino acid permease, was upregulated in the absence of PUG1. The purpose of this study is to characterize the role of these genes that interact with PUG1, by deleting them, and characterizing associated phenotypes. We first generated CRISPR-Cas9 mediated mutagenesis vectors that targeted GAP2. These plasmids were then transformed into the C. albicans pug1∆ along with a repair template that introduced a stop codon in the GAP2 open reading frame. This process results in a pug1/gap2 double knockout. We are now in the process of creating additional knockout strains, including a GAP2 single deletion. These strains will then be used in comparative experiments that will characterize viability and virulence of the pug1/gap2 double knockout.
References:
Kabir, M Anaul et al. Candida albicans: A Model Organism for Studying Fungal Pathogens. ISRN microbiology vol. 2012 538694. 29 Sep. 2012
Spenkuch, F., Y. Motorin, and M. Helm, Pseudouridine: still mysterious, but never a fake (uridine)! RNA Biol, 2014. 11(12): p. 1540-54.
Huang, S., et al., Pseudouridine monophosphate glycosidase: a new glycosidase mechanism. Biochemistry, 2012. 51(45): p. 9245-55.
Seidel, A., et al., Modified nucleosides: an accurate tumour marker for clinical diagnosis of cancer, early detection and therapy control. Br J Cancer, 2006. 94(11): p. 1726-33.
Sun T, Bi F, Liu Z, Yang Q. SLC7A2 serves as a potential biomarker and therapeutic target for ovarian cancer. Aging (Albany NY). 2020 Jul 9;12(13):13281-13296. doi: 10.18632/aging.103433. Epub 2020 Jul 9. PMID: 32647070;
Keywords: Knockout, C albicans
55. Recognition of RNA duplexes by the tRNAHis guanylyltransferase
Jarrett Wilson (The Ohio State University), Malithi Jayasinghe (The Ohio State University), BrianSmith (The Ohio State University), Maria Abad (The Ohio State University), Jane E. Jackman (The Ohio State University)
Abstract:
tRNAs are fundamental to the central dogma of biology, as they have a long-established role in protein synthesis. Known as the most highly modified RNA molecule, we lack a complete understanding of the many enzymes that have a role in their post-transcriptional maturation. tRNAHis guanylyltransferase (Thg1) is a part of a large family of enzymes that play an important role in the tRNA maturation process. Thg1 has the unique ability to synthesize RNA in the 3’-5’ direction, catalyzing the non-templated addition of a G-1 nucleotide to the 5’ end of tRNAHis in eukaryotes. Thg1s ability to polymerize in the 3'-5' direction is the opposite canonical RNA polymerases. G-1 is an essential identity element for histidyl-tRNA synthetase (HisRS) to recognize tRNAHis from other tRNAs. Recent studies show that despite its high specificity for its known biological substrate (tRNAHis), Thg1 also binds in vitro to varying RNA duplexes with even higher affinity than for tRNA. This raises questions about the molecular basis for Thg1 recognition of these small RNA duplexes that lack the typical structural features of a tRNA. Previous assays identified highly conserved residues in human Thg1 that reduce tRNA binding, including a catalytically important Glu that exhibits the most significant reduction in affinity for tRNAHis when altered to alanine. In this research, I probe whether yeast and human Thg1 utilize the same essential conserved residue to recognize the RNA duplexes as is used to bind to tRNAHis. Filter binding assays are used to measure RNA duplex binding to WT and mutant versions of yeast and human Thg1, providing important insight into Thg1 RNA recognition properties that are currently unknown.
References:
"Mechanistic insights into a reverse polymerase",Brian Smith, PhD Thesis
"Exploration of broader substrate specificity, applications, and mechanism of tRNAHis guanylyltransferase-like proteins (TLPs)",Malithi Jayasinghe, PhD Thesis
Jane E. Jackman and Eric M. Phizicky 2008
Maria Abad et al. 2011
Keywords: Thg1, tRNAHis
56. Proximity labeling to investigate protein dynamics mediating nonsense-mediated mRNA decay in yeast
Sarah L.H. Nock (Dept of Genetics & Genome Sciences, Case Western Reserve University), Romeo Gallego (Dept of Genetics & Genome Sciences, Case Western Reserve University; Bridges to the Baccalaureate Research Training Program at Cuyahoga Community College, Cleveland, Ohio), DaJuan L. Whiteside (Dept of Genetics & Genome Sciences, Case Western Reserve University), Vishwum Kapadia (Youth Engaged in Science/Summer Enrichment and Opportunities Program, Case Western Reserve University), Kristian E. Baker (Dept of Genetics & Genome Sciences, Case Western Reserve University)
Abstract not available online - please check the booklet.
57. Improved long non-coding RNA annotation and detection in human transcriptomic profiles
Phillip J. McCown (Department of Internal Medicine, University of Michigan), Felix Eichinger (Department of Internal Medicine, University of Michigan), Matthew Manninen (Department of Internal Medicine, University of Michigan), Fadhl Alakwaa (Department of Internal Medicine, University of Michigan), Damian Fermin, Abhijit S. Naik, Sean Eddy (Department of Internal Medicine, University of Michigan), Matthias Kretzler (Department of Internal Medicine, University of Michigan)
Abstract:
The importance of long non-coding (lnc) RNA involvement in biology is increasingly recognized. By remapping transcriptomic datasets from human kidneys, we can identify lncRNAs involved in disease processes with cell type selectivity. To accomplish this, we created the lEG (lncRNAKB-Ensembl GTF), a non-redundant, modified Gene Transfer File (GTF) that incorporates lncRNAs from the lncRNAKB and Ensembl. The lEG was then applied to bulk RNA-seq and single cell RNA-seq (scRNA-seq) analyses and processing pipelines.
Keywords: lncRNA, Single cell RNA-seq, Kidney
58. Differential m5C in embryonic mRNA reveals methyltransferase specificity
Taylor N. Ayers, (University of Pittsburgh, Department of Biological Sciences), Wesley A. Phelps (University of Pittsburgh, Department of Biological Sciences), Siddharth V. Mahesh (University of Pittsburgh, Department of Biological Sciences), Hannah E. Moore (University of Pittsburgh, Department of Biological Sciences), Joel C. Rosenbaum (University of Pittsburgh, Department of Biological Sciences), Anne E. Carlson, Miler T. Lee (University of Pittsburgh, Department of Biological Sciences)
Abstract not available online - please check the booklet.
59. Investigating the formation and function of 2’-O-methylation in the anticodon loop of tRNAPhe
Samuel D. Seibert (Chemistry & Biochemistry; Northern Kentucky University), Thao Linh Le (Chemistry & Biochemistry; Northern Kentucky University), Holly M. Funk (Chemistry & Biochemistry; Northern Kentucky University), Michael P. Guy (Chemistry & Biochemistry; Northern Kentucky University)
Abstract not available online - please check the booklet.
60. Spectra of ribonucleosides in hyperglycemic mammalian cells and diabetic murine cardiac models
Chase N. Morse (Pharmacy and Pharmaceutical Sciences, Department of Medicinal and Biological Chemistry, University of Toledo), Taylor A. Dodson (Pharmacy and Pharmaceutical Sciences, Department of Medicinal and Biological Chemistry, University of Toledo), Stephen Nieuwoudt, Chao Liu, Valinteshley Pierre (Department of Biomedical Engineering, Case Western Reserve University), Samuel E. Senyo (Department of Biomedical Engineering, Case Western Reserve University), Erin G. Prestwich (Pharmacy and Pharmaceutical Sciences, Department of Medicinal and Biological Chemistry, University of Toledo)
Abstract not available online - please check the booklet.
61. A non-canonical RNA-binding domain of the fragile X protein, FMRP, elicits translational repression independent of mRNA G-quadruplexes
MaKenzie R. Scarpitti (1,2), Julia E. Warrick (1,2), Evelyn L. Yoder (1,2), Michael G. Kearse (1. Department of Biological Chemistry and Pharmacology, 2. Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract:
Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome, the leading form of inherited intellectual disability and the most common monogenic cause of autism spectrum disorders. FMRP is an RNA-binding protein that controls neuronal mRNA localization and translation. FMRP is thought to inhibit translation elongation after being recruited to target transcripts via binding RNA G-quadruplexes (G4s) within the coding sequence. Here, we directly test this model and report that FMRP inhibits translation independent of mRNA G4s. Furthermore, we found that the RGG box motif together with its natural C-terminal domain forms a non-canonical RNA-binding domain (ncRBD) that is essential for translational repression. The ncRBD elicits broad RNA binding ability and binds to multiple reporter mRNAs and all four homopolymeric RNAs. Serial deletion analysis of the ncRBD identified that the regions required for mRNA-binding and translational repression overlap but are not identical. Consistent with FMRP stalling elongating ribosomes and causing the accumulation of slowed 80S ribosomes, transcripts bound by FMRP via the ncRBD co-sediment with heavier polysomes and were present in puromycin-resistant ribosome complexes. Using RNA-seq to probe steady state mRNA levels, we also found that FMRP-mediated ribosome stalling controls mRNA stability by triggering the No-Go Decay (NGD) pathway. Using RNA-seq and stringent statistical cut-offs, we identified that 16 direct FMRP targets are putative NGD substrates caused by FMRP-mediated stalling. Biochemical analysis of FMRP-stalled ribosomes to validate interaction with NGD pathway factors is currently ongoing. Nevertheless, these data are among the first to demonstrate FMRP directly regulates target mRNA levels post-transcriptionally. Together, this work identifies a ncRBD and translational repression domain that shifts our understanding of how FMRP inhibits translation independent of mRNA G4s.
Keywords: RNA binding protein, Translation elongation, G-quadruplexes
62. New insights into the alternative translation initiation factor eIF2A
Daisy J. DiVita (Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology), Michael G. Kearse (Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology)
Abstract:
Canonical eukaryotic mRNA translation uses the heterotrimeric eukaryotic initiation factor 2 (eIF2) to deliver the initiator tRNA. However, eIF2A (not to be confused with the alpha subunit of eIF2) was the first initiator tRNA carrier discovered in eukaryotes, but further insights into its molecular mechanism dwindled once eIF2 was identified as the primary initiator tRNA carrier. The exact role of eIF2A in translation still remains a mystery despite it having nearly identical affinity for initiator tRNA and being present at equal concentrations in cells as eIF2. Recent work has shown that eIF2A is required for cancer progression and proper long-term lipid metabolism in mice. Atypical of a translation factor, eIF2A primarily localizes to the nucleus, but can shuttle to the cytoplasm during cell stress. How and why eIF2A is kept away from the translation machinery is unclear but it may be a mechanism for cells to only use eIF2A in specific conditions. To answer these questions, we first supplemented mammalian in vitro translation extracts with recombinant human eIF2A and found that excess recombinant eIF2A inhibits mRNA translation. To decipher which step of translation is inhibited, we used sucrose gradient ultracentrifugation along with various translation inhibitors to capture and measure the levels of translation complexes at different stages in vitro. Using the non-hydrolyzable GTP analog GMPPNP to capture 48S initiation complexes, we identified that excess eIF2A reduces the abundance of small subunits at start codons. Excess eIF2A also inhibited translation directed by all four types of IRESs, including those that do not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters the 40S subunit. Importantly, reactions supplemented with additional 40S subunits rescued translation and pull-down assays show evidence of direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering the 40S ribosomal subunit.
Keywords: translational control, ribosome, initiation factors
63. Determining the Influence of PUS7 localization on RNA Substrate Selection
Minli Ruan (Biological Chemistry, University of Michigan), Daniel E. Eyler (Chemical Biology, University of Michigan), Kristin S. Koutmou (Chemical Biology, University of Michigan)
Abstract:
Pseudouridine (Ψ), one of the most abundant post-transcriptional RNA modifications in cells, broadly affects RNA structure and function. The insertion of Ψ into RNAs (tRNA, snRNA, rRNA, mRNA) is catalyzed by Pseudouridine Synthase (Pus) enzymes, which can be categorized into six families: TruA, TruB, RluA, RsuA, TruD, and Pus10. Pus enzymes are important for gene expression, as the misregulation of Ψ incorporation is associated with a variety of human diseases and cancers.1-3 For example, mutants of the TruD family member, Pus7, are linked with several inherited diseases, including speech delay, intellectual disability, and microcephaly. Recently we have begun to gain insight onto the dynamic regulation of Pus7, but how it selects its specific substrates is still poorly understood. Elucidating the mechanism of Pus7 substrate selection is important for understanding pathologies linked to Pus7 dysfunction. Pus7 recognizes RNA substrates that possess a consensus UGUAR (R = A/G) sequence. However, transcriptome-wide sequencing demonstrates that < 3% of RNAs containing this consensus sequence are pseudouridylated in cells under normal-growth conditions. However, the fraction of UGUAR sequences modified under heat shock conditions, where Pus7 relocaliezes from the nucleus into the cytoplasm, increases to more than 20%. These data indicate that the consensus sequence alone cannot explain Pus7 substrate selection and further mechanistic investigations, such as enzyme relocalization, are warranted. My work investigates the possibility that Pus7 localization impacts RNA substrate selection to influence protein synthesis in response to specific cellular stresses. I am developing Pus7 constructs capable of being localized in either the nucleus or cytoplasm and by mapping pseudouridylation dynamic changes together with translation efficiency changes in yeast to determine this molecular mechanism.
References:
1. Eyler, D. et al. (2019). Proceedings Of The National Academy Of Sciences, 116(46), 23068-23074.
2. Carlile, T. et al.(2014). Nature, 515(7525), 143-146.
3. Garcia, D. et al. (2021). Elife, 10.
4. Purchal, M. et al. (2022). Proceedings Of The National Academy Of Sciences, 119(4).
5. de Brouwer, A. et al. (2018). The American Journal Of Human Genetics, 103(6), 1045-1052.
6. Carlile, T. et al.(2019). Nature Chemical Biology, 15(10), 966-974.
7. Schwartz, S. et al. (2014). Cell, 159(1), 148-162.
Keywords: Pseudouridine Synthase 7 (Pus7), Pseudouridine, Localization
64. Mutate2test: New Algorithm and Software for Mutational Design
Sara E. Ali (Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester), Yan Sun (Department of Microbiology & Immunology, University of Rochester), Ela Kierzek (Institute of Bioorganic Chemistry Polish Academy of Sciences), Ryszard Kierzek (Institute of Bioorganic Chemistry Polish Academy of Sciences), David H. Mathews (Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester)
Abstract:
It is common that a hypothesized RNA secondary structure needs to be tested. A traditional method is the site-directed mutation of a sequence to abolish function, followed by restoration of the structure with compensating changes. This approach, however, is complicated by the need to carefully design the mutants, which can be especially difficult when the sequence falls within an open reading frame. It is essential to maintain the amino acid identity so that the structure is the only changing variable in the test. Manual mutation design can be complicated, nonoptimal and time-consuming.
The goal of this study is to write new software to automate the design of sequences to test putative RNA structures. We created a new program, mutate2test, that, given an RNA secondary structure, designs mutants to abolish the structure and designs the set of compensating mutations to restore the structure. The program iterates over all possible disrupting and restoring mutations within an open reading frame that maintain the amino acid identity of the input sequence. The objective function used to accept or reject mutations is based on Ensemble Defect (ED). For a candidate sequence and a given target secondary structure, the ensemble defect is the average number of incorrectly paired nucleotides at equilibrium evaluated over the ensemble of secondary structures. Thus, it is used to quantify the level of disruption or restoration for a sequence given a model structure. It is key that the sequence restoring the original structure by compensating mutations have low ensemble defect to the hypothesized structure, the loss of function mutants have high average ensemble defect and the total number of mutations be minimized to facilitate the experiments. The mutations are then ranked to find the most optimal disrupting and restoring mutations. We are benchmarking mutate2test by designing mutants for a conserved RNA secondary structure model we developed in respiratory syncytial virus (RSV).
Keywords: RNA secondary structure, mutation design, open reading frame (ORF)
65. Knockout of three U5 snRNA sequence variants in 293T cells alters splicing and expression of an overlapping set of genes
Justin W. Mabin (Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health), Peter W. Lewis (Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health), David A. Brow (Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health)
Abstract not available online - please check the booklet.
66. Inosine mRNA modifications impact translation elongation
Rachel N. Giles (Chemistry, University of Michigan), Kristin S. Koutmou (Chemistry, University of Michigan)
Abstract not available online - please check the booklet.
67. Title not available online - please see the booklet.
Gabrielle Caito (Department of Chemistry and Biochemistry, The Ohio State University), Isobel Bowles (Department of Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the booklet.
68. Analysis of photo-oxidation induced products of post-transcriptional modifications in tRNA using LC-MS/MS technique
Tulsi Bhandari (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)
Abstract:
Translation function is highly dependent on the post-transcriptional modifications (PTM) on tRNA molecule. PTM play crucial role in maintaining the structure, stability, and function of tRNA molecules. Absence or aberrant presence of PTM at different position in RNA has been linked with diseases including cancer. It has been proved that UVA rays as exogenous source of reactive oxygen species affected PTMs in tRNA molecules causing oxidative degradation of PTMs. Literature tends to serve the knowledge that ribonucleosides undergo photo reaction to give photoproducts, it is not well studied about the conversion products of all the PTMs that degrades after UVA exposure. By using Neutral Loss Scan (NLS) technique of mass-spectrometry, we unearthed photoproducts of some modified ribonucleosides produced after photosensitizer induced UV damage, and with the tools like stable isotope labelling, high mass accuracy LC-MS/MS, and spectral matching we did the structural characterization. We intend to probe into biological significance of such photoproducts detected.
Keywords: post-transcriptional modifications, tRNA, UVA rays, oxidative degradation, Neutral Loss Scan, spectral matching, LC-MSMS
69. FRET-PAINT for Studying Long Telomeric DNA Overhangs and Their Interactions with Shelterin Proteins
Sajad Shiekh (Department of Physics, Kent State University, Kent Ohio), Golam Mustafa, Sineth G Kodikara, Eric Yokie, Prabesh Gyawali (Department of Physics, Kent State University, Kent Ohio), John J. Portmana, Hamza Balcia (Department of Physics, Kent State University, Kent Ohio), Mohammed Enamul Hoque (Department of Chemistry and Biochemistry, Kent State University, Kent Ohio), Amanda Jack, Ahmet Yildiz (Department of Molecular and Cell Biology, University of California, Berkeley, Physics Department, University of California, Berkeley)
Abstract:
I will be presenting the single molecule FRET-PAINT measurements and computational modeling studies where we investigated the accessibility of a range of different human telomeric overhangs. These overhangs can form 1-7 tandem G-quadruplex (GQ) structures, which, to our knowledge, is the most comprehensive range studied to date and covers a significant portion of the physiologically relevant telomeric overhang length scale. Our measurements demonstrate novel accessibility maps where certain regions of the telomeric overhang are significantly more accessible than others. We also observe folding frustration patterns with a well-defined periodicity and constructs with a certain number of telomeric repeats demonstrate elevated levels of frustration compared to others. These patterns have significant implications for telomere organization, protection of free 3’-end against exonuclease activity and telomerase-catalyzed extension, and folding cooperativity between neighboring GQ structures. We further investigated the impact of POT1 and a four-protein Shelterin complex on the accessibility of human telomeric DNA constructs with physiologically relevant overhang lengths (28-150 nt). To quantify telomere accessibility, we monitored transient binding events of a Cy5-labeled, short peptide nucleic acid strand to available sites on the telomere using the FRET-PAINT methodology. We observed approximately 2.5-fold reduced accessibility in the presence of POT1 (compared to DNA-only case) and about 5-fold reduced accessibility in the presence of Shelterin. These results suggest the protection of exposed telomeric segments by POT1 was effective enough to dominate over its mild GQ unfolding activity. The more effective protection in the presence of Shelterin compared to the POT1-only case suggests the restructuring of the junction region between single and double-stranded telomere, which is otherwise the most accessible part of the overhang, serves an important function in telomere maintenance.
References:
1. Sajad Shiekh, G Mustafa, Mohammed Enamul Hoque, Eric Yokie, John J. Portman, H Balci."Emerging Accessibility Patterns in Long Telomeric Overhangs"(https://doi.org/10.1073/pnas.2202317119)
1. G Mustafa, S Shiekh, K Gc, S Abeysirigunawardena, H Balci. "Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction" NAR, Vol 49, Issue 6, Pages 3371–3380
Keywords: FRET-PAINT, G-quadruplexes, Telomere
70. Title not available online - please see the booklet.
Abdul McKinney (Genetics and Genome Sciences, Case Western Reserve Uneversity), Dr. Hua Lou (Genetics and Genome Sciences, Case Western Reserve Uneversity), Jenell Betts (Genetics and Genome Sciences, Case Western Reserve Uneversity)
Abstract not available online - please check the booklet.
71. Modulating insulin receptor alternative splicing for therapeutic benefit in Ewing sarcoma
Akila S. Venkataramany (Medical Scientist Training Program and Biomedical Sciences Training Program, OSUCOM; Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Safiya Khurshid, PhD (Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Pin-Yi Wang, PhD (Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Timothy P. Cripe, MD PhD (Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital; Div. of Hematology, Oncology and Blood and Marrow Transplant, Department of Pediatrics, OSUCOM), Dawn S. Chandler, PhD (Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital; Department of Pediatrics, OSUCOM; Molecular, Cellular and Developmental Biology Graduate Program, OSU)
Abstract not available online - please check the booklet.
72. Investigating molecular interactions with 3’-5’ RNA polymerases in Dictyostelium Discoideum
Madison Allegretti (The Ohio State University Chemistry and Biochemistry), Brandon Iwaniec (The Ohio State University Biochemistry Program), Jane E. Jackman (The Ohio State University Chemistry and Biochemistry)
Abstract:
The Thg1/TLP family of enzymes catalyze a number of diverse 3’-5’ nucleotide addition reactions that are involved in processes such as tRNA-His maturation and 5’ end repair of tRNAs. While Thg1 has a strong preference to add a G-1 nucleotide to the 5’ end of tRNA-His in a non-Watson-Crick pair, TLPs, or Thg1-like-proteins, kinetically prefer to repair truncated RNA substrates in a Watson-Crick dependent manner and are able to act upon a variety of tRNAs beyond tRNA-His. The eukaryotic slime mold Dictyostelium discoideum encodes four enzymes from the Thg1/TLP family: DdiThg1, DdiTLP2, DdiTLP3, and DdiTLP4. Previous work has revealed distinct biological functions for three out of the four DdiTLPs. Because of the complexity of tRNA editing and the diversity of the reactions catalyzed by the different TLP enzymes expressed in Dictyostelium, we hypothesize that these enzymes may interact with one or more cellular macromolecules in order to fulfill their respective biological functions. In this investigation we are focusing on DdiTLP3, which is targeted to the mitochondria of the cell where it repairs the 5’ end of mitochondrial tRNAs, and DdiTLP4, whose true in vivo function remains unclear but has demonstrated editing activity on a variety of RNA substrates. By encoding a FLAG epitope tag to a terminus of these Ddi TLPs, interacting proteins that participate in the reactions catalyzed by the TLPs in D. discoideum cells can be immunoprecipitated and identified. This approach will uncover unknown mechanistic details of the reactions that these 3’-5’ polymerases are involved in and will help us further distinguish the roles that each enzyme plays in the cell. By further understanding the interactions that occur between DdiTLP3 and DdiTLP4 and other candidate molecules, we can understand the exact roles these enzymes play in Dictyostelium and possibly apply our findings to the roles of TLPs in other eukaryotes where their function has not yet been demonstrated.
References:
Abad MG, Long Y, Willcox A, Gott JM, Gray MW, Jackman JE. A role for tRNAHis guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5′-tRNA editing. RNA. 2011;17(4):613-623. doi:10.1261/rna.2517111
Jackman JE, Gott JM, Gray MW. Doing it in reverse: 3′-to-5′ polymerization by the Thg1 superfamily. RNA. 2012;18(5):886-899. doi:10.1261/rna.032300.112
Abad MG, Rao BS, Jackman JE. Template-dependent 3′–5′ nucleotide addition is a shared feature of tRNAHis guanylyltransferase enzymes from multiple domains of life. Proc Natl Acad Sci U S A. 2010;107(2):674-679. doi:10.1073/pnas.0910961107
Long Y, Abad MG, Olson ED, Carrillo EY, Jackman JE. Identification of distinct biological functions for four 3′-5′ RNA polymerases. Nucleic Acids Research. 2016;44(17):8395-8406. doi:10.1093/nar/gkw681
Keywords: tRNA editing, 3-5 polymerase
73. A crucial role for the leader-trailer helix and minor role for the antitermination complex in biogenesis of the 30S ribosomal subunit
Benjamin Warner (Department of Microbiology, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University), Kurt Fredrick (Department of Microbiology, The Ohio State University)
Abstract:
Ribosome biogenesis occurs co-transcriptionally and entails ribosomal (r) RNA folding, r protein binding, rRNA processing, and rRNA modification. In most bacteria, rRNAs are transcribed with tRNAs, and the primary transcript contains leader, trailer, and spacer sequences. Transcription of these long operons involves a modified RNA polymerase, called the antitermination complex, which is recruited by cis-acting RNA elements (boxB, boxA, and boxC) in the leader and spacer regions of the operon. Sequences flanking the rRNAs are complementary and form long helices known as leader-trailer helices. In this work, we interrogate the functional roles of these RNA elements in 30S subunit biogenesis. We find that the structure of the leader-trailer helix is absolutely essential, although its primary sequence can vary. By contrast, loss of boxA only modestly impacts 30S production, indicating a relatively minor role for the antitermination complex. Two adjacent leader helices, termed here hA and hB, are also largely dispensable, consistent with the variable presence of these elements across the Enterobacteriaceae. The fact that the leader-trailer helix is crucial for 30S biogenesis in vivo but unnecessary for efficient reconstitution of the 30S subunits in vitro implies important differences between the two processes.
Keywords: ribosome biogenesis, RNA structure, precursor RNA
74. Characterization of alternative polyadenylation in roots of red clover
Caleb Gooden (Plant and Soil Sciences, University of Kentucky ), Randy Dinkins (Plant and Soil Sciences, University of Kentucky), Arthur Hunt (Plant and Soil Sciences, University of Kentucky )
Abstract:
Root nodulation is a process recognized among legumes in which host roots are infected with a soil bacterium and form nodules that better fix nitrogen from the atmosphere. In agriculture, nodules help to reduce the need for fertilizer and are thus part of sustainable practice. Studies in Medicago truncatula have shown the process of root nodulation to be suppressed by the expression of a regulatory RNA (TAS3). Over-expression of TAS3 and the miR390 pathway with which it interacts resulted in prioritization of lateral root formation over nodulation. Thus, TAS3 is an attractive target for manipulation to improve nitrogen fixation in legumes and enhance their agronomic value.
In Arabidopsis, TAS3a transcripts may end at one of two alternative polyadenylation sites, the choice of which determines whether functional or non-functional TAS3a RNAs are made. We have confirmed through 3’ RACE and bioinformatic analysis that two poly(A) sites are present in the TAS3 region of Trifolium pratense. We also showed that a miR390 site is present in between these two sites through BLAST. We hypothesize that differential use of these sites will determine the production of either lateral roots or nodules, similar to the module shown in Medicago and Arabidopsis, and that use of the proximal site (which produces non-functional TAS3) will be responsible for nodule formation.
Current research focuses on measuring the differential use of these sites through Poly(A) Tag Sequencing (PATseq) and bioinformatic mapping. It is our hypothesis that red clover plants introduced to rhizobia, and therefore exhibiting nodulation, will contain higher numbers of reads at the proximal poly(A) site indicating the expression of the short TAS3 isoform. Conversely, we hypothesize wild-type clover will show higher numbers of reads at the distal poly(A) site indicating expression of full-length, functional TAS3 and production of lateral roots as a result. Control samples of Medicago roots and nodules will be included for comparison.
Keywords: Alternative polyadenylation, red clover, microRNA
75. Title not available online - please see the booklet.
Asif Rayhan (Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Chemistry, University of Cincinnati), Patrick A. Limbach (Chemistry, University of Cincinnati)
Abstract not available online - please check the booklet.
76. Role of the sarcin-ricin loop (SRL) of 23S rRNA in assembly of the 50S ribosomal subunit
Sepideh Fakhretaha Aval (Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA), 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.), 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 and 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 of SRL remains unclear. Lancaster et al. (2008) showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in E. coli. These ΔSRL ribosomes were purified from cells and 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. These reconstituted ΔSRL particles exhibit full activity, in contrast to the mutant ΔSRL subunits from which the rRNA came. These data suggest that the assembly defect conferred by ΔSRL is specific to 50S biogenesis in the cell. During the assembly of large subunit, the SRL folds early on as part of block 1. We hypothesize that one or more trGTPases then transiently bind via the SRL and facilitate subsequent folding events.
References:
Moazed D, Robertson JM, Noller HF. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature. 1988;334(6180):362-364. doi:10.1038/334362a0
García-Ortega L, Alvarez-García E, Gavilanes JG, Martínez-del-Pozo A, Joseph S. Cleavage of the sarcin-ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding. Nucleic Acids Res. 2010;38(12):4108-4119. doi:10.1093/nar/gkq151
Lancaster L, Lambert NJ, Maklan EJ, Horan LH, Noller HF. The sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit. RNA. 2008;14(10):1999-2012. doi:10.1261/rna.1202108
Davis JH, Williamson JR. Structure and dynamics of bacterial ribosome biogenesis. Philos Trans R Soc Lond B Biol Sci. 2017;372(1716):20160181. doi:10.1098/rstb.2016.0181
Keywords: Ribosome biogenesis, Sarcin-ricin loop, Ribosomal RNA
77. Biochemical and structural analysis of Pus4 mRNA target selection
Amelia Cochran (Chemistry, University of Michigan), Markos Koutmos (Chemistry, University of Michigan), Kristin S. Koutmou (Chemistry, University of Michigan)
Abstract:
Pseudouridine synthase (Pus) enzymes catalyze an isomerization reaction that converts a uridine to a pseudouridine (Ψ), impacting the stability and structure of the modified RNA. Originally characterized as modifiers of tRNA and non-coding RNAs, it is now known that these enzymes also modify mRNA. Pus4, a member of the Trub family, was originally identified as the enzyme responsible for the formation of Ψ55 in most tRNAs. Recent studies have shown that Pus4 is one of the Pus enzymes (along with Pus1 and Pus7) to have many mRNA targets.1 mRNA modifications are likely to impact processing, translation, and degradation of the mRNA molecule, making Pus4 a potential regulator of gene expression. Understanding how Pus4 selects its mRNA targets will be essential in deconvoluting the role of pseudouridine synthases in gene expression regulation. Recent results from a study of Pus7,2 as well as previous characterization of TruB as a tRNA chaperone,3 suggest that Pus enzymes can bind to RNAs with structures and sequences different from their tRNA and ncRNA targets. This has led us to hypothesize that Pus enzymes select their mRNA targets based on consensus sequence, but substrate availability and localization – factors that depend less on Pus4’s ability to selectively bind the substrate, may play a bigger role in target selection. We aim to characterize Pus4’s ability to bind to and modify RNAs of different sequences and structures, and to use crystallography to understand how structural characteristics of Pus4 impact substrate selection.
References:
1.Carlile, T. M.; Rojas-Duran, M. F.; Zinshteyn, B.; Shin, H.; Bartoli, K. M.; Gilbert, W. V. Pseudouridine Profiling Reveals Regulated MRNA Pseudouridylation in Yeast and Human Cells. Nature 2014, 515 (7525), 143–146.
2.Purchal, M. K.; Eyler, D. E.; Tardu, M.; Franco, M. K.; Korn, M. M.; Khan, T.; McNassor, R.; Giles, R.; Lev, K.; Sharma, H.; Monroe, J.; Mallik, L.; Koutmos, M.; Koutmou, K. S. Pseudouridine Synthase 7 Is an Opportunistic Enzyme That Binds and Modifies Substrates with Diverse Sequences and Structures. Proceedings of the National Academy of Sciences 2022, 119 (4), e2109708119.
3.Keffer-Wilkes, L. C.; Veerareddygari, G. R.; Kothe, U. RNA Modification Enzyme TruB Is a TRNA Chaperone. Proceedings of the National Academy of Sciences 2016, 113 (50), 14306–14311.
Keywords: RNA modifications, RNA binding proteins , pseudouridine
78. Role of HTLV-1 Gag-host factor interactions in chaperoning tRNA-Pro primer annealing
Yu-Ci Syu (Molecular, Cellular, and Developmental Biology Graduate Program, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University), Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University), Yingke Tang (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University), Amanda R. Panfil (Molecular, Cellular, and Developmental Biology Graduate Program, Department of Veterinary Biosciences, Center for Retrovirus Research, The Ohio State University), Patrick L. Green (Molecular, Cellular, and Developmental Biology Graduate Program, Department of Veterinary Biosciences, Center for Retrovirus Research, The Ohio State University), Karin Musier-Forsyth (Molecular, Cellular, and Developmental Biology Graduate Program, Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University)
Abstract:
Human T-cell leukemia virus type 1 (HTLV-1) is the only oncogenic human retrovirus discovered to date. In contrast to HIV-1, several critical details of HTLV-1 reverse transcription remain unclear. All retroviruses use a host cell tRNA to prime reverse transcription. In HTLV-1, the primer binding site (PBS) in the genomic RNA (gRNA) is complementary to the 3'-18-nucleotides of human tRNAPro. Structure-probing of the gRNA revealed that the PBS is embedded in a highly structured hairpin.1 Neither HTLV-1 nucleocapsid (NC) nor matrix (MA) proteins are capable of annealing tRNAPro to the stable PBS domain in vitro. We hypothesize that HTLV-1 Gag, the polyprotein made up of MA, capsid, and NC domains, may have more robust chaperone activity than mature NC or MA and that a cellular co-factor may be required to facilitate primer tRNA annealing. We successfully purified recombinant HTLV-1 Gag for the first time and performed primer-annealing assays. Relative to NC and MA, HTLV-1 Gag is only slightly more effective at chaperoning the annealing of tRNAPro to the PBS. To identify potential HTLV-1 Gag interacting partners and co-chaperones of tRNA annealing in cells, we performed affinity tagging/purification mass spectrometry (AP-MS). Three significant AP-MS hits, DDX21, RPL7, and YBX1, were further validated by reciprocal co-IP studies in both HEK293T and MT-2 cells. Domain mapping studies revealed that the zinc fingers in the NC domain of HTLV-1 Gag interact with both the N-terminal basic leucine zipper (bZIP) and C-terminal domains (CTD) of RPL7. In addition, we showed that the interactions depend on the intact zinc finger structures but not on the presence of RNA, Gag myristoylation, or Gag oligomerization. RPL7 alone was more effective than either HTLV-1 MA or Gag at annealing tRNAPro to the PBS. Domain deletion studies showed that both bZIP and CTD of RPL7 are involved in facilitating primer annealing. Current studies are focused on investigating the annealing activity of another HTLV-1 Gag-interacting partner, the DDX21 helicase, and the influence of RPL7 and DDX21 knockdown on viral infectivity. Taken together, these studies will lead to mechanistic insights that could be exploited for new therapeutic strategies.
References:
1. Wu, W., Hatterschide, J., Syu, Y.C., Cantara, W.A., Blower, R.J., Hanson, H.M., Mansky, L.M. and Musier-Forsyth, K. (2018) Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization. J Biol Chem, 293, 16261-16276.
Keywords: Gag, RPL7, DDX21
79. Title not available online - please see the booklet.
Kurtis Breger (University of Notre Dame), Jessica A. Brown (University of Notre Dame)
Abstract not available online - please check the booklet.
80. Title not available online - please see the booklet.
Joseph G. Kanlong (The Ohio State University, Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, Columbus OH), Christina Ross (The Ohio State University, Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, Columbus OH), Heewon Seo (The Ohio State University, Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, Columbus OH), Michael G. Kearse (The Ohio State University, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, Columbus OH), Karin Musier-Forsyth (The Ohio State University, Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, Columbus OH)
Abstract not available online - please check the booklet.
81. Plant-exclusive domain of trans-editing enzyme ProXp-ala confers dimerization and enhanced tRNA binding
Jun-Kyu Byun (Department of Chemistry and Biochemistry, The Ohio State University), John A. Vu (Department of Chemistry and Biochemistry, The Ohio State University), Siou-Luan He (Department of Horticulture and Crop Science and Center for Applied Plant Sciences, The Ohio State University), Jyan-Chyun Jang (Department of Horticulture and Crop Science and Center for Applied Plant Sciences, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University), Alezandra M. Hernandez Marquez (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Faithful translation of the genetic code is critical for the viability of all living organisms. The trans-editing enzyme ProXp-ala, encoded by bacteria and eukaryotes, prevents Pro to Ala mutations during translation by hydrolyzing misacylated Ala-tRNAPro that has been synthesized by prolyl-tRNA synthetase. Plant ProXp-ala sequences contain a conserved C-terminal domain (CTD) that is absent in other organisms; the origin, structure, and function of this extra domain are unknown. To characterize the plant-specific CTD, we performed bioinformatics and computational analyses that were consistent with a conserved α-helical structure. We also expressed and purified wild-type Arabidopsis thaliana (At) ProXp-ala in Escherichia coli, as well as variants lacking the CTD or containing only the CTD. Circular dichroism spectroscopy confirmed a loss of α-helical signal intensity upon CTD truncation. Size-exclusion chromatography with multi-angle laser light scattering revealed that wild-type At ProXp-ala was primarily dimeric and CTD truncation abolished dimerization in vitro. Bimolecular fluorescence complementation assays in At protoplasts support a role for the CTD in homodimerization in vivo. The deacylation rate of Ala-tRNAPro by At ProXp-ala was significantly reduced in the absence of the CTD, and kinetic assays indicated that the reduction in activity is primarily due to a tRNA binding defect. Overall, these results broaden our understanding of eukaryotic translational fidelity to the plant kingdom. Our studies revealed that the plant-specific CTD plays a significant role in substrate binding and canonical editing function. Through its ability to facilitate protein-protein interactions, the CTD may also provide expanded functional potential to trans-editing enzymes in plants. Ongoing studies using mutant and transgenic plants are aimed at further exploring the function and interacting partners of At ProXp-ala in vivo.
Keywords: aminoacyl-tRNA, aminoacyl-tRNA synthetase, Arabidopsis thaliana
82. Exploring structural determinants of ligand selectivity in cobalamin riboswitches
Ingrid Kilde (Chemistry, University of Michigan Ann Arbor), Elizabeth Tidwell (Biophysics, University of Michigan Ann Arbor), Suada Leskaj (Biochemistry, University of Michigan Ann Arbor), Markos Koutmos (Chemistry and Biophysics, University of Michigan Ann Arbor)
Abstract:
Cobamides are a family of complex organometallic coenzymes involved in numerous metabolic pathways across all domains of life. Within microbial communities, some bacterial strains are capable of cobamide synthesis (cobamide producers) whereas others (cobamide scavengers) must rely on environmental sources of cobamides including cross-feeding, resulting in a complex and dynamic network of nutrient sharing that is incompletely understood. Both producers and scavengers employ riboswitches (RSs) as a strategy to attenuate gene expression based on environmental cobamide concentrations. The first cobamide RS identified was found to bind adenosylcobalamin and thus RSs identified with similar aptamer domains were classified as cobalamin (Cbl) RSs. However, further study is required to assess the ability of RSs in this class to respond to or differentiate between cobamides besides Cbl. In order to further our understanding of ligand specificity and molecular recognition by Cbl RSs, we aim to characterize Cbl RSs likely to differentiate between cobamides. We seek to validate putative RSs and determine their ligand specificity via binding assays. Following this, we aim to identify the structural foundation of this selectivity using high resolution methods including x-ray crystallography and Cryo-EM. Additionally, we are beginning to use differential scanning fluorimetry as a label-free, highly parallel method to monitor the impact of different cobamides on RS stability. This work could help gain insight into the mechanisms which regulate cobamide metabolism in prokaryotes, further subclassify Cbl RSs, and answer fundamental questions about molecular recognition by RNA.
Keywords: Riboswitch, Cobalamin, Structure
83. Characterizing the function of the human CCR4-NOT complex using rapid degron systems.
Shardul Kulkarni (Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.), Cheryl Keller (Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.), Belinda Giardine (Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.), Courtney Smith (Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.), Alexei Arnaoutov (Division of Molecular and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.), Joseph Reese (Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.)
Abstract not available online - please check the booklet.
84. Investigating the Role of ADARs and A-to-I RNA editing in Germline RNA Regulation
Emily A. Erdmann (Department of Biology, Indiana University ), Heather A. Hundley (Department of Biology, Indiana University)
Abstract not available online - please check the booklet.
85. tRNA family-specific turnover pathways and environmental stresses regulate tRNA intron degradation
Katherine Clark (Molecular Genetics, Ohio State University), Sara Metcalf (Molecular Genetics, Ohio State University), Alicia Bao (Molecular Genetics, Ohio State University), Lauren Peltier (Molecular Genetics, Ohio State University), Anita K. Hopper (Department of Molecular Genetics, Center for RNA Biology, OSU, Columbus, OH)
Abstract:
In S. cerevisiae, 10 tRNA families are encoded by intron-containing genes. Precursor tRNAs are processed into mature functional tRNAs via several steps, including intron removal by the tRNA splicing endonuclease complex. After splicing, free introns are rapidly and efficiently degraded. The mechanism for tRNA intron turnover has been delineated for two of the 10 families (Wu and Hopper 2014); tRNAIleUAU and tRNALeuCAA linear introns are degraded by the 5’ to 3’ exonuclease Xrn1 after 5’ phosphorylation by the tRNA ligase/kinase Rlg1. There are at least 4 additional pathways for the remaining 8 tRNA families: Rlg1-dependent/Xrn1-independent, Rlg1-independent/Xrn1-dependent, Xrn1 and Rlg1-independent, and an endonuclease-dependent pathway for the circular form of the tRNATrp intron. We identified several candidate genes encoding proteins that function in tRNA family-specific intron turnover. From the identified candidates, three pathways for intron degradation have been uncovered: 1) competing pathways, 2) parallel pathways, and 3) an endonuclease pathway. We also investigated the effects of various environmental stresses on tRNA intron levels and uncovered stress-specific, tRNA family-specific intron accumulation. Treatment of wild-type cells with 3mM H2O2 results in 15- and 8-fold elevations of tRNATrpCCA and tRNALeuCAA intron levels, respectively. Accumulation of these tRNA introns occurs immediately and is maintained for at least 2 hours, despite immediate termination of tRNA transcription upon oxidative stress. tRNA introns spliced from tRNASer GCU and tRNALys UUU initially accumulate with the addition of H2O2, but level out after 1 hour. Inversely, most tRNA intron levels decrease with 42 ̊ C heat treatment. Our studies provide insight into family-specific endogenous and stress-induced intron turnover pathways that indicate possible novel regulatory functions of tRNA introns.
Keywords: tRNA turnover, intron degradation, environmental stress
86. Post-transcriptional silencing of the nuclear poly(A) binding protein N1 is critical for postnatal maturation and function of the heart
Sandip Chorghade,Joe Seimetz,Bo Zhang,Chaitali Misra,Subhashish Natua (Department of Biochemistry,University of Illinois Urbana-Champaign.), Qinyu Hao,Kananganattu V. Prasanth (Department of Cell and Developmental Biology,University of Illinois Urbana-Champaign.), Xander Wehrens (Department of Molecular Physiology and Biophysics, Baylor College of Medicine), Jiwang Chen (Department of Medicine, University of Illinois Chicago), Auinash Kalsotra (Department of Biochemistry,Carl R. Woese Institute for Genomic Biology, 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.
Importantly, 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. 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: PABPN1, Cardiac maturation, Cardiac Hypertrophy , Intron Retaintion
87. Temporally controlled expression of a splicing factor in single cells coordinates the metabolic and proliferative activities of regenerating livers
Nick Baker (Department of Biochemistry, UIUC), Sushant Bangru (Department of Biochemistry, UIUC), Ullas V. Chembazhi (Department of Biochemistry, UIUC), Auinash Kalsotra (Department of Biochemistry, UIUC)
Abstract:
The liver has an extensive ability to regenerate following injury, typically via cellular proliferation of hepatocytes. However, the exact mechanics of regeneration, such as how quiescent hepatocytes transition into a proliferative state and how regenerating livers sustain normal metabolic activities while the tissue recovers from injury, are largely unknown. The role of alternative splicing and RNA binding proteins during liver regeneration has remained completely uninvestigated. Epithelial splicing regulator protein 2 (ESRP2) is an RNA splicing factor that acts as the developmental switch for splicing targets during postnatal liver maturation, including in the Hippo signaling pathway, which is critical for organ development and regeneration. ESRP2 promotes the production of adult Hippo pathway splice variants thereby limiting hepatocyte proliferation in a quiescent mature liver. We have previously demonstrated that ESRP2 and its splicing targets are transiently reprogrammed to the fetal stage during active phases of regeneration. We further hypothesized that ESRP2 is temporally controlled within regenerating hepatocytes to promote a proliferative or metabolic state. We used single-cell RNA sequencing to determine altered cell states and gene expression changes in ESRP2 KO cells compared to WT mice during the initiation and termination stages of liver regeneration. Here we show that regenerating hepatocytes bifurcate into proliferating or metabolically-hyperactive cell states, such that ESRP2 is highly expressed in the metabolically active hepatocytes and is nearly absent in the proliferating hepatocytes. Remarkably, the adult quiescent ESRP2 KO hepatocytes exhibit an altered starting cell state, which is more closely related to the fetal stage. We further show that forced expression of ESRP2 in the mouse liver inhibits the proliferation of hepatocytes, whereas ESRP2 deletion in hepatocytes increases their proliferative index during liver regeneration. Taken together, these data imply that tightly controlled expression of ESRP2 coordinates the metabolic and proliferative activities of regenerating livers.
References:
Dysregulated activation of fetal liver programme in acute liver failure. Hyun et al. Gut. (2019)
Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration. Bangru et al. Nat Struct Mol Biol. (2018)
Keywords: Liver, Regeneration, scRNA-seq
88. Transcriptional and post-transcriptional landscapes of alcohol-associated human liver diseases
Ullas V. Chembazhi (Department of Biochemistry, University of Illinois, Urbana-Champaign, IL, USA), Sushant Bangru (Department of Cell Biology, Duke University School of Medicine), Rajesh Dutta (Division of Gastroenterology, Department of Medicine, Duke University Health System, Durham, NC, USA), Zhaoli Sun (Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA), Anna Mae Diehl (Division of Gastroenterology, Department of Medicine, Duke University Health System, Durham, NC, USA), Auinash Kalsotra (Department of Biochemistry, University of Illinois, Urbana-Champaign, IL, USA)
Abstract not available online - please check the booklet.
89. Cardiomyocyte-specific knockout of nuclear poly(A)-binding protein leads to heart failure and death
Subhashis Natua (Department of Biochemistry, University of Illinois at Urbana-Champaign), Joseph Seimetz (Department of Biochemistry, Chemical Biology Training Program, University of Illinois at Urbana-Champaign), Bo Zhang (Department of Biochemistry, University of Illinois at Urbana-Champaign), Sandip Chorghade (Department of Biochemistry, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, Chemical Biology Training Program, Cancer Center at Illinois, and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign)
Abstract not available online - please check the booklet.
90. Discriminator Base is a Critical Recognition Element for Trypanosoma Brucei Ala-tRNAPro Editing Domains
Anna Vradi (Center for RNA Biology, Department of Microbiology, The Ohio State University Columbus, Ohio 43210), Rylan Watkins (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University Columbus, Ohio 43210), Irina Shulgina (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University Columbus, Ohio 43210), Karin Musier-Forsyth (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University Columbus, Ohio 43210)
Abstract:
Aminoacyl-tRNA synthetases (ARSs) are responsible for charging tRNA with the proper amino acid. However, ARSs are error-prone and rely on editing mechanisms to lower the rate of misincorporation. Previous studies have shown prolyl-tRNA synthetase (ProRS) mischarges Ala onto cognate tRNAPro—this error can be corrected by a cis-editing site (INS) or by a freestanding INS homolog, ProXp-ala. Trypanosoma brucei (Tb) ProRS encodes an appended ProXp-ala domain, which is a putative cis-acting Ala-tRNAPro editing domain, as well as a distinct free-standing ProXp-ala homolog that we have named ProXp-ala2. Tb infection causes human African Trypanosomiasis (HAT), which is fatal; current treatments have serious side-effects and are relatively ineffective. ProXp-ala2 is not found in humans making it a potential drug target. Tb may have two Ala-tRNAPro editing domains due to its unusual metabolism. When in the procylic form, Tb’s main source of carbon is Pro, with Ala as a metabolic byproduct. With high concentrations of Ala, robust editing mechanisms may avoid misincorporation of Ala at Pro codons. Previous studies have shown that Homo Sapiens (Hs) ProXp-ala recognizes nucleotides in the tRNA acceptor stem for optimal function. In contrast, the E. coli INS domain, which is appended to the synthetase, relies exclusively on ProRS recognition of the anticodon. Our goal is to elucidate the acceptor stem specificity of Tb ProXp-ala, both free and when appended to ProRS, as well as of ProXp-ala2. We have mutated the acceptor stem of Tb tRNAPro to mimic a bacterial acceptor stem (G1C:C72G, C73A) and performed deacylation assays. Preliminary data suggest that both freestanding Tb ProXp-ala and ProXp-ala2 have strong specificity for the tRNAPro discriminator base (C73) and more relaxed specificity for the first base pair (G1:C72) when compared to similar experiments with Hs ProXp-ala. Surprisingly, ProXp-ala2 deacylated triple mutant (G1C:C72G, C73A) tRNA better than the single (C73A) mutant. Thus, the context of the discriminator base is important for this editing domain. Ongoing studies are aimed at elucidating the Tb ProXp-ala/2 residues responsible for acceptor stem binding. Results of these studies may have implications for new therapeutic strategies.
Keywords: tRNA Synthetase, tRNA, Trypanosoma Brucei
91. RRP44 contributes to the unusually short half-life of tRNATyr in T. brucei
Gabriel Silveira DAlmeida (Department of Microbiology; The Center for RNA Biology; The Ohio State University), Ananth Casius (Department of Microbiology; The Center for RNA Biology; The Ohio State University), Jeremy Henderson (Department of Microbiology; The Center for RNA Biology; The Ohio State University), Ruslan Afasizhev (Department of Molecular and Cell Biology; The Boston University School of Dental Medicine), Inna Afasizheva (Department of Molecular and Cell Biology; The Boston University School of Dental Medicine), Juan D. Alfonzo (Department of Microbiology; The Center for RNA Biology; The Ohio State University)
Abstract not available online - please check the booklet.
92. Translational regulation of photoreceptor-specific mRNA by Musashi 1 and Musashi 2 proteins for photoreceptor maintenance
Bohye Jeong (Department of Molecular and Biochemistry Medicine, West Virginia University), Fatimah Matalkah (Receptor Biology Laboratory, Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, CDC, Morgantown, WV), Visvanathan Ramamurthy (Department of Molecular and Biochemistry Medicine, West Virginia University; Department of Ophthalmology, West Virginia University School of Medicine, Center for Neuroscience, West Virginia University), Peter Stoilov (Department of Molecular and Biochemistry Medicine, West Virginia University)
Abstract:
The Musashi (MSI) RNA binding protein family consists of two paralogs, Musashi 1 (MSI1) and Musashi 2 (MSI2) that are highly conserved across species. We and others have shown that these two proteins are essential for the photoreceptor's survival and morphogenesis and maintenance. Double knockout of the Msi1 and Msi2 in developing or mature photoreceptor cells leads to progressive vision loss accompanied with photoreceptor degeneration. MSI1-CLIP seq (crosslinking and immunoprecipitation) in mouse retina showed binding to introns and 3’-UTRs. The transcripts whose 3’-UTRs are bound by MSI1 were enriched for genes involved in phototransduction, suggesting that Musashi is involved in the differentiation and maintenance of photoreceptor cells. Our proteomics analysis of Musashi double knockout retina demonstrates that Musashi predominantly promotes protein expression of its targets. The upregulation of protein expression likely is achieved through stimulation of mRNA translation as it is not accompanied with increase in mRNA levels both in retina and when model Gnat1 transcripts were tested in 3T3 cells. However, MSI binding to the target transcript alone is not sufficient to confer regulation. Therefore, we hypothesize that there are additional factors that cooperate with the MSI proteins to regulate translation. Currently, we are investigating potential interactions with the RNA interference machinery in vivo and in transient transfections that use the rod transducin beta subunit (Gnb1) transcript as a model. Our most recent results using chimeric AGO2-eCLIP show that MSI stabilizes binding of RISC to the target 3’-UTRs. Paradoxically the increased binding of RISC to the 3’-UTRs results in elevated levels of protein expression. Gnb1 is bound by MSI and this binding stabilizes the binding of RISC complexes carrying the photoreceptor-specific miR-183 microRNA. We hope that our ongoing studies on the regulation of model Gnb1 transcripts in 3T3 cells will shed light on the mechanism by which Musashi and RISC cooperate to promote protein expression.
References:
1. Sundar, J., Matalkah, F., Jeong, B., Stoilov, P. & Ramamurthy, V. The Musashi proteins MSI1 and MSI2 are required for photoreceptor morphogenesis and vision in mice. J Biol Chem 296, 100048 (2021).
2. Xiang, L. et al. miR-183/96 plays a pivotal regulatory role in mouse photoreceptor maturation and maintenance. Proc Natl Acad Sci U S A 114, 6376–6381 (2017).
3. Sanuki, R. et al. miR-124a is required for hippocampal axogenesis and retinal cone survival through Lhx2 suppression. Nat Neurosci 14, 1125–1134 (2011).
4. MacNicol, M. C., Cragle, C. E. & MacNicol, A. M. Context-dependent regulation of Musashi-mediated mRNA translation and cell cycle regulation. Cell Cycle Georget. Tex 10, 39–44 (2011).
Keywords: Photoreceptor, Translational regulation, RNA-binding protein
93. Characterizing the Role of ADARs in Regulating Insulin Signaling and Inter-Organ Communication
Ananya Mahapatra (Genome, Cellular and Developmental Biology, Indiana University), Heather A. Hundley (Department of Biology, Indiana University )
Abstract:
Communication between organs is a vital factor that affects several physiological processes and is important for proper homeostasis. A major way in which organs communicate with each other is through insulin signaling. Since the nervous system is a master regulator of inter-organ communication, insulin signaling within the nervous system has the potential to affect insulin signaling in other organs. Even though neural insulin signaling can affect other organs, the molecular mechanism involved in the inter-organ communication through insulin signaling is unknown. Novel mechanisms of insulin signaling regulation have been attributed to RNA binding proteins since these proteins can control post-transcriptional gene expression. However, how this occurs molecularly is unclear. These gaps in the field emphasize the need to explore mechanisms of neural gene regulation and inter-organ communication.
My long-term goal is to understand how RNA binding proteins (RBPs) regulate gene expression to impact inter-organ communication and how misregulation could lead to disease. My preliminary data demonstrates that RBPs called adenosine deaminases that act on RNA (ADARs) regulate insulin signaling in the nervous system, which subsequently regulates insulin signaling in other tissues. My research uses the model system C. elegans since ADARs are essential in mammals and insulin signaling is conserved in C. elegans. C. elegans has two ADAR proteins- ADR-1 and ADR-2. My preliminary studies show that compared to wildtype, adr-2 lacking animals show decreased expression of genes regulated by insulin signaling. Interestingly, in animals that express ADR-2 solely in the nervous system, gene expression is like wildtype, suggesting insulin signaling is regulated cell-nonautonomously. My studies also suggest that loss of ADR-2 causes aberrant binding of ADR-1, thus impacting insulin signaling. My initial experiments testing neural ADR-1 binding suggest that ADR-1 binds irld-24, an insulin-receptor L domain protein, specifically in the absence of ADR-2. My proposed model is that in the absence of ADR-2, ADR-1 binds irld-24 aberrantly in the nervous system, which is responsible for causing downregulation of downstream genes regulated by insulin signaling throughout the animal.
Keywords: ADARs, insulin signaling, nervous system
94. Role of ADARs in the pathogen response of C. elegans
Alfa Dhakal (School of Medicine), Chinnu Salim (Department of Biology), Heather Hundley (Department of Biology)
Abstract:
All organisms must be able to fight infection from pathogen to survive. To be prepared to fight 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 editing (modifying). 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 role of ADARs in bacterial infection and host-defense mechanisms have not been studied.
There are two ADARs in C. elegans ADR-1 and ADR-2 with double stranded RNA binding domains and a deaminase domain. Using a standard slow killing assay with Pseudomonas aeruginosa as a pathogen, we observed that both adr-1(-) and adr-2(-) single mutants and adr-1(-); adr-2(-) double mutant animals are susceptible. Interestingly, an ADR-1 mutant that is unable to bind to dsRNA also showed a sensitive phenotype suggesting that ADARs are regulating certain transcripts by binding. On doing a pilot RNA sequencing, with WT, adr-1(-) and the ADR-1 dsRNA binding mutant worms after pathogen exposure, we observed that many genes that were downregulated in adr-1(-) and ADR-1 dsRBM1 mutant were also down in normal food suggesting that ADR-1 is regulating these genes basally. Further, RNA sequencing and differential gene expression analysis of WT, adr-1(-), adr-2(-) and adr-1(-); adr-2(-) worms grown on normal food was done. Among the genes overlapping between double mutant and single mutant worms compared to WT, we found “collagen” genes to be significantly down. Collagens are involved in epithelial tissue integrity and form a first line of defense against pathogen. This led to our hypothesis that ADARs are regulating the collagen genes to provide organismal survival to pathogen.
Ongoing studies are focused on finding the cuticular structure changes on ADAR mutant worms using SEM and finding the tissue specific contributions and molecular mechanisms of ADARs in pathogen response.
Keywords: RNA binding protein, pathogen, gene expression
95. Understanding the function of the La protein in Trypanosoma brucei
G. Lankani Gunaratne (Ohio State Biochemistry Program, The Ohio State University), Ananth Casius (Department of Microbiology, The Ohio State University), Gabriel Silveira dAlmeida (Department of Microbiology, The Ohio State University), Richard J. Maraia (Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health), Juan D. Alfonzo (Department of Microbiology, The Ohio State University)
Abstract:
La is a ubiquitous nuclear RNA binding protein found in almost all eukaryotes investigated to date. Although this conservation suggests that La participates in an important process, its role in eukaryotic cells is not completely clear. Trypanosoma brucei, the causative agent of African Trypanosomiasis in humans, encodes a ‘classical’ genuine La protein (TbLa) with a La motif closely followed by an RNA recognition motif (RRM1), an additional RRM (RRM2α) and a C terminal short basic motif (SBM) along with a nuclear localization signal (NLS). We have shown that TbLa is essential for the survival of T. brucei through knocking down endogenous La by RNA interference (RNAi). A synonymous codon swapped version of myc-tagged TbLa, which is impervious to RNAi, (TbLa Rec) has been stably expressed in T. brucei and rescues a growth defect caused by RNAi down-regulation of endogenous La. Immunofluorescence microscopy showed that TbLa Rec resides mainly in the nucleus and partly in the cytoplasm, similar to endogenous La. Stable expression of an NLS-deleted TbLa Rec (TbLa NLSΔRec) in La RNAi cells rescues the growth phenotype similar to TbLa Rec. Immunofluorescence microscopy coupled with treatment of cells with Leptomycin B confirmed that TbLa with the NLS deletion indeed leads to exclusively a cytoplasmic localization of TbLa, but by rescuing the growth defect of endogenous La, it leads to the conclusion that the essential function of La is not necessarily due to its nuclear localization. Through polysome sedimentation analysis we show that polysome formation gets disrupted in the absence of TbLa which is rescued by TbLa Rec and TbLa NLSΔRec. This confirms TbLa plays an important role in ribosome biogenesis in T. brucei, a function TbLa is able to carry out while being limited to the cytoplasm. We have also constructed a series of myc-tagged recoded La mutants with each of the RRM, SBM and the NLS deleted. Through in vivo expression of individual RRM-deleted La mutants in endogenous La RNAi cells, we show that whereas RRM1 is essential for function, the second putative RRM2α and the SBM are dispensable. The latter raises questions about why RRM2α and SBM are maintained, other than for structural rather than functional reasons.
References:
Blewett,N.H.; Maraia,R.J. La Involvement in tRNA and Other RNA Processing Events. Biochem Biophys. Acta - Gene Regul. Mech. 2018, 1861(4)
Maraia,R.J.; Mattijssen,S.; Cruz-Gallardo,I.; Conte,M.R. La and Related RNA-Binding Proteins. Wiley Interdiscip. Rev. RNA 2017, 8(6)
Foldynová-Trantírková,S.; Paris,Z.; Sturm,N.R.; Campbell,D.A.; Lukeš,J. Trypanosoma brucei La Protein Is a Candidate Poly(U) Shield That Impacts Spliced Leader RNA Maturation and tRNA Intron Removal. Int. J. Parasitol. 2005, 35(4)
Marchetti,M.A.; Tschudi,C.; Kwon,H.; Wolin,S.L.; Ullu,E. Import of Proteins into the Trypanosome Nucleus and Their Distribution at Karyokinesis. J. Cell Sci. 2000, 906
Shan,F.; Mei,S.; Zhang,J.; Zhang,X. et al. A Telomerase Subunit Homolog La Protein from Trypanosoma brucei Plays an Essential Role in Ribosomal Biogenesis. FEBS J. 2019, 286(16)
Keywords: La protein, Trypanosoma brucei, RNA binding proteins
96. Investigating the Role of Trypanosoma brucei Prolyl-tRNA Synthetase and MCP3 in Maintaining tRNAPro Translational Fidelity
Rylan Watkins (Chemistry and Biochemistry, The Ohio State University), Irina Shulgina (Chemistry and Biochemistry, The Ohio State University), Juan D. Alfonzo (Microbiology, The Ohio State University ), Karin Musier-Forsyth (Chemistry and Biochemistry, The Ohio State University)
Abstract:
Trypanosoma brucei (Tb) are unicellular pathogens that cause Human African Trypanosomiasis (HAT)—a disease that is fatal if left untreated. Currently, HAT lacks a safe and effective treatment option; thus, new therapeutic targets encoded in the Tb genome are sought. Prolyl-tRNA synthetase (ProRS) attaches proline to cognate tRNAPro in a two-step aminoacylation reaction. ProRSs across all domains of life are error prone and often mischarge alanine onto cognate tRNAs. In most bacteria, ProRSs encode a dedicated editing domain (INS) that is responsible for hydrolyzing Ala-tRNAPro. Organisms that lack an INS domain within their ProRS often encode a homologous editing factor, ProXp-ala, to perform the same proofreading function in trans. Tb ProRS has an appended N-terminal ProXp-ala domain and belongs to a Multi-tRNA Synthetase Complex (MSC) along with an uncharacterized protein, MCP3, which has significant structural homology to ProXp-ala. MCP3 homologs and Tb-like ProRSs are highly conserved in human pathogens and are not encoded in the human genome. The Kinetoplastid Informatics Resource (TriTrypDB) reports MCP3 and ProRS are essential for parasite fitness. Here, we recombinantly expressed and purified Tb ProRS and MCP3 for the first time. We performed in vitro kinetic assays and showed that MCP3 has more robust Ala-tRNAPro editing activity than ProXp-ala enzymes from other organisms investigated to date. We performed site-directed mutagenesis and have identified several residues critical for efficient catalysis by MCP3. Multiple sequence alignments revealed that MCP3 has a unique N-terminal extension and we showed that this sequence is required for MCP3 dimerization. Preliminary kinetic data show that when expressed as a freestanding domain, Tb ProXp-ala displays reduced Ala-tRNAPro deacylase activity compared to full-length Tb ProRS. Prior to mammalian infection, trypanosomes rely on proline for ATP production. A consequence of this metabolic strategy is the excess side production of alanine. We hypothesize the exaggerated alanine pool in Tb results in more tRNAPro mischarging events and a need for two robust Ala-tRNAPro editing mechanisms. Our structural and mechanistic insights into tRNAPro editing in Tb serve as the foundation for exploring Tb ProRS and MCP3 as drug targets.
Keywords: aminoacyl-tRNA synthetase, translation fidelity, Multi-tRNA synthetase complex (MSC)
97. Gathering insights into the importance of ADR-1/ADR-2 interaction in RNA editing in vivo
Boyoon Yang (Molecular and Cellular Biochemistry, Indiana University Bloomington), Pranathi Vadlamani (Biology departement, Indiana University Bloomington), Heather Hundley (Biology departement, Indiana University Bloomington)
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 mRNAs and select an individual adenosine for editing in vivo.
For ADARs to target specific adenosines, ADARs have to dimerize, bind RNA, and catalyze deamination. While residues crucial for RNA binding and editing are extensively studied, residues important for protein-protein interaction of ADARs are poorly understood. In Caenorhabditis elegans, RNA editing is mediated by ADR-2, whereas ADR-1 is an editing-deficient ADAR family member. Our lab showed that the majority (80%) of ADR-2 bound transcripts require the help of ADR-1, and ADR-1 and ADR-2 heterodimerize independent of dsRNA binding. To investigate which domain of ADARs plays a role in both RNA editing and protein-protein interaction in vivo, we have generated ADAR mutants lacking the deaminase domain by CRISPR. In vivo assays of these novel mutants demonstrated that the dsRBD of ADR-2 is sufficient for ADR-1/ADR-2 interaction. Furthermore, the lack of the ADR-1 deaminase domain, while not affecting the heterodimerization, results in defects in RNA editing in vivo, suggesting that an inactive deaminase domain might play a role in editing efficiency. Elucidating the mechanism of how ADR-1 and ADR-2 dimerize to edit target mRNAs will provide new insights into how A-to-I editing is mediated in vivo.
Keywords: RNA editing, ADAR, protein-protein interaction
98. Characterization of novel TRMT1 variants linked to intellectual disability disorders in the human population
Cailyn P. Leo 1 (Biology; University of Rochester), Chenghong Deng 1 (Biology; University of Rochester), Kejia Zhang (Biology, University of Rochester), Dragony Fu (Biology; University of Rochester), 1Contributed equally to work
Abstract not available online - please check the booklet.
99. Long noncoding RNAs are abundant targets of ADAR3 in glioblastoma
Priyanka Mukherjee (Cell, Molecular and Cancer Biology Program, Indiana University School of Medicine), Heather A. Hundley (Department of Biology, Indiana University Bloomington)
Abstract not available online - please check the booklet.
100. AtRTD2 versus AtRTD3: how Serine/Arginine-rich 45 affects the sperm transcriptome in Arabidopsis thaliana based on 2 versions of transcriptome reference
Arden Bui (Biochemistry Program, Department of Biology, St. Bonaventure University, St. Bonaventure, NY 14778), Christopher Chin (Biochemistry Program, Department of Biology, St. Bonaventure University, St. Bonaventure, NY 14778), Xiao-Ning Zhang (Biochemistry Program, Department of Biology, St. Bonaventure University, St. Bonaventure, NY 14778)
Abstract:
In flowering plant, pollen grains are the male gametophyte that produces sperms. Studies have shown that there is a transcriptome switch during pollen mitosis II when producing mature pollen grains. Arabidopsis Serine/Arginine-rich 45 (SR45) is a splicing regulator that is highly expressed during pollen development. The sr45-1 null mutant plant exhibits pleiotropic phenotypes throughout the life cycle, including a mild sterility and a reduced seed set. To investigate SR45’s effects in reproduction, sperm cells were isolated from wild-type and the sr45-1 mutant mature pollen using FACS. Using the 3D RNA-seq App, the bulk sperm transcriptome was sequenced and compared between the two genotypes using 2 versions of Arabidopsis transcriptome reference: AtRTD2 and AtRTD3. From the RNA-seq data analyses, the majority of differentially expressed (DE) genes overlapped between AtRTD2 and AtRTD3. The analyses suggest that SR45 promoted the expression of genes for pollen tube development and suppressed the expression of genes involved in pollen sperm cell differentiation. Several sperm markers are used to validate this transcriptome profile. Differentially expressed transcripts are overrepresented in mRNA splicing and phosphorelay signal transduction using both AtRTD2 and AtRTD3. This suggests that SR45-dependent AS events may preferentially contribute to the sperm identity in mature pollen grains via maintaining a homeostasis in alternative splicing and phosphorelay signal transduction. About 30% overlap (28 genes) was found between differential alternatively spliced (DAS) genes using AtRTD2 versus AtRTD3. Some of the DAS genes showed significant switches in isoform abundance and potential structural changes in the predicted protein products. Among genes exhibiting isoform switches, both RAD4 and RAD51 are of interest. Transgenic lines with a constitutive promoter and transgenic lines with the native promoter were developed to explore possible isoform-specific functional differences between RAD4 and RAD51 isoforms.
References:
Honys, D. & Twell, D. (2004). Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biology, 5(11), R85.
Guo,W. et al. (2020) 3D RNA-seq: a powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists. RNA biology, 1–14.
Zhang, R., et al. (2022). A high-resolution single-molecule sequencing-based arabidopsis transcriptome using novel methods of iso-seq analysis. Genome Biology, 23(1).
Zhang R, et al. (2017) A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing. Nucleic Acids Res.45(9):5061-5073.
Keywords: transcriptome analysis, alternative splicing, Arabidopsis thaliana
101. The Interplay within the ASAP Complex in Arabidopsis thaliana
Serena Fan (Biochemistry Program, Department of Biology, St. Bonaventure University), Anna Hu (Biochemistry Program, Department of Biology, St. Bonaventure University), Xiao-Ning Zhang (Biochemistry Program, Department of Biology, St. Bonaventure University)
Abstract:
The evolutionary conserved apoptosis-and splicing associated protein (ASAP) complex is involved in the regulation of different aspects of RNA metabolism. In Arabidopsis thaliana, the ASAP complex is comprised of Serine/Arginine-rich45 (SR45), Acinus, and Sin3-associated protein 18 (SAP18). Previous studies suggest that the ASAP complex binds to the flowering suppressor FLC gene to reduce its expression and allow normal flowering. In the sr45-1 null mutant, there is a higher FLC expression and a delayed flowering. In addition, SAP18 protein abundance is lower in sr45-1, particularly in the nucleus, suggesting that SR45 is required for a normal level of SAP18 protein. Taken together, we hypothesize that SR45 regulates SAP18 at the protein level to keep a low FLC expression and normal flowering. To test this hypothesis, we first examined the rate of translation and degradation of SAP18 protein in both wildtype (Col-0) and sr45-1 mutant. The results showed a greater rate of degradation of SAP18 protein in sr45-1 . To see if restoring nuclear SAP18 in sr45-1 would lead to a decrease in FLC expression and the normal flowering, transgenic plants were created by overexpressing a DEX-inducible gSAP18-GFP. The T2 generation was screened to confirm the expression of the transgene in the wildtype and the sr45-1 mutant. Under the confocal microscope, the Col-0 transgenics displayed GFP signals in the nucleus after treatment as expected. However, the sr45-1 transgenic had results that did and did not follow the expectations. This may be due to the transgene being inserted to different genome locations that affected its behavior. Next, we are crossing the confirmed Col-0 transgenic lines with sr45-1 to see how the transgene responds to DEX without SR45. Also from confocal microscopy, we observed distinct GFP spots along the edge of the nucleus suggesting that SAP18 may be deposited to condensed chromosomes. Further research is needed to identify the locations of SAP18 on these chromosomal loci.
References:
Chen, S. L., Rooney, T. J., Hu, A. R., Beard, H. S., Garrett, W. M., Mangalath, L. M., Powers, J. J., Cooper, B., & Zhang, X. N. (2019). Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana. Frontiers in plant science, 10, 1116.
Kim, D. H., & Sung, S. (2013). Coordination of the vernalization response through a VIN3 and FLC gene family regulatory network in Arabidopsis. The Plant cell, 25(2), 454–469.
Rosin, L. F., Crocker, O., Isenhart, R. L., Nguyen, S. C., Xu, Z., & Joyce, E. F. (2019). Chromosome territory formation attenuates the translocation potential of cells. eLife. 8: e49553.
Keywords: ASAP, protein regulation
102. Crosstalk and compensation between the Hog1 and Gcn2 pathways in C. neoformans stress adaptation.
David Goich (SUNY Buffalo, Department of Microbiology and Immunology), Amanda LM Bloom (SUNY Buffalo, Department of Microbiology and Immunology), Murat C Kalem (SUNY Buffalo, Department of Microbiology and Immunology), John C Panepinto (SUNY Buffalo, Department of Microbiology and Immunology)
Abstract:
Cryptococcus neoformans is an environmental fungus that causes severe opportunistic infections in immunocompromised individuals, particularly people living with HIV/AIDS. Upon entry into the host, C. neoformans rapidly alters its proteome to accommodate a variety of stressors, including elevated temperature, reactive oxygen species (ROS) in phagocytes, nutrient limitation, and more. Our previous work demonstrated that these processes are dependent in part on the p38 MAPK, Hog1, which couples the translational response to stress-sensing signal transduction. Our preliminary data found that signaling via the Gcn2 pathway, another stress-induced repressor of translation, is upregulated in a hog1∆ mutant strain. We generated a hog1gcn2∆∆ strain to investigate whether defects in translation regulation in hog1∆ are a result of, or masked by, hyperactivity of the Gcn2 pathway. First, we investigated whether loss of both kinases resulted in exacerbated growth defects under thermal and oxidative stresses, measured by spot dilution assays and kinetic growth curves. Using polysome profiling and northern blots, we also investigated whether loss of both kinases resulted in synergistic defects in global translation and repression of homeostatic mRNAs during stress. Lastly, we used RNA-seq to investigate whether the Gcn2-dependent transcriptional response was enhanced in hog1∆. Compared to either single knockout, we found that hog1gcn2∆∆ had exacerbated growth defects under thermal and oxidative stress, and that this was associated with defects in both global translation and repression of homeostatic transcripts. Surprisingly, hyperactivity of the Gcn2 pathway in hog1∆ led to enhanced transcriptional response in the context of thermal, but not oxidative stress. Our data suggest that crosstalk between Hog1 and Gcn2 is compensatory or redundant in regulation of translation and mRNA decay, but that the coupling of this crosstalk to transcription is stress-specific. Ongoing investigations will determine whether transcriptional and translational defects of hog1gcn2∆∆ are associated with attenuated virulence.
Keywords: translation, transcriptome, signaling
103. Upf1 is required for translational response to oxidative stress in Cryptococcus neoformans
Sean R. Duffy (Department of Microbiology and Immunology, SUNY Buffalo), John C. Panepinto (Department of Microbiology and Immunology, SUNY Buffalo)
Abstract:
Nonsense-mediated decay (NMD) is an evolutionarily conserved pathway that is important for mRNA quality control and is initiated by the RNA helicase Upf1. NMD targets mRNAs which possess premature termination codons (PTCs) resulting from DNA damage, RNA damage, transcriptional errors, or PTCs programmed into the mRNA sequence. The fungal pathogen Cryptococcus neoformans must adapt to the host-lung environment, which includes temperature and oxidative stress. In response to stress, C. neoformans represses translation by decreasing translation initiation, clearing homeostatic mRNAs, and promoting the translation of mRNAs necessary for stress adaptation. Previous studies have investigated the role of Upf1 in the retention of introns and the control of translation through start codon context in C. neoformans. However, the role of Upf1 during stress adaptation remains unknown. Utilizing a upf1Δ mutant we found that translation is more readily repressed by oxidative stress in the absence of NMD. In polysome profiles, the upf1Δ mutant showed a greater decrease in high molecular weight complexes than the WT strain after exposure to hydrogen peroxide. Western blotting also revealed that upf1Δ displays increased eIF2α phosphorylation, a known hallmark for global translation inhibition, at lower peroxide concentrations. Northern blot assessment of the ribosomal protein transcript, RPL2, also demonstrated slower decay of homeostatic transcripts in the upf1Δ mutant following oxidative stress. Upf1 along with the other core NMD factors Upf2 and Upf3 are conserved in both humans and yeast. However, the additional cofactors involved in targeting NMD-regulated transcripts for decay vary. BLAST analyses revealed two genes, CNAG_05233 and CNAG_07351, in C. neoformans which encode for proteins that have sequence similarity and domain conservation to the human NMD cofactors, hSmg5/7, and hSmg6, respectively, than to the yeast NMD cofactors, Ebs1p and Nmd4p, respectively. Future work will aim to explore the NMD complex in C. neoformans and understand the mechanism by which Upf1 contributes to translational regulation and translatome reprogramming in response to oxidative stress.
Keywords: Translation, NMD, Stress
104. Ribosomal protein bL38 a uniquely conserved component of the Bacteroidia ribosome
Md Siddik Alom (1Ohio State Biochemistry Program, 2Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Zakkary A. McNutt (1Ohio State Biochemistry Program, 2Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Kurt L. Fredrick (1Ohio State Biochemistry Program, 2Center for RNA Biology, 3Department of Microbiology The Ohio State University, Columbus, OH 43210, USA)
Abstract not available online - please check the booklet.
105. Title not available online - please see the booklet.
Jillian Wagner (Biological Chemistry and Pharmacology; The Ohio State University), Ian Price (Biological Chemistry Pharmacology; Center for RNA Biology; The Ohio State University), Benjamin Pastore (Biological Chemistry Pharmacology; Center for RNA Biology; The Ohio State University), Hannah L. Hertz (Biological Chemistry and Pharmacology; The Ohio State University), Wen Tang (Biological Chemistry Pharmacology; Center for RNA Biology; The Ohio State University)
Abstract not available online - please check the booklet.
106. C. neoformans casein kinase Yck2 contributes to translational reprogramming and cell wall remodeling during temperature stress
Amanda L.M. Bloom (State University of New York University at Buffalo), John C. Panepinto (State University of New York University at Buffalo)
Abstract not available online - please check the booklet.
107. The dichotomy of MDM2 splicing regulation in response to genotoxic stress
Zac R. LaRocca-Stravalle (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital), Hannah Ackerman (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital), Andrew K. Goodwin (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital), Dawn S. Chandler (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital)
Abstract:
MDM2 is a negative regulator of the tumor suppressor protein p53. Under genotoxic stress, MDM2 alternative splicing is upregulated to produce MDM2-ALT1. MDM2-ALT1 differs from its full-length form by the skipping of exons 4 though 11 and retention of the outermost coding exons 3 and 12. However, it is unclear how the skipping of these exons is regulated. We hypothesize two models of exon skipping regulation in MDM2 pre-mRNA: (1) exon skipping is independently regulated such that exons contain binding sites that regulate their own splicing within the MDM2 transcript (exon autonomous model), (2) and skipping occurs as a single event (cassette regulon model).
To test these two models, we used a damage-inducible MDM2 minigene system. Previously, we identified conserved binding sites of splicing regulatory proteins within exon 11 that induce alternative splicing under stress – an exon autonomous event. To assess splicing regulation of other intervening exons, we used MDM2 minigenes containing an exon 4 to 10 and exposed them to genotoxic stressors, UV and cisplatin, in vitro.
We show that the minigene containing exon 4 undergoes damage-inducible alternative splicing, like exon 11. However, exons 5 to 10 were not differentially spliced in response to stress, suggesting a cassette regulon model in which exons 4 and 11 include sequences important for the splicing regulon of their intervening exons. However, these minigenes excluded intronic sequences flanking their exons. Importantly, intronic sequence mutations are known to alter splicing in cancer. Thus, to further interrogate the regulon model and test the importance of adjacent intronic sequences, we created the same minigenes but with flanking intronic regions.
Our current results support the first evidence of a cassette regulon model, which has important implications on how we understand exon recognition and alternative splicing regulation, and may serve to provide insights into new therapeutic targets.
Keywords: alternative splicing, MDM2, genotoxic stress
108. Regulation of pd-l1 expression through splice-switching oligonucleotide treatment.
John Kipp (Center for Life Science EducationCenter for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Akila S. Venkataramany (Medical Scientist Training Program and Biomedical Sciences Graduate Program, OSUCOM; Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Kevin Cassady, MD (Dept. of Pediatrics, Div. of Pediatric Infectious Diseases, Nationwide Childrens Hospital; Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Dawn S. Chandler, PhD (Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital; Dept. of Pediatrics, OSUCOM; Molecular, Cellular and Developmental Biology Graduate Program, OSU)
Abstract:
Programmed death-ligand (PD-L1), a transmembrane protein that interacts with the PD-1 receptor, is highly expressed in various pediatric malignancies and serves as a mechanism for tumor cells to evade immune response and develop resistance against endogenous apoptotic pathways and current immunotherapies. Anti-PD-L1 monoclonal antibody therapies have been clinically successful, yet limited response in other cancers and debilitating side effects in patients highlight a need for novel PD-L1-based therapies. Targetable regions include the IgV-like extracellular domain in exon 2, which is responsible for correct cellular localization and interaction with the PD-1 receptor. We hypothesize that down-regulation of PD-L1 expression by forcing defective pre-mRNA splicing of exon 2 will be a viable approach to ablate PD-L1 immunosuppressive activity in the tumor.
To test our hypothesis, we designed splice-switching oligonucleotides (SSOs) that block the 5’ and 3’ splice sites of PD-L1 exon 2. Skipping PD-L1 exon 2 would promote the expression of a non-functional PD-L1 pre-mRNA transcript and down-regulate the PD-L1 protein. We transfected Rh30 cells, which detectably express PD-L1, with either 5’, 3’, or 5’+3’ SSOs for 24 hours and harvested RNA to measure endogenous PD-L1 RNA levels and exon 2 skipping via RT-PCR. Simultaneously, we designed and successfully cloned a chimeric PD-L1 minigene, which includes exon 2 and the flanking intronic regions. We will transfect this minigene in HeLa cells to further validate our SSOs and study the regulation of PD-L1 alternative splicing.
While this project is in its beginning stages, the results from our study will be the foundation for developing an SSO therapy targeting PD-L1 expression in pediatric cancers. In addition, understanding the utility of RNA-based therapies that manipulate alternative splicing will be beneficial for novel therapies for any cancer with PD-L1-induced immunotherapy resistance.
Keywords: PD-L1, splice-switching oligonucleotides
109. Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay
Paul J Russell (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210 USA), Jacob A. Slivka (Department of Biological Chemistry and Pharmacology, Department of Computer Science and Engineering, The Ohio State University, Columbus, OH 43210 USA), Elaina P. Boyle (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210 USA), Arthur H. M. Burghes (Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43210 USA), Michael G. Kearse (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210 USA)
Abstract:
It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5′ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5′ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.
Keywords: ribosome, translational control, mRNA stability
110. Title not available online - please see the booklet.
Rushdhi Rauff (Department of Chemistry and Biochemistry, Kent State University), Paul DAmico (Department of Chemistry and Biochemistry, Kent State University), Sudeshi Abedeera (Department of Chemistry and Biochemistry, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)
Abstract not available online - please check the booklet.
111. Title not available online - please see the booklet.
Sudeshi Abedeera (Department of Chemistry and Biochemistry, Kent State University), Mohamed Rushdhi (Department of Chemistry and Biochemistry, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)
Abstract not available online - please check the booklet.
112. Selective mRNA oxidation of the mitochondrial electron transport chain complex subunits in human neuronal cells dysregulate energy production in Multiple Sclerosis
Thulasi Mahendran (Chemistry and Biochemistry), Soumitra Basu (Chemistry and Biochemistry)
Abstract not available online - please check the booklet.
113. Title not available online - please see the booklet.
Jiale Xie (Chemistry & Biochemistry, Kent State University ), Rushdhi Rauff (Chemistry & Biochemistry, Kent State University ), Sanjaya Abeysirigunawardena (Chemistry & Biochemistry, Kent State University )
Abstract not available online - please check the booklet.
114. mRNA modifications impact stop codon recognition
Zezhong Wan (Department of Chemistry, University of Michigan), Tyler J. Smith (Department of Chemistry, University of Michigan), Kristin S. Koutmou (Department of Chemistry, University of Michigan)
Abstract:
As templates for protein synthesis by the ribosome, mRNAs use the four standard nucleosides to record the sequence of amino acids. In addition to A, C, G and U, they can also possess modified nucleobases that impact protein expression. Present in all phylogenetic domains, there is significant chemical diversity among modified RNA nucleosides 1, including the addition of atoms (e.g. H), functional groups (e.g. methyl-, acetyl-, or carboxyl-)2 or nucleoside isomers (e.g. pseudouridine)3. At the molecular level, mRNA modifications have the potential to affect the lifespan and activity of transcripts by altering post-transcriptional splicing, translation, and decay4. Among the entire process, translation is central to protein synthesis. The initiation and elongation steps of translation are dependent on mRNA/tRNA interactions, while termination requires recognition of stop codon by release factors (RF)5. However, how and when these steps in the protein synthesis pathway can be affected by chemical modifications to single nucleobases still remain unclear. Here we explored the impact of modified bases on translation termination. We utilized a fully-reconstituted E.coli in vitro translation system to examine how the inclusion of a series of guanosine modifications in stop codons (UGA and UAG) influences the kinetics of peptide release. Our results suggest that the rate of peptide release is highly dependent on H-bond donor/acceptor positions around the purine ring, and indicate that the H-bond between N1 site on the purine ring and residues in RF domains is potentially crucial for RF recognition of stop codons.
References:
1.Roundtree, Ian A., et al. "Dynamic RNA modifications in gene expression regulation." Cell 169.7 (2017): 1187-1200.
2.Cantara, William A., et al. "The RNA modification database, RNAMDB: 2011 update." Nucleic acids research 39.suppl_1 (2010): D195-D201.
3.Limbach, Patrick A., Pamela F. Crain, and James A. McCloskey. "Summary: the modified nucleosides of RNA." Nucleic acids research 22.12 (1994): 2183-2196.
4.Li, Sheng, and Christopher E. Mason. "The pivotal regulatory landscape of RNA modifications." Annu Rev Genomics Hum Genet 15.1 (2014): 127-150.
5.Korkmaz, Gürkan, et al. "Comprehensive analysis of stop codon usage in bacteria and its correlation with release factor abundance." Journal of Biological Chemistry 289.44 (2014): 30334-30342.
Keywords: mRNA modification, translation, stop codon
115. Structural and kinetic characterization of multiple oligomeric states in a minimal protein-only RNase P
Catherine A. Wilhelm (Department of Chemistry, University of Michigan), Abigail L. Kelly, Shayna B. Brotzman, Johnny Mendoza, Suada Leskaj (Department of Chemistry, University of Michigan), Leena Mallik (Center for Structural Biology, Life Sciences Institute, University of Michigan), Markos Koutmos (Departments of Chemistry and Biophysics, University of Michigan)
Abstract:
Transfer RNA (tRNA) genes are often transcribed as precursor tRNAs (pre-tRNAs) with additional leader and trailer sequences. In one of the first steps of the extensive processing to convert pre-tRNA to mature tRNA, RNase P enzymes cleave the 5’-end of pre-tRNA in all domains of life. Homologs of Aquifex RNase P (HARPs) are a newly discovered class of RNase P consisting of only a metallonuclease domain. HARPs are minimal proteins compared to their ribonucleoprotein (RNP) and protein-only RNase P (PRORP) counterparts and they appear to lack the usual substrate recognition domain. Despite these features, HARPs maintain the ability to bind to and cleave the 5’ leader sequence of tRNAs.
To improve our understanding of how this minimal RNase P enzyme, we used a combination of x-ray crystallography, cryo-EM, and biochemical assays. In this work, we solved the cryo-EM structure of A. thaliana HARP, which is a ring-like hexamer of dimers, and the crystal structure of H. thermophilus HARP, a ring-like heptamer of dimers. These structures reveal the active site is approximately 42 Å away from a helical region of positively charged residues – the perfect distance for the acceptor stem of a pre-tRNA. Curious about the potential for multiple oligomeric states of HARP, we used native ion-mobility mass spectrometry to identify multiple oligomeric states in solution we had previously not detected. We also discovered that HARPs bind pre-tRNAs in multiple oligomeric states, likely as both a tetramer and a dodecamer. To round out our studies, we determined that HARPs specifically cleave and bind pre-tRNA from multiple species with activity similar to other PRORPs. This data provides a closer look at the similarities in tRNA binding recognition and processing across disparate classes of RNase P.
References:
Nickel et al. (2017) Minimal and RNA-free RNase P in Aquifex aeolicus PNAS 114(42) 11121-11126.
Feyh et al. (2021) Structure and mechanistic features of the prokaryotic minimal RNase P eLife 10:e70160.
Keywords: RNase P, tRNA, RNA processing
116. Ablation of the imprinted PWS-orthologous domain in a rat insulinoma cell line reveals cell-autonomous transcriptional deficiencies in peptide hormones and ER chaperones independent of ER stress
Erik A. Koppes (Department of Pediatrics, Division of Genetic & Genomic Medicine, School of Medicine, University of Pittsburgh), Marie A. Johnson (Department of Pediatrics, Division of Genetic & Genomic Medicine, School of Medicine, University of Pittsburgh), Hyun J. Park (Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh), Peter Drain (Department of Cell Biology, School of Medicine, University of Pittsburgh), Robert D. Nicholls ( Department of Pediatrics, Division of Genetic & Genomic Medicine, School of Medicine, University of Pittsburgh)
Abstract:
The Prader-Willi syndrome (PWS) imprinted domain spans multiple protein-coding and non-coding paternally expressed genes, including extended lncRNAs encompassing bicistronic SNURF-SNRPN and five classes of box-C/D snoRNAs. Characteristic PWS clinical features of neonatal hypotonia, juvenile onset hyperphagia and obesity, hormone deficiencies, and additional comorbidities manifest when this domain is paternally deleted or bimaternally inherited. Previously, we established a neonatally lethal hypoglycemia phenotype subsequent to hypoinsulinemia and hypoglucagonemia in a PWS mouse model (1, 2). To delineate pathophysiological mechanisms, we engineered a 3-Mb deletion of the PWS-orthologous domain in rat INS-1 cells and identified cell-autonomous deficits in basal and glucose-stimulated insulin secretion (3). Transcriptomic analysis by RNA-Seq revealed decreased expression of peptide hormones Iapp, Npy and an Insulin::mCherry transgene and for numerous ER-chaperones, critical to their production. These changes were validated by congruent proteomic and targeted RT-ddPCR and western blot experiments. While PWS INS-1 lines had a propensity for a more robust early chemically induced activation of ER-stress pathways, including IRE1α/XBP1, ATF6 and eIF2α, there was no basal activation of ER-stress or the unfolded protein response. Additionally, both control and PWS β-cells similarly activated profound ER-stress transcriptome-wide changes at 5 hours of thapsigargin treatment and had widespread changes in alternative splicing. Genes with robust activation on ER-stress in INS-1 cells included Atf3, Trib3, Ddit3 and Rcn1. Intriguingly, differential RNA-splicing or intron-retention events occurred for the Snrpn ancestral paralog Snrpb, the SR-protein splicing factor Srsf3, and multiple ribosomal protein genes with embedded snoRNA genes. Current studies are further assessing molecular events underlying transcriptional alterations in PWS β-cells and in β-cells under ER stress.
References:
1. Stefan M, et al. (2005) Hormonal and metabolic defects in a Prader-Willi syndrome mouse model with neonatal failure to thrive. Endocrinology 146, 4377-85
2. Stefan M, et al. (2011) Global deficits in development, function, and gene expression in the endocrine pancreas in a deletion mouse model of Prader-Willi syndrome. AJP-Endo 300, E909-22
3. Koppes EA, et al. (2022) Insulin secretion deficits in a Prader-Willi syndrome β-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones. https://www.biorxiv.org/content/10.1101/2021.12.16.473032v3
Keywords: RNA-Seq, ncRNA, Alternative Splicing
117. Investigating the relationship between structure and function of htrA RNA Thermometer
JaeEun Lee (Department of Chemistry and Biochemistry, Denison University), Rachel Mitton-Fry (Department of Chemistry and Biochemistry, Denison University)
Abstract not available online - please check the booklet.
118. Stress-induced translational control induces the ISR in the human pathogen Cryptococcus neoformans
Corey M. Knowles (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA), David Goich (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA), Gemma E. May (Department Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA), Joel McManus (Department Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA), John C. Panepinto (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA)
Abstract:
Cryptococcus neoformans is a ubiquitous environmental fungus and opportunistic human pathogen, primarily impacting immunocompromised hosts such as those living with HIV/AIDS. One of C. neoformans’ key virulence traits is its ability to rapidly adapt to the harsh environment inside the human lung. Here, it is subject to both a sudden temperature shift to 37°C and oxidative stress from resident lung macrophages, among other stressors. Exposure to these stressors is met with, and combated by, an initial repression of translation and induction of the integrated stress response (ISR), resulting in a reprogrammed stress-responsive translatome. To better understand the mechanisms involved in the translational response to host stress, we’ve begun to characterize the C. neoformans response to these stressors in culture. We find oxidative stress causes robust eIF2α phosphorylation, Gcn2-dependent polysome collapse, and mild decay of ribosomal protein (RP) mRNAs. Conversely, temperature stress is met with robust RP mRNA decay and only moderate eIF2α phosphorylation, yet leads to similar polysome collapse that is Gcn2-independent. Interestingly, ribosome collisions, as visualized by disome profiling, are induced in response to temperature stress, while no increase in disomes is seen in response to oxidative stress. This difference was unexpected given recent work linking ribosome collisions to eIF2&alpha phosphorylation. Although the amount of eIF2α phosphorylation varies in response to each stressor, translational activation of C. neoformans Gcn4 is comparable, suggesting similar induction of the ISR. Despite comparable production of Gcn4 protein, the magnitude of ISR target mRNA induction varies with the type and intensity of the stressor. Future work will seek to identify mRNAs whose translation changes in response to these stresses and evaluate mRNA features that specify their fates.
Keywords: translation regulation, stress response
119. Sequestration of the anti-Shine-Dalgarno (ASD) causes reduced efficiency of prfB programmed frameshifting in Flavobacterium johnsoniae
Fawwaz Naeem (Ohio State Biochemistry Program, The Ohio State University), Zakkary A. McNutt (Ohio State Biochemistry Program, The Ohio State University), Kurt Fredrick (Microbiology Department, The Ohio State University)
Abstract:
In bacteria, two release factors are responsible for translation termination—RF1 and RF2. Production of RF2 is autoregulated in most bacteria (> 80%) via programmed frameshifting. In short, this entails a competition between RF2-dependent termination at an in-frame UGA codon and +1 frameshifting of peptidyl-tRNALeu on the slippery sequence CUUU. The frameshifting event is promoted by a SD-like sequence closely juxtaposed to the slippery sequence. Ribosomes of Bacteroidia fail to recognize SD sequences due to sequestration of the 3’ tail of the 16S rRNA on the 30S platform. Yet, in these organisms, the prfB gene typically contains the frameshift site with its characteristic SD-like sequence. Here we investigate prfB autoregulation in Flavobacterium johnsoniae, a member of the Bacteroidia. We show that the efficiency of prfB frameshifting in F. johnsoniae is low (5-6%) relative to that in E. coli (~50%). Mutation or truncation of bS21 in F. johnsoniae increases frameshifting substantially, suggesting that ASD sequestration is responsible for the reduced efficiency. The frameshift site of certain Flavobacteriales, such as Winogradskyella psychrotolerans, has lost the SD-like sequence. In F. johnsoniae, this W. psychrotolerans sequence supports frameshifting at 3-5%, which is unaffected by mutation of bS21. Collectively, these data suggest that prfB frameshifting is largely independent of SD-ASD pairing in the Bacteroidia and consequently tuned down.
Keywords: Flavobacterium johnsoniae , Ribosome, prfB frameshifting
120. Identification of inhibitors of Methanobrevibacter smithii RNase P using small molecule microarrays
Vaishnavi Sidharthan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Kara Dunne-Dombrink (Chemical Biology Laboratory, National Cancer Institute), Walter J. Zahurancik, Ila A. Marathe, Lien B. Lai (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), John S. Schneekloth Jr. (Chemical Biology Laboratory, National Cancer Institute), Venkat Gopalan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University)
Abstract not available online - please check the booklet.
121. Reversible Acylating Reagents to Dictate RNA’s Fate
Kaizheng Liu (Department of Chemistry, Carnegie Mellon University), Marta Rachwalak (Department of Chemistry, Carnegie Mellon University), Anna M. Kietrys (Department of Chemistry, Carnegie Mellon University)
Abstract:
RNA transcripts appear in various forms and play diverse roles in cellular processes[1,2]. New methods to manipulate RNA function and interactions increase the potential of revealing its new biochemical significances[3–5]. Recently, the 2′-OH acylation has gained wide attention due to its one-step procedure, high efficiency, and reversibility. Taking advantage of these findings, we have been working on the development of universal, effective, and biocompatible acylating reagents for understanding RNA interactome and gaining control over the biological function of various RNAs.
Herein, we present two approaches of molecular tools designed to acylate 2′-OH groups in single-stranded regions of RNA. The first strategy is based on the possibility of linking two RNA strains which would significantly disrupt RNA structure and function. Each probe contains two photocleavable groups connected by linkers of different chemical structures. Thanks to photosensitive units, the RNA function may be fully restored by UV light. The RNA will first be silenced by acylation in vitro, followed by its transfection into cells. To further develop this idea and find a fully biocompatible approach for in vivo applications, we have developed another set of reagents sensitive to enzyme. In these molecules, the disulfide bond acts as a thioredoxin reductase (TrxR) cleavable site, and self-immolative group enables the recovery of 2′-OH. Such design allows us to correlate the RNA activity with TrxR concentration. In cells with an elevated level of TrxR, treated RNA can be uncaged and alter gene expression. We have tested both approaches and optimized conditions for in vitro acylation of oligo RNAs. To show the potential biological application of proposed strategies, we plan to test these probes in cellulo. We believe that these molecules will serve as chemical tools to dictate RNA’s fate in cells. Additionally, designed reagents may help to map short and long-distance interactions in RNA complexes.
References:
(1) Hsiao, K. Y.; Sun, H. S.; Tsai, S. J. Circular RNA – New Member of Noncoding RNA with Novel Functions. Exp Biol Med 2017, 242 (11), 1136–1141.
(2) Cech, T. R.; Steitz, J. A. The Noncoding RNA Revolution—Trashing Old Rules to Forge New Ones. Cell 2014, 157 (1), 77–94.
(3) Velema, W. A.; Park, H. S.; Kadina, A.; Orbai, L.; Kool, E. T. Trapping Transient RNA Complexes by Chemically Reversible Acylation. Angew Chem Int Ed Engl 2020, 59 (49), 22017–22022.
(4) Knutson, S. D.; Sanford, A. A.; Swenson, C. S.; Korn, M. M.; Manuel, B. A.; Heemstra, J. M. Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging. J Am Chem Soc 2020, 142 (41), 17766–17781.
(5) Kadina, A.; Kietrys, A. M.; Kool, E. T.; Kadina, [ A; Ietrys, A. M. K.; Kool, E. T. RNA Cloaking by Reversible Acylation. Angewandte Chemie International Edition 2018, 57 (12), 3059–3063.
Keywords: RNA acylation, RNA Interactome , Gene regulation
122. Understanding canonical T-box riboswitches: A study of the Bacillus subtilis thrS RNA
Alexander T. Runyon (Microbiology Department, The Ohio State University), Jane K. Frandsen (Ohio State Biochemistry Program, The Ohio State University), Rebecca N. Wiliams-Wagner (Microbiology Department, The Ohio State University), Tina M. Henkin (Microbiology Department, The Ohio State University)
Abstract:
T-box riboswitches regulate many amino acid-related genes in Gram-positive bacteria. These cis-acting RNA structures utilize an uncharged tRNA corresponding to the downstream gene as a regulatory ligand. Roughly 95% of T-box RNAs are predicted to adopt a canonical structure, composed of several RNA motifs. Previous biochemical and structural studies of T-box riboswitches have focused primarily on T-box RNAs that lack one or more conserved structural elements, as in vitro work with a canonical T-box RNA has not been possible. We have demonstrated specific tRNAThr binding to the canonical Bacillus subtilis thrS T-box RNA and tRNAThr-dependent antitermination in a purified in vitro system. Point mutations were generated in conserved RNA motifs of the thrS riboswitch, and the structural and functional effects were investigated using SHAPE, in vitro transcription assays, tRNA binding assays, and in vivo RT-qPCR assays. These studies revealed that all conserved elements, including those absent from the noncanonical T-box RNAs, are required for function both in vivo and in vitro. Our analysis of the thrS riboswitch has provided a more robust understanding of how all of the key structural elements interact in canonical T-box RNAs, which represent the majority of these RNAs found in nature.
References:
1 Grundy FJ, Henkin TM. 1993. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74:475–482.
2 Sherwood AV, Frandsen JK, Grundy FJ, Henkin TM. 2018. New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch. Proc Natl Acad Sci USA 115:3894–3899.
3 Putzer H, Condon C, Brechemier-Baey B, Brito R, Grunberf-Manago M. 2002. Transfer RNA‐mediated antitermination in vitro. Nucleic Acids Res. 30:3026–3033.
4 Suddala KC, Zhang J. 2019. High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nat Struct Mol Biol 26:1114–1122.
5 Rollins SM, Grundy FJ, Henkin TM. 1997. Analysis of cis-acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene. Mol Microbiol 25:411–421.
Keywords: tRNA, Riboswitch, T-box
123. RNP granule condensation: a potential mechanism for post-transcriptional regulation of the yeast-to-hyphal transition in Candida albicans
Melissa A. Tosiano (Biological Sciences, Carnegie Mellon University ), Fred Lanni (Biological Sciences, Carnegie Mellon University ), Gemma E. May (Biological Sciences, Carnegie Mellon University ), C. Joel McManus (Biological Sciences, Carnegie Mellon University )
Abstract:
Localization of mRNA to ribonucleoprotein granules (RNPs) such as P-bodies (PBs) and stress granules (SGs) is an integral part of the eukaryotic stress response. RNPs have been extensively studied in S. cerevisiae where they are important for translational repression as well as reprogramming the transcriptome. In diverse yeast species, DEAD-box helicase Ded1p acts as a translation initiation factor during active growth1. Conversely, heat shock induces Ded1p condensation, selectively repressing housekeeping mRNAs in SGs1. However, the extent to which these functions of RNPs and Ded1p are conserved in other fungi remains opaque.
The human opportunistic fungal pathogen Candida albicans responds to heat stress and starvation by forming filamentous hyphae, which are strongly linked to virulence2. Prior work demonstrated that C. albicans forms PBs and SGs in response to various stressors3-4, and the PB component Edc3p is required for typical filamentation4. However, the molecular mechanisms by which PBs promote filamentation have not been determined. Additionally, the importance of SGs and Ded1p in this phenotypic shift has not been investigated.
To examine RNP formation during C. albicans filamentation, we fluorescently tagged canonical SG (PAB1) and PB (EDC3) markers as well as DED1 in the genome and examined their condensation. We found that PBs, SGs, and Ded1p condense in response to acute heat shock. Interestingly, SGs and Ded1p fail to form condensates during physiologically relevant filamentation conditions, while abundant PBs condense along the developing germ tube. In the future we will examine the transcriptome, morphology, and virulence of an edc3/edc3 strain to elucidate the contribution of PBs to C. albicans filamentation.
References:
1. Iserman, C. et al. Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production. Cell 181, 818-831.e19 (2020).
2.Thompson, D. S., et al. Coevolution of morphology and virulence in Candida species. Eukaryot. Cell 10, 1173–1182 2011.
3. O’Meara, T. R. et al. Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen. PLoS Biology 17, 1-38 (2019).
4. Jung, J. H. & Kim, J. Accumulation of P-bodies in Candida albicans under different stress and filamentous growth conditions. Fungal Genet. Biol. 48, 1116–1123 (2011).
Keywords: P-Bodies, C albicans, post-transcriptional regulation
124. The influence on transcription and translation by the TRMT1 tRNA modification enzyme
Chenghong Deng (Biology, University of Rochester), Kejia Zhang (Biology, University of Rochester), Dragony Fu (Biology, University of Rochester)
Abstract:
A series of post-transcriptional modifications are placed on transfer RNAs (tRNA) in all organisms. As one example, tRNA methyltransferase 1 (TRMT1) is a tRNA modifying enzyme which methylates a guanine residue at position 26 to form dimethylguanosine (m2,2G). Pathogenic variants in TRMT1 cause intellectual disability disorders in the human population. However, the link between TRMT1 and gene expression is unknown. To understand how TRMT1 affects gene expression and protein translation, we performed transcriptomics and ribosome profiling on wild-type or TRMT1-deficient human cells. At the transcriptional level, we identified a gene called BASP1 that is upregulated by 2-fold in TRMT1-deficient human cells. BASP1 encodes a brain-abundant membrane-attached signal protein. By upregulation of BASP1, the signal transmission will also be upregulated in TRMT1 – deficient cells. The results indicate that TRMT1 is important for maintaining the expression level for a subset of genes. In the future, we plan to use a TRMT1-deficient mouse model to further investigate the genes and pathways linked to TRMT1 in human cells.
References:
Dewe, Joshua M., Benjamin L. Fuller, Jenna M. Lentini, Stefanie M. Kellner, and Dragony Fu. 2017. “TRMT1-Catalyzed TRNA Modifications Are Required for Redox Homeostasis To Ensure Proper Cellular Proliferation and Oxidative Stress Survival.” Molecular and Cellular Biology 37 (21): e00214-17. https://doi.org/10.1128/MCB.00214-17.
Zhang, Kejia, Jenna M Lentini, Christopher T Prevost, Mais O Hashem, Fowzan S Alkuraya, and Dragony Fu. 2019. “A Missense Variant Impairing TRMT1 Function in TRNA Modification Is Linked to Intellectual Disability.” Preprint. Molecular Biology. https://doi.org/10.1101/817247.
Keywords: tRNA modification, TRMT1
125. PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing
Katherine Tucker (Department of Cancer Biology and Genetics and the Comprehensive Cancer Center, The Ohio State University, 460 West 12th Avenue, Columbus, OH 43210, USA), Swetha Rajasekaran, Eshan Khan, Misbah Khan, Jalal K. Siddiqui, Chenyu Lin (Department of Cancer Biology and Genetics and the Comprehensive Cancer Center, The Ohio State University, 460 West 12th Avenue, Columbus, OH 43210, USA), Wayne O. Miles (Department of Cancer Biology and Genetics and the Comprehensive Cancer Center, The Ohio State University, 460 West 12th Avenue, Columbus, OH 43210, USA), Samuel R. Ching, Stephanie J. Bouley, James A. Walker (Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA), Dayna L. Mercadante, Amity L. Manning (Department of Biology and Biotechnology and Bioinformatics and Computational Biology Program, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609, USA), Dayna L. Mercadante, Andr P. Gerber (Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, UK; Department of Genetics, Federal University of Parana, Curitiba, Brazil)
Abstract not available online - please check the booklet.
126. Transcription regulation by CRISPR/dCas9 complex in the context of G-quadruplex structure
Mohammed Enamul Hoque (Department of Chemistry and Biochemistry, Kent State University), Hamza Balci (Department of Physics, Kent State University), Soumitra Basu (Department of Chemistry and Biochemistry, Kent State University)
Abstract:
CRISPR-Cas is an RNA-mediated adaptive immune system found in bacteria and archaea that protects host cells from foreign DNA invasion. CRISPR-Cas9 is guided by a single guide RNA (sgRNA), which hybridizes with complementary genomic sequences and induces a double-stranded break (DSB). The use of an engineered nuclease-deficient Cas9, known as dCas9, allows the system to be repurposed for targeting genomic DNA without cleaving it. According to recent studies, dCas9 is a versatile, RNA-guided DNA recognition platform that enables precise, scalable, and robust RNA-guided transcription regulation. There is a prevalence of guanosine-rich sequences in the human genome and transcriptome and such sequences have a propensity to adopt secondary structures called G-quadruplexes (GQ). Despite abundant information and practical knowledge about the CRISPR-Cas9 technology, its capabilities and limitations in targeting DNA GQ structures have not yet been systematically investigated. To address this knowledge gap, we utilized CRISPR/dCas9 system for targeting in the vicinity of GQ forming sequence of human tyrosine hydroxylase (TH) gene promoter and discovered that TH mRNA expression levels could both be up or down regulated. This interesting observation still needs a mechanistic explanation which would require an understanding of how the interactions between dCas9 and RNA polymerase (RNAP) is affected by the neighboring GQ structures. When GQ was present in the non-template strand, only a full-length RNA band was observed. However, when GQ was present in the template strand, we observed some truncated RNA bands in addition to the full-length RNA. The truncated RNA products could be due to the GQ-mediated stalls on the RNAP progression. Then, we targeted the same GQ containing sequence by using CRISPR/dCas9 complex in four different sites of the PQS, two in the template and the remaining two in the non-template strand. When target site was on the non-template strand of T7 RNAP, we found that dCas9 served as the dominant block compared to the GQ stall. Interestingly, when target site was on the template strand, GQ served as the dominant block, with no dCas9 blockade. Furthermore, smFRET experiment will be performed to gain the mechanistic and dynamic understanding of the interactions among RNAP, dCas9 and GQ.
Keywords: Transcription Regulation, CRISPR-dCas9, G-quadruplex
127. Deciphering the principles of RNA binding specificity of multi-domained proteins using non-canonical amino acids
Vikas Kaushik (Biochemistry and Molecular Biology, Saint Louis University, School of Medicine), Rahul Chadda (Biochemistry and Molecular Biology, Saint Louis University, School of Medicine), Abhinav Vayyeti, Sonia Patel (Biochemistry and Molecular Biology, Saint Louis University, School of Medicine), Richard Cooley (Biochemistry and Biophysics, Oregon State University), Ryan Mehl (Biochemistry and Biophysics, Oregon State University), Edwin Antony (Biochemistry and Molecular Biology, Saint Louis University, School of Medicine)
Abstract:
RNA binding proteins (RBPs) regulate the life cycle of RNA by recognizing the correct substrate and appropriately directing their nuclear export, translation/degradation, and intracellular localization of target transcripts. Here, we focus on the family of multi-domained onco-fetal RNA binding Insulin-like Growth Factor 2 mRNA-Binding Proteins (IGF2BP or IMP). IMP1, IMP2, and IMP3 are the three orthologous members in this family and are structurally composed of six RNA binding domains (RBDs): four KH and two RRM domains. While structurally homologous, they recognize several hundred individual transcripts and define their cellular fates. Yet, how they impart functional specificity is poorly understood. We propose that individual RBDs are differentially regulated and positioned to drive specific outcomes. To test this hypothesis, we use non-canonical amino acid (ncAA) tools to probe the dynamics of individual RBDs while keeping the full-length protein intact. Here, results from positioning a site-specific fluorophore within a single RBD and site-specific phospho-Ser in IMP1 and IMP3 will be presented.
Keywords: IGF2BP, RBDs, ncAA
128. Deciphering the function of MDM2-ALT1 splice isoform using a novel mouse model.
Safiya Khurshid (Nationwide Childrens Hospital/ Ohio State University), Matias Montes (Nationwide Childrens Hospital/ Ohio State University), Claudia Kelly (Nationwide Childrens Hospital/ Ohio State University), Vincenzo Coppola (Ohio State University), Dawn Chandler (Nationwide Childrens Hospital/ Ohio State University)
Abstract not available online - please check the booklet.
129. Modulation of archaeal RNase P ribozyme activity by temperature- and metal-ion–dependent phase separation
Walter J. Zahurancik (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Gable M. Wadsworth, Priya R. Banerjee (Department of Physics, The State University of New York at Buffalo, Buffalo, NY 14260), Xiangze Zeng, Rohit Pappu (Department of Biomedical Engineering, Center for Science and Engineering of Living Systems, Washington University in St. Louis, St. Louis, MO 63130), Lien B. Lai, Vaishnavi Sidharthan, Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract not available online - please check the booklet.
130. A method for isolation of target circular RNAs through specific biotinylated-DNA probes
Megan L. Van Horn (Department of Chemistry, Carnegie Mellon University), Marta Rachwalak (Department of Chemistry, Carnegie Mellon University), Anna M. Kietrys (Department of Chemistry, Carnegie Mellon University)
Abstract:
Circular RNAs (circRNA) are a class of long, endogenous RNAs possessing a covalently closed continuous loop structure and are formed through a combination of precursor mRNA (pre-mRNA) linear splicing and backsplicing1. This creates a unique junction of the downstream exon’s 3’-end spliced to the upstream exon’s 5’-end. Circular RNAs have extended life span2 and versatile interaction with small RNAs3,4 and proteins, and are promising biomarkers for various diseases and pathologies.
While there is a growing number of methods that can be used to identify a particular circRNA within a given population, the majority of these methods are destructive or non-isolating. In other words, the circular RNA cannot be used in downstream applications or not without further processing. This includes various RNA-sequencing methods5, as well as 2D gel electrophoresis and gel traps, among others6. However, the ability to isolate a particular circRNA of interest in a non-destructive manner is desirable for various experimental applications, such as modification/labeling, RNA sequencing, or biochemical assays.
Our group is working on a novel experimental tool to isolate particular circular RNAs from total RNA content. From prior research, we are using two previously-identified circRNAs as targets for the initial round of testing: a mitochondrial circRNA originating from the COX3 coding region and a chromosome 19 circRNA originating from the MYH14 gene that is found to be expressed in the brain7. Synthetic fluorescently-tagged circRNA have been generated and validated. Biotinylated-DNA probes have been developed to specifically bind to the backsplice junction of a target circular RNA. The binding and pull-down efficiency of the biotinylated-DNA probes is being tested within the pool of synthetic circRNA and cellular ones. We believe that this method can be a useful tool for future studies utilizing populations of circular RNA.
References:
1. Xiao, J, Cohen, IR, Lajtha, A, et al. Circular RNAs, Biogenesis and Functions 2018, Vol. 1
2. Piwecka M, Glažar P, Hernandez-Miranda LR, et al. Science 2017.
3. Hansen TB, Jensen TI, Clausen BH, et al. Nature 2013. doi:10.1038/nature11993
4. Memczak S, Jens M, Elefsinioti A, et al. Nature 2013. doi:10.1038/nature11928
5. Panda, AC et al. Nucleic Acids Res. 2017.
6. Jeck, WR, Sharpless, NE, Nature Biotechnology 2014.
7. Rybak-Wolf, A, Stottmeister, C, Glažar, P, et al. Molecular Cell 2015. doi:10.1016/j.molcel.2015.03.027
Keywords: circular RNA, DNA probe
131. Establishing robust calibration curves for mass photometry-based measurements of RNAs and ribonucleoprotein complexes
Ila A. Marathe (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry-Guided Structural Biology, Center for RNA Biology; The Ohio State University), Stella M. Lai (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry-Guided Structural Biology, Center for RNA Biology; The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology; The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry-Guided Structural Biology, Center for RNA Biology; The Ohio State University)
Abstract:
Mass photometry (MP) measures masses of biomolecules using interferometric scattering microscopy1. This method can be used to rapidly assess sample heterogeneity, composition, and macromolecular interactions. Typically, molecules with known masses and similar optical properties as the analytes (e.g., proteins, nucleic acids) are used for calibration. However, calibrations generated with either RNA or protein may lead to incorrect mass assignments for ribonucleoprotein (RNP) complexes2. We sought to develop alternatives that would allow greater accuracy during MP studies of RNPs; mass measurements were independently validated by high-resolution native mass spectrometry (nMS). Preliminary studies were conducted using archaeal RNase P, an essential, multi-subunit, catalytic RNP3-5, consisting of one RNase P RNA (RPR) and five RNase P proteins (RPPs). RPR can be reconstituted with different suites of RPPs, to form RNPs of varying RNA:protein ratios. First, protein- and RNA-only calibrations were generated from the ratiometric contrasts obtained by MP-based measurements of biomolecules with known molecular masses. Beta-amylase and thyroglobulin (present as multimeric complexes) were used for protein calibration. In vitro transcribed RNAs of known sizes (including some larger RNAs generated by RNA engineering) were used for RNA-based calibrations. Masses of protein and RNA standards were measured with MP using appropriate calibrations and were found to be in good agreement (within 0 – 6%) of expected and nMS-generated masses. To evaluate the suitability and appropriateness of RNA and protein calibrations in estimating masses of RNP complexes, RPR was reconstituted with either two or five RPPs (67% and 49% RNA content respectively). Despite significant fractional mass contribution of the RPR, protein calibration provided more accurate mass measurements (0.5 – 2.6% of expected mass) compared to the RNA calibration (5 – 7% of expected mass). Studies with a more structurally diverse set of RNPs, followed by mass validation using nMS, are needed to establish optimal mass calibration protocols for RNPs.
References:
1. Young G and Kukura P. (2019), Annu. Rev. Phys. Chem., 70: 301-322.
2. Lai SH et al. (2021), iScience, 24: 103211.
3. Zahurancik WJ et al (2021), Meth. Enzymol., 649: 71-103.
4. Marathe IA et al (2021), Nucleic Acids Res., 49: 9444-9458.
5. Phan HD et al. (2022), Nucleic Acids Res, 50: 8154-8167.
Keywords: Mass photometry, Native mass spectrometry, Ribonucleoproteins
132. Entropy-driven Phase Transition of RNA
Gable M. Wadsworth (Department of Physics, The State University of New York at Buffalo), Xiangze Zeng (Department of Biomedical Engineering and Center for Biomolecular Condensates CBC, Washington University in St. Louis), Walter J. Zahurancik (Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, ), Lien B. Lai, Vaishnavi Sidharthan, Venkat Gopalan (Department of Chemistry & Biochemistry, Center for RNA Biology, The Ohio State University, ), Rohit V. Pappu (Department of Biomedical Engineering and Center for Biomolecular Condensates CBC, Washington University in St. Louis), Paul Pullara, Priya R. Banerjee (Department of Physics, The State University of New York at Buffalo)
Abstract:
RNAs are ubiquitously found alongside protein cofactors as components of ribonucleoprotein (RNPs) granules, which are thought to form and dissolve under the influence of reversible phase transitions. Recently, RNAs, specifically sequences with different repeat expansions have been observed to form independent foci in cells. The formation of protein-free, RNA foci is thought to be driven primarily by multivalent base pairing and stacking interactions. Utilizing temperature-controlled titrations, we uncover the surprising result that RNA molecules can undergo reversible phase transitions upon heating above RNA-specific threshold temperatures. This type of thermo-responsive phase transition, known as phase behavior with a Lower Critical Solution Temperature (LCST), is an entropy-driven transition. An assortment of systems including RNA homopolymers, inorganic polyphosphate, RNAs with trinucleotide repeats, catalytic RNase P RNA, and enhancer RNA all demonstrate LCST-type phase behavior in the presence of Mg2+. These observations suggest that the coupling of associative and segregative transitions known as phase separation coupled to percolation (PSCP) is driven, at least in part, by entropic factors. Such transitions cannot be ascribed to base pairing and base-stacking, which diminish as temperature increases. Instead, using molecular simulations we find that the LCST-type phase behavior in the simplest case of polyphosphate chains, which lack base pairing and stacking interactions, is driven by a combination of desolvation to minimize the entropic penalty associated with solvation, and Mg2+ ion-mediated bridging interactions among phosphate moieties within and across chains. Further, we find that the heat-induced RNA phase transition is tuned by the nucleobase composition and sequence patterning, giving rise to a set of generalizable rules to encode and manipulate thermo-responsive PSCP transitions of RNA molecules.
Keywords: phase separation, LCST transition, RNA condensates
133. Title not available online - please see the booklet.
Evan Corden (Carnegie Mellon University), Joel McManus (Carnegie Mellon University)
Abstract not available online - please check the booklet.
134. The use of RNA nanoparticles as vectors for ocular drug delivery
Mohammed Hassan (Chemistry Department / University of Cincinnati), Kevin Li (College of Pharmacy / University of Cincinnati), Patrick Limbach (Chemistry Department / University of Cincinnati)
Abstract not available online - please check the booklet.
135. Identification of Circular RNAs in Early Human Immunodeficiency Virus Infection
Jian Shi (Department of Chemistry at Carnegie Mellon University), Megan L. Van Horn (Department of Chemistry at Carnegie Mellon University), Marta Rachwalak (Department of Chemistry at Carnegie Mellon University), Anna M. Kietrys (Department of Chemistry at Carnegie Mellon University)
Abstract:
The Human Immunodeficiency Virus (HIV) targets the human immune system and compromises defense against many other infections. It affected 38.4 million people worldwide at the end of 2021 and had a high mortality rate of 4.7% [1, 2]. Circular RNA (circRNA) is an understudied molecule involved in the pathogenesis of early HIV infection (EHI), a critical period that determines disease severity and progression to AIDS [3]. Understanding the role of circRNAs in the etiology of EHI may contribute to the development of more effective HIV therapeutics.
Our group focused on HIV type 1 (HIV-1), the more prevalent and pathogenic type worldwide [4]. To identify potential circRNAs correlated with EHI, our group used bioinformatic methods to conduct differential expression analysis on multiple existing human RNA-Seq data collected from CD4+ T cells at different times post-infection from 18 EHI and 17 healthy controls. Characteristics and gene ontology (GO) of the differentially expressed (DE) circRNAs are analyzed. To compare the role of circRNA in EHI and latent HIV infection, we also performed a differential analysis of circRNA on 51 latent HIV samples and 4 healthy controls collected from whole blood.
We identified a total of 56 DE circRNAs related to EHI, 49 of which are downregulated. The median length of the 56 DE circRNAs is 456nt, close to the average length of circRNA, which is 500nt. There are 7 DE circRNAs shown up in more than two sampling windows post-infection. We believe these circRNAs are important in EHI. Through GO analysis, we found these circRNAs may affect HIV-1 infectivity by altering HIV binding, transport, integration, and viral reservoirs. Finally, 3 DE circRNAs are shown to be involved in latent phase HIV infections. However, none of these circRNAs is differentially expressed in EHI, suggesting that some circRNA-regulated events are unique to EHI.
References:
[1] HIV. https://www.who.int/data/gho/data/themes/hiv-aids (accessed Sep 10, 2022).
[2] HIV-related death rate in U.S. fell by half from 2010 to 2017 press release. https://www.cdc.gov/nchhstp/newsroom/2020/hiv-related-death-rate-press-release.html (accessed Sep 10, 2022).
[3] Zhang, Y.; Zhang, H.; An, M.; Zhao, B.; Ding, H.; Zhang, Z.; He, Y.; Shang, H.; Han, X. Crosstalk in Competing Endogenous RNA Networks Reveals New Circular RNAS Involved in the Pathogenesis of Early HIV Infection. Journal of Translational Medicine 2018, 16 (1).
[4] Gilbert, P. B.; McKeague, I. W.; Eisen, G.; Mullins, C.; Guéye-NDiaye, A.; Mboup, S.; Kanki, P. J. Comparison of HIV-1 and HIV-2 Infectivity from a Prospective Cohort Study in Senegal. Statistics in Medicine 2003, 22 (4), 573–593.
Keywords: Circular RNA, HIV
136. The catalytic activity of H1 circRNA
Avleen K. Chawla (Department of Chemistry, Carnegie Mellon University), Anna M. Kietrys (Department of Chemistry, Carnegie Mellon University)
Abstract:
RPPH1 complex has been long known to cleave pre-tRNA from its 5´-end leading to the formation of its mature form. In human cells, it consists of a catalytic RNA subunit, known as H1 RNA, and various protein subunits, which assist the RNA in folding and interacting with the substrate pre-tRNA. In 2006, H1 RNA was found to be able to independently catalyze this cleavage in vitro under a high Mg2+ concentration. This ribozyme is crucial for proper translation and cell functioning.
In our work, we analyzed a pool of RNAs with altered expression in the human brain. Interestingly, we found that one of overexpressed circular RNAs has the partial sequence of the H1 RNA. We experimentally validated this circRNA and also found a circular RNA having the same sequence as the linear H1 RNA. This circular H1 RNA is expressed among cell lines such as HEK293T, HeLa, and U87MG cells. The predicted structures (using RNAfold) for both the linear and circular isoforms were found to be identical, which suggests catalytic activity of circRPPH1. Circular isoforms have been known to be more stable than linear isoforms. Circular H1 RNA present in cells may have a better catalytic efficiency comparing to the linear H1 RNA. To investigate this, we have synthesized the linear RNA using in vitro transcription and circularized it using T4 Ligase I. Further studies involve studying the catalytic activity of circular RNA in vitro and in vivo, along with the mechanistic and kinetic studies of the reaction. Interactions of this circular RNA will be investigated with the RPPH1 protein subunits to map the function of this H1 circRNA.
References:
1. Altman, S., Gold, H. A. & Bartkiewicz, M. Ribonuclease P as a snRNP. Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles 183–195 (1988) doi:10.1007/978-3-642-73020-7_7.
2. Kikovska, E., Svärd, S. G. & Kirsebom, L. A. Eukaryotic RNase P RNA mediates cleavage in the absence of protein. Proceedings of the National Academy of Sciences 104, 2062–2067 (2007).
3. Rybak-Wolf, A. et al. Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed. Mol Cell 58, 870–885 (2015).
4. Maass, P. G. et al. A map of human circular RNAs in clinically relevant tissues. J Mol Med 95, 1179–1189 (2017).
5. Cocquerelle, C., Mascrez, B., Hétuin, D. & Bailleul, B. Mis-splicing yields circular RNA molecules. The FASEB Journal 7, 155–160 (1993).
Keywords: circular RNA, ribozymes, H1 RNA
137. Title not available online - please see the booklet.
Raahul Sriram (Chemistry, Carnegie Mellon University), Megan Van Horn (Chemistry, Carnegie Mellon University), Erika Kim (Chemistry, Carnegie Mellon University), Anna M. Kietrys (Chemistry, Carnegie Mellon University)
Abstract not available online - please check the booklet.
138. Stability and functional effect of RNA G-quadruplex within the open reading frame of NAT8L mRNA under oxidative stress
Brintha Croos (Chemistry and Biochemistry), Sajad Shiekh (Physics), Soumitra Basu (Chemistry and Biochemistry), Hamza Balci (Physics)
Abstract not available online - please check the booklet.
139. Title not available online - please see the booklet.
Bereketab N. Abeje (Biology Department, Swarthmore College), Christina A. Rabeler (Biology Department, Swarthmore College), Dawn M. Carone (Biology Department, Swarthmore College)
Abstract not available online - please check the booklet.
140. Refinement of the Amber Force Field For RNA: Improving the Description of Non-Bonded Interactions
Anees Mohammed Keedakkatt Puthenpeedikakkal (Department of Biochemistry and Biophysics, University of Rochester), Chapin E. Cavender (Department of Biochemistry and Biophysics, University of Rochester), Louis G. Smith (Department of Biochemistry and Biophysics, University of Rochester), Alan Grossfield (Department of Biochemistry and Biophysics, University of Rochester), David H. Mathews (Department of Biochemistry and Biophysics, University of Rochester)
Abstract:
RNA carries out a broad range of functions including coding, regulation and expression of genes. Molecular dynamics (MD) simulation can be used to investigate the time evolution of RNA conformations and be used to complement or interpret experimental observations. Amber uses a fixed charge forcefield model to estimate the potential energy of a molecule from the atomic coordinates. Studies suggest that stacking free energies observed from the current RNA forcefield are too favorable with respect to the experimental data. The non-bonded interactions are modeled by a Lennard Jones potential and Coulomb's law; these parameters have not been updated since 1995.
We are fitting the Amber parameters to potential energies determined by quantum mechanics. Structures from the Protein Data Bank and MD simulation of RNA duplexes were used to assemble a database of conformations representing base pairs, base stacks, base-phosphates and base-sugar interactions. We clustered these structures by interatomic distances using density peak clustering, and cluster centers were chosen as representative structures. To include the polarization from solvent, we fit charges to represent the time-averaged electrostatic field from solvent sampled through molecular dynamics trajectory of these cluster centers with OPC water. Symmetry adapted perturbation theory calculations (SAPT) were used with these representative structures and solvation charges to estimate the non-bonded interaction energy. We fit the Amber non-bonded parameters for the fixed-charge potential form by nonlinear regression to the observed interaction energies.
Keywords: RNA, forcefield, QM
141. Role of Dhx36/G4R1 in coronary angiogenesis and heart development
Evan M. Rogers (Department of Biology, Ball State University), Frank Bogan (Department of Biology, Ball State University), Kamal Baral (Department of Biology, Ball State University), Brendan W. Jones, Michael Reisinger, Bryce Kushel (Department of Biology, Ball State University), Philip J. Smaldino and Bikram Sharma (Department of Biology, Ball State University)
Abstract:
Coronary vessels are blood vessels that deliver oxygen and nutrients to the heart muscle, and they develop by angiogenesis, the process of new blood vessel formation. Since coronary vessels feed the heart muscle, proper formation of these blood vessels is essential for heart development. Dysfunctional or damaged coronary arteries are the primary cause of cardiac failure, the leading cause of death in adults, so it is vital to understand the molecular underpinnings of coronary vessel formation. The embryonic heart is an excellent model to study this because it allows us to capture the molecular details of how coronary vessels are first established in the heart. Using an embryonic mouse heart as a model system, we study the developmental mechanisms of coronary vessel formation. In our most recent investigation into the molecular mediators of this process, we identified Dhx36 as a potential regulator. Dhx36 (aliases: G4R1 and RHAU) encodes an RNA helicase that unwinds G-quadruplex structures in DNA and RNA. Previously, substantial cardiovascular defects have been shown to arise when Dhx36 is knocked out in mice embryos, however its exact role in coronary vessel formation is still unknown. Our preliminary study from a Dhx36 conditional knockout in the vasculature of a mouse embryo revealed significant delay in coronary vessel expansion and coronary endothelial differentiation. In addition, we observed a reduced thickness of the myocardium. Our future studies are aimed at characterizing this exact role and the underlying molecular mechanisms by which Dhx36 regulates coronary angiogenesis. An improved understanding of the mechanisms of coronary vessel formation could be beneficial to strategizing the repair and regeneration of damaged coronary vessels within the adult heart.
Keywords: Dhx36G4R1, coronary angiogensis, heart development
142. Title not available online - please see the booklet.
Sushant Bangru (Department of Biochemistry and Cancer Center, University of Illinois, Urbana-Champaign), Jackie Chen, Ullas V. Chembazhi, Waqar Arif (Department of Biochemistry, University of Illinois, Urbana-Champaign), Frances Alencastro, Andrew Duncan (Department of Pathology, University of Pittsburgh), Russ P. Carstens (Perelman school of medicine, University of Pennsylvania), Auinash Kalsotra (Department of Biochemistry, Carl R. Woese Institute of Genomic Biology, Cancer Center, University of Illinois, Urbana-Champaign)
Abstract not available online - please check the booklet.
143. Regulation of the Oncogenic Androgen Receptor Variant 7 by micro and long noncoding RNAs in Prostate Cancer
Samantha Gargasz (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Science, Cleveland State University), Eviania Likos (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Science, Cleveland State University), Girish C. Shukla (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Science, Cleveland State University)
Abstract not available online - please check the booklet.
144. Single-Cell and Spatial Mapping Identify Cell Types and Signaling Networks in the Human Ureter with Regards to its Regenerative Potential
Surbhi Sona (Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland,, OH 44195, USA), Emily E. Fink, Matthew Bradley, Angela H. Ting (Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Pierre-Emmanuel Desprez, Mohamed Eltemamy, Georges-Pascal Haber (Glickman Urological & Kidney Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Uyen Tran, Hong Qiu, Madison Wolkov, Oliver Wessely,Byron H. Lee (Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Marlo Nicolas (Pathology & Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH 44195, USA ), Booki Min (Department of Microbiology and Immunology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA)
Abstract:
Developmental defects or damage due to high levels of inflammation or radiation therapy in cancer can often lead to ureteral pathologies. Ureter reconstruction surgeries utilizing tissue engineering remain a lucrative treatment avenue in such scenarios. Here, we utilized scRNA-seq data generated from 10 normal human ureters to understand the cellular architecture and signaling pathways that contribute to the regenerative potential of the ureter. We identified several subtypes of the 3 major cell types in the urothelium, which is functionally the most important tissue layer in ureter. Among the urothelial cell types, we identified Sonic Hedgehog (SHH)-expressing basal cells that are most likely the stem cell population in the ureter - setting these cells as the root in the trajectory analysis using monocle3, accurately predict the general differentiation trajectory across the different urothelial cells. This was experimentally validated, using ureter organoid generation assay, the rate of which positively correlated with SHH expression. Further, the spatial transcriptomics analysis on this cohort’s ureter sections revealed that peri-urothelial fibroblasts reside right next to the urothelium. We then utilized, the Cellphonedb analysis to query the potential cellular signaling between basal cells and peri-urothelial fibroblasts, which included WNT, BMP, EGF and TGF pathways (likelihood test, p value <0.00001). We observed WNT4 signaling from these fibroblasts to frizzled class (FZD) receptors on all basal cell population, including SHH-expressing basal cells. This is synonymous to a previous mice study that suggested an important role of interaction between WNT7B and FZD1 working in parallel to SHH signaling. In conclusion, our study describes the cellular composition of the human ureter at a single cell resolution, identifies a potential SHH-expressing basal stem cell subpopulation and offers useful insights to the regenerative potential of the human ureter which could contribute to tissue engineering endeavors for the treatment of ureteral pathologies.
Keywords: Human ureter, Single-cell RNA sequencing, sonic hedgehog
145. Title not available online - please see the booklet.
Ying Xu (Department of Chemistry, Case Western Reserve University), Na Tian (Department of Chemistry, Case Western Reserve University), Huaxia Shi (Department of Chemistry, Case Western Reserve University), Chenwei Zhou (Department of Chemistry, Case Western Reserve University), Yufan Wang (Department of Chemistry, Case Western Reserve University), Fu-Sen Liang (Department of Chemistry, Case Western Reserve University)
Abstract not available online - please check the booklet.
146. High throughput screening of small molecule binding partners for the FMN riboswitch by a multidisciplinary approach
Elizabeth D Tidwell (University of Michigan Department of Biophysics; University of Michigan Chemical Biology Interface Training Program), Anna Anders (University of Michigan Department of Chemistry; University of Michigan Cellular Biotechnology Training Program), Varun Gadkari (University of Michigan Department of Chemistry), Brandon T Ruotolo (University of Michigan Department of Chemistry; University of Michigan Department of Biophysics), Aaron T Frank (ARRAKIS Therapeutics, Waltham, MA, ), Markos Koutmos (University of Michigan Department of Chemistry; University of Michigan Department of Biophysics)
Abstract not available online - please check the booklet.
147. Dynamics of RNA Conformational Sampling by the SMK box riboswitch
Haoyun Yang (Department of chemistry and biochemistry, OSU), Mark Foster (Department of Chemistry and Biochemistry, OSU), Tina Henkin (Department of Microbiology, OSU)
Abstract:
The conformational dynamics of non-coding RNAs are coupled to vital cellular processes such as metabolite sensing, site-specific RNA catalysis and the hierarchy of ordered ribonucleoprotein assemblies. However, the detailed mechanistic description of the structures and RNA functional conformational dynamics remains poorly understood. To fill this knowledge gap, we propose to study a model system, the SMK box, S-adenosylmethionine (SAM) metabolite-binding/SAM-III riboswitch RNA, a compact RNA that undergoes ligand-mediated conformational changes to alter ribosome binding and thereby regulate translation1,2. Previously, X-Ray crystallography analysis of the SMK aptamer bound to SAM established the structure of the ligand-bound, repressed state3. In the absence of SAM, SMK is proposed to be in equilibrium between an alternative fold (ISO) that has no obvious ligand binding site and a ligand binding competent state (PRIMED) evidenced by mutation and chemical probing experiments4. Thus, a conformation selection model is speculated. To understand how the SMK box riboswitch is able to undergo conformational fluctuations between alternative structures in order to enable its function. We propose to first use stopped-flow and 2-aminopurine labeled RNA to reveal the ligand binding model that best describes this riboswitch and extract thermodynamics parameters that can provide structural insight of the binding steps. From this, we can use NMR relaxation dispersion (RD) experiments to investigate the conformational fluctuations of the RNA with atomic resolution.
References:
1. Fuchs RT, Grundy FJ, Henkin TM. S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA. PNAS U S A. 2007;104(12):4876-4880.
2. Fuchs RT, Grundy FJ, Henkin TM. The S(MK) box is a new SAM-binding RNA for translational regulation of SAM synthetase. Nat Struct Mol Biol. 2006;13(3):226-233.
3. Lu C, Smith AM, Fuchs RT, et al. Crystal structures of the SAM-III/S(MK) riboswitch reveal the SAM-dependent translation inhibition mechanism. Nat Struct Mol Biol. 2008;15(10):1076-1083.
4. Wilson RC, Smith AM, Fuchs RT, Kleckner IR, Henkin TM, Foster MP. Tuning riboswitch regulation through conformational selection. J Mol Biol. 2011;405(4):926-938.
Keywords: Riboswitch , Dynamics
148. Title not available online - please see the booklet.
Christina Haddad (Department of Chemistry, Case Western Reserve University ), Srinivasa Penumutchu (Department of Chemistry, Case Western Reserve University ), Josephine King (Department of Chemistry, Case Western Reserve University ), Blanton Tolbert (Department of Chemistry, Case Western Reserve University )
Abstract not available online - please check the booklet.
149. Investigating the effectiveness of HILIC-type column chemistries for oligonucleotides
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:
Analyzing DNA and RNA has become increasingly important as more is learned about the transcriptome and more RNA-based drugs are being produced in an attempt combat various diseases. Traditionally, liquid chromatography – mass spectrometry (LC-MS) analysis of nucleic acids is done by digestion of the sample into nucleotides and oligonucleotides and then building the sequence from the bottom-up. The gold standard for oligonucleotide chromatography has been ion pairing reverse phase liquid chromatography (IP-RP-LC). IP-RP-LC utilizes mobile phase additives that aid the chromatographic retention and gas-phase ionization of oligonucleotides. While this method has been very effective, ion pairing reagents are inconvenient to work with and difficult to eliminate from HPLC instruments. Additionally, ion-pairing reagents can lead to increased background noise in mass spectrometry when trying to change assays or modes. Thus, most oligonucleotide assays can require dedicated instruments, which is not practical and can be very costly. Recently, hydrophilic interaction liquid chromatography (HILIC) for oligonucleotide separation during LC-MS analysis has been shown to provide competitive chromatographic and mass spectrometric figures of merit. Currently, assays have been reported for a diol and amide functionalized column. The focus of this project is to further understand the retention mechanism as well as the limitations of HILIC-type columns for oligonucleotide LC-MS assays.
Keywords: HILIC, Oligonucleotides
150. Utilization of high-pressure NMR for the study of allosteric regulation of hnRNP A1
Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University), Julien Roche (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University), Sebla Kutluay (Department of Molecular Biology, Washington University School of Medicine), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)
Abstract:
The hnRNP A1 protein is involved in numerous processes of nascent RNA transcripts. These roles include, but are not limited to, translational control, splicing regulation, and mRNA stabilization. The protein performs its functions by binding RNA at a high affinity site before recruiting other proteins to aid in its RNA processing activities.
hnRNP A1 is composed of three domains, two structurally identical RNA recognition motifs (RRMs) that form the N-terminal protein UP1, and a disordered, glycine rich C-terminal domain (G-CTD). The UP1 domain is responsible for binding the nucleic acid substrates while the G-CTD assembles the multi-protein complexes the protein utilizes to carry out its various functions. It is also a low-complexity domain (LCD) capable of mediating liquid-liquid phase separation (LLPS).
A crystal structure of UP1 bound to a trinucleotide showed RRM1 to be the primary domain for binding. The structure presents the opportunity for allosteric regulation if RNA binding at RRM1 causes conformational changes that can alter the conformations of the other two domains.
High-pressure NMR spectroscopy is a powerful technique for examining the biophysical nature of proteins. Application of pressure to a protein increases solvent density and pushes the protein towards a lower molar volume, causing folded proteins to unfold. For disordered domains, pressure has been shown to have just minor effects on NMR parameters.
Applying pressure to UP1 revealed the protein to be remarkably stable. Variants of UP1 in which the interdomain interactions between the two RRMs are disrupted were much less stable than the wild type protein. Similar studies with full length hnRNP A1 revealed UP1 to be unaffected by the presence of the LCD. The application of pressure to the LCD caused it to adopt an elongated conformation, in contrast to its resting compact nature. This conformational change is not effected by the presence of the UP1 domain.
References:
Levengood, J.D., and Tolbert, B.S. (2019) Idiosyncrasies of hnRNP A1-recognition: Can binding mode influence function. Semin Cell Develop Biol. 86:150-161.
Levengood, J.D., Peterson, J., Tolbert, B.S., and Roche, J. (2021) Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR. Proteins 89(7):781-791.
Keywords: RRM, NMR, Splicing
151. Semi-enzymatic Synthesis of Pseudouridine
Melanie Clawson (Department of Biological Science, Southern Illinois University Edwardsville), Tristan Sanford (Department of Chemistry, Southern Illinois University Edwardsville), Andrew Riley (Department of Chemistry, Southern Illinois University Edwardsville), Kylie Raasch (Department of Chemistry, Southern Illinois University Edwardsville), Ridwan Oyebamiji (Department of Biological Science, Southern Illinois University Edwardsville), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville)
Abstract:
Modifications of RNA molecules have a significant effect on their structure and function. One of the most common modifications is pseudoruidine (Ψ) which isomerizes from uridine. Recently, pseudouridine is in high demand due to the COVID-19 pandemic because a derivative of pseudouridine (N1-methylpseudouridine) has been used for the development of mRNA vaccines against SARS-CoV-2. However, organic synthesis of pseudouridine has been challenging because of the stereochemistry requirement and the sensitivity of reaction steps to moisture. Herein, a semi-enzymatic synthetic route is developed for the synthesis of pseudouridine using a reverse reaction catalyzed by a pseudouridine monophosphate glycosidase. This synthetic route has fewer steps and high yield production of pseudouridine
References:
Huang, S.; Mahanta, N.; Begley, T. P.; Ealick, S. E. Pseudouridine Monophosphate Glycosidase: A New Glycosidase Mechanism. Biochemistry 2012, 51 (45), 9245–9255.
Preumont, A.; Snoussi, K.; Stroobant, V.; Collet, J. F.; Van Schaftingen, E. Molecular Identification of Pseudouridine-Metabolizing Enzymes. J. Biol. Chem. 2008, 283 (37), 25238–25246.
Riley, A.T.; Sanford, T. C.; Woodard, A. M.; Clerc, E. P., Sumita, M. Semi-enzymatic Synthesis of Pseudouridine. Bioorg. Med. Chem. Lett. 2021, 44, 128105
Keywords: RNA modification, modified nucleotide synthesis, pseudouridine
152. Purification with
Nicole Burbach (Department of Chemistry, Southern Illinois University Edwardsville), Anthony DeGregorio (Department of Chemistry, Southern Illinois University Edwardsville), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville)
Abstract:
Purification is an important step of organic synthesis. Column chromatography is a typical method of purifying products; however, it is difficult when there are several side products and if the Rf values are similar to one another. The early step of 2-aminopyridine nucleoside synthesis faces this challenge. At the base protection step, the unused 1,2-bis(chlorodimethylsilyl)ethane was difficult to separate from the target product. Because of the high boiling point of the protecting group, we decided to use Kugelrohr distillation. We successfully house-built a cost-effective Kugelrohr distillation apparatus using parts of an old rotovap. We achieved the desired purified product and are now ready for the next step of the synthetic scheme.
Keywords: RNA modification, Nucleoside Synthesis, Kugelrohr distillation
153. Synthesis of 1-Aminopyridine for Fluorescent Studies of RNA
Anthony DeGregorio (Department of Chemistry, Southern Illinois University Edwardsville), Nicole Burbach (Department of Chemistry, Southern Illinois University Edwardsville), Ashlynn Winkler (Department of Chemistry, Southern Illinois University Edwardsville), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville)
Abstract:
RNA has multiple different functions and compositions, which allow it to have a major role in many important biological processes. Fluorescence spectroscopy has advantages including its sensitivity. However, regular nucleosides are not fluorescence active at room temperature. Therefore, it is required to synthesize fluorescent modified nucleotides in studying RNA function and composition. While there is an available fluorescent modified purine nucleoside analog, there is not a fluorescent nucleoside analog for pyrimidine nucleosides. In this research project, a fluorescent pyrimidine analog, 2-aminopyridine nucleoside, is developed for further fluorescence studies of RNA.
References:
Jones, A.C.; Neely, R.K., 2-Aminopurine as a fluorescent probe of DNA conformation and the DNA-enzyme interface. Quarterly reviews of Biophysics 2015, 48(2), 244-79.
Hanessian, S., and Machaalani, R. A Highly Stereocontrolled and Efficient Synthesis of Alpha- and Beta-Pseudouridine, 2003, 44, 2049-2052.
Keywords: RNA modification, Fluorescence Spectroscopy, Nucleoside Synthesis
154. The role of pumilio homolog 1 (PUM1) in human hemoglobin switching and erythroid differentiation
Anita R. Dhara, (Department of Biological, Geological, and Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, OH-44115), Dr. Mahesh Ramamoorthy and (Department of Biological, Geological, and Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, OH-44115), Dr. Merlin Nithya Gnanapragasam (Department of Biological, Geological, and Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, OH-44115)
Abstract:
The conversion from fetal to adult hemoglobin occurs around birth, accompanied by a change in an expression level from γ-globin to the β-globin chain. Patients with high fetal hemoglobin levels (HbF) have a milder clinical manifestation of β-hemoglobinopathy, such as sickle cell anemia and β-thalassemia. Therefore, γ-globin (β-type of globin gene) provides a promising therapeutic option for replacing mutated or absent β-globin, thereby increasing fetal hemoglobin levels. However, in contrast to transcriptional and epigenetic control, post-transcriptional regulation of β-globin switching is poorly known, with few physiological and clinical studies. We have shown that PUM1, an RNA-binding protein, is a novel direct candidate for Erythroid Krüppel-like factor (EKLF), a master and transcriptional regulator of erythropoiesis. PUM1 binds to the fetal γ-globin mRNA and decreases γ-globin protein levels, demonstrating post-transcriptional regulation of fetal γ-globin. Similar results were observed with human primary CD34+ erythroid cells. Furthermore, we showed that PUM1 knockdown does not affect the progression of terminal erythroid differentiation. Our unpublished data for the PUM1 knockout showed similar results for γ-globin protein levels, corroborating our previous results. PUM1 knockout does not impair but rather augments human terminal erythroid differentiation. We will further characterize the PUM1 knockout cells to check any dose-dependent effects on erythroid terminal differentiation. Also, we will be investigating the impact of two putative binding sites at the 3’-UTR of HBG1 fetal γ-globin mRNA in regulating fetal hemoglobin profile.
References:
Elagooz R, Dhara AR, et al., Blood Advances, 2022 (Online ahead of print)
Keywords: Pumilio homolog 1 (PUM1), Hemoglobin switching, Erythroid terminal differentiation
155. Ectopic expression of HSATII RNA results in nuclear focal accumulations and chromatin defects
Christina A. Rabeler (Department of Biology, Swarthmore College), Emily K. Ferrari (University of Pennsylvania), Lia R. DAlessandro (Broad Institute), Dawn M. Carone (Department of Biology, Swarthmore College)
Abstract:
Human Satellite II (HSATII) is a tandemly repeated pericentric satellite sequence that is transcriptionally silenced in normal cells yet is aberrantly expressed in many types of cancer cells, where the RNA forms large nuclear foci in cis. These focal accumulations of HSATII RNA have been shown to recruit and sequester regulatory proteins including methyl-CpG binding protein 2 (MeCP2). In order to examine the role of HSATII RNA, cells which do not normally transcribe HSATII were transfected to stably express this RNA. Both HeLa and primary fibroblast cells which ectopically expressed HSATII RNA were seen to aggregate the RNA in nuclear foci in cis and also accumulate MeCP2 onto those foci. Additionally, an increase in cellular division defects including lagging chromosomes and chromatin bridges was observed in cells after long-term ectopic expression of HSATII. These results suggest that forced expression of HSATII RNA leads to nuclear accumulation and recruitment of protein binding partners that ultimately results in cell division defects, thus suggesting a role for HSATII expression in cancer.
Keywords: HSATII, lncRNA, chromatin instability
156. Ribosomes lacking bS21 gain function to regulate protein synthesis
Bappaditya Roy (Department of Microbiology, Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Zakkary A. McNutt (Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Bryan T. Gemler, Elan A. Shatoff (Department of Physics, Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Kyung-Mee Moon, Leonard J. Foster (Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada), Ralf Bundschuh (Department of Physics, Department of Chemistry & Biochemistry, Division of Hematology, Department of Internal Medicine, Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Kurt Fredrick (Department of Microbiology, Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA)
Abstract not available online - please check the booklet.
157. Investigating TERRA functions in VSG switching and telomere maintenance in Trypanosoma brucei
SK ABDUS SAYEED (Cleveland State University), Bibo Li (Cleveland State University)
Abstract:
Human African Trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei, which regularly switches its major surface antigen (variant surface glycoprotein, VSG) to evade the human immune response. VSGs are expressed from subtelomeric loci in a monoallelic manner. Work from our lab has shown that telomere proteins play important roles in VSG monoallelic expression and VSG switching. The active VSG gene is flanked by upstream 70 bp repeats and downstream telomeric repeats. The telomeric repeats downstream of the active VSG are transcribed into the telomeric repeat-containing RNA (TERRA), a long non-coding RNA, as a result of read-through transcription by RNA polymerase I. It has been shown that TbRAP1 and TbTRF, two telomere proteins, suppress TERRA and telomeric R-loop (RNA: DNA hybrid) levels, thereby suppressing DNA recombination mediated VSG switching events. We previously showed that excessive amounts of TERRA and the telomeric R-loops cause more DNA breaks at the telomere and threatens cell viability. Recently, we successfully generated T. brucei strains with LacO repeats targeted to the subtelomeric region immediately downstream of the active VSG and immediately upstream of the telomere repeats. We observed that inducing the LacI-GFP expression significantly reduces the TERRA level, presumably because binding of LacI-GFP to the LacO repeats blocks the read-through transcription of the telomeric repeats downstream of the active VSG gene. This is the first conditional repression system established in trypanosomes, which effectively solves the problem of inefficient depletion of TERRA by RNAi. TERRA depletion does not affect T. brucei growth. However, TERRA depletion increases the TbTRF protein level and the amount of telomere bound TbTRF. Whether TERRA depletion affects the VSG switching frequency and/or telomere length maintenance are under investigation now.
Keywords: TERRA, Telomere
158. Unraveling the roles of 5’ transcript leaders in gene regulation
Christina Akirtava (Carnegie Mellon University, Biological Sciences), Hunter Kready (Carnegie Mellon University, Biological Sciences), Lauren Nazzaro (Carnegie Mellon University, Biological Sciences), Matt Agar-Johnson (Carnegie Mellon University, Biological Sciences), Gemma E May (Carnegie Mellon University, Biological Sciences), C. Joel McManus (Carnegie Mellon University, Biological Sciences)
Abstract:
Eukaryotic translation initiation typically follows a cap-dependent directional scanning model, and thus is limited by the 5’ transcript leader (TL) and start codon. The 5’ TL contains a variety of cis -regulatory sequences, structures and trans -acting factors which influence translation initiation efficiency (Kozak, 1984; Rojas-duran & Gilbert, 2012). Previous studies using in vivo reporters of fixed-length synthetic TLs identified upstream AUGs as major repressors of gene expression (Cuperus et al, 2017; Dvir et al, 2013; Sample et al, 2018). However, the relative contributions of these features to translation regulation in natural TLs have not been directly determined. To address this, we used two massively parallel reporter systems (MPRAs) (Noderer et al, 2014) to quantify in vivo regulation from 86% of natural yeast TLs and systematically identify the relative impacts of their sequence features on initiation in a systematic manner. First, we compared expression from genes containing alternative start sites and found huge differences in initiation regulation. We also, experimentally determined the relative strengths of all yeast Kozak contexts and used these values to develop a leaky scanning model that predicts initiation efficiency. Next, we use computational modeling to explain ~64% of translation initiation, detect key cis-acting sequence features, and quantify their effects in vivo. Additionally, we tested the TLs in an eIF2a phosphomimetic mutant that limits translation initiation. Most TLs exhibit a decrease in translation efficiency; however, a subset of TLs increase YFP expression with changes up to 4-fold. Machine learning models suggest preinitiation complexes scan the TL less effectively and are highly impacted by TL length and structure in the eIF2a mutant. Finally, we compared our results to sequence features found in a recent massively parallel in vitro study of translation initiation (Niederer et al, 2022). Together, our results identify the range and relative influence of cis-acting sequences and structures on translation initiation from native yeast TLs in vivo.
Keywords: translation, modeling, UTR
159. The basis of multi-step tRNA recognition by a eukaryotic tRNA editing deaminase
Luciano G. Dolce (European Molecular Biology Laboratory, Grenoble), Aubree A. Zimmer (Ohio State Biochemistry Program), Felix Weis (European Molecular Biology Laboratory, Heidelberg), Henry Arthur, Mary Anne T. Rubio, Katherine M. McKenney, Jessica L. Spears (Department of Microbiology, The Center for RNA Biology), Laura Tengo, Eva Kowalinksi (European Molecular Biology Laboratory, Grenoble), Juan D. Alfonzo (Department of Microbiology, The Center for RNA Biology)
Abstract:
Transfer RNAs (tRNAs) are central to protein synthesis by converting genomic information of protein-coding regions into a precise polypeptide chain, as dictated by codons in mRNA. tRNAs require extensive chemical modifications for structural stability, translation fidelity, as well as decoding functions. One such modification is the deamination of adenosine to inosine at the first position of the anticodon, which is essential for viability in bacteria and eukaryotes. Inosine formation at this wobble position in both bacteria and eukaryotes increases the decoding capabilities of a tRNA to recognize multiple different synonymous codons. The deaminase responsible for A-to-I activity in bacteria specifically deaminates a single tRNA while the eukaryotic counterpart has evolved to deaminate 7-8 tRNAs independent of any sequence specificity. How the eukaryotic enzyme recognizes multiple substrate tRNAs from non-substrate molecules has been a long-standing question in the field. In collaboration with the Kowalinski laboratory at the EMBL, we have described a model for tRNA recognition based on biochemical assays and the first structure of a eukaryotic deaminase bound to substrate tRNA. Our model describes a multi-step tRNA recognition mechanism using domains distal from the active site as well as an essential cation- π molecular gate guarding the catalytic site.
Keywords: tRNA editing, deaminase, Trypanosoma brucei
160. Single-Molecule Study of Telomeric Overhangs Using FRET Method
Sineth G Kodikara (Kent State University, Department of Physics ), Sajad Shiekh (Kent State University, Department of Physics ), Kylie Merkel (Kent State University, Department of Physics), Simon Haas (Kent State University, Department of Physics), Hamza Balci (Kent State University, Department of Physics)
Abstract:
Single-molecule FRET was used to analyze multiple telomeric overhang conformations and find the potassium chloride (KCl) dependence of binding frequency. This was accomplished by using surface-immobilized partial duplex DNA labeled with the donor fluorophore, Cy3, and a short Peptide Nucleic Acid (PNA) labeled with the acceptor fluorophore, Cy5. It was found that when the binding zones for the Cy5-PNA are increased, the width of the FRET energy transfer peak also increases. It was also found that the binding frequency of the Cy5-PNA increases as binding zones increase, unless three binding zones repeat in a row, then a possible triplex may form and prevent binding at higher salt concentrations. This was tested at varying KCl concentrations, and it was found that there appears to be an initial decrease in the binding frequency at low salt concentrations followed by an increase after about 20 mM KCl as higher salt concentrations possibly increase the binding affinity of PNA to DNA.
Keywords: Triplex