Talk abstracts
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Friday 01:15-01:30pm: Kinetic modeling of chemical probing data enables extraction of intrinsic nucleotide energetics
Edric K. Choi (Chemical and Biological Engineering, Northwestern University), Ritwika Bose, Anthony M. Mustoe, Julius B. Lucks
Abstract:
Chemical probing has long been indispensable in studying RNA structures within diverse biological systems, leading to significant insights into RNA functionality. However, the full potential of chemical probing data from these experiments remains obscured due to the complexities of interpreting raw reactivity data. Here we address this limitation by developing a conceptual framework for interpreting reactivities that is analogous to the framework used in analyzing hydrogen exchange experiments for protein structures. Using this model, we show that raw reactivities, r, are a non-linear convolution between intrinsic RNA structural information, denoted K, and experimental parameters related to probe modification kinetics. We validate the model through non-linear fitting of time-course DMS probing experiments conducted on the extensively studied Salmonella fourU thermometer at a range of temperatures. Analysis of extracted K values show that they exhibit Arrhenius-like temperature dependence, allowing us to extract an effective ΔG value from the K values for each nucleotide. These extracted ΔG values are significantly correlated with published NMR-derived base pair dissociation energies (R2 = 0.78) and ΔΔG (R2 = 0.54), supporting that they represent physical measurements of base-pair stability. We suggest a path forward for further study for a deeper understanding of the aspects of RNA structure being revealed by this analysis. These findings demonstrate the capability to extract nucleotide-resolution RNA structure thermodynamic parameters from RNA chemical probing experiments, and a framework for analyzing reactivity data that can be extended into a robust standard for comparing RNA structures across varied experimental conditions.
Keywords: RNA chemical probing, Nucleotide thermodynamics, Quantitative data analysis
Friday 01:30-01:45pm: Identification of leader-trailer helices of precursor ribosomal RNA in all phyla of bacteria and archaea
Bryan T. Gemler (Interdisciplinary Biophysics Graduate Program, The Ohio State University), Benjamin R. 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:
Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli, complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function.
References:
Gemler BT, Warner BR, Bundschuh R, Fredrick K. Identification of leader-trailer helices of precursor ribosomal RNA in all phyla of bacteria and archaea. RNA. 2024 Sep 16;30(10):1264-1276. doi: 10.1261/rna.080091.124. PMID: 39043438; PMCID: PMC11404451.
Keywords: leadertrailer helix, rRNA processing, ribosome biogenesis
Friday 01:45-02:00pm: How are RNA helicases recruited to their RNA substrates?
Taylor N. Ayers (Carnegie Mellon University), Fiona Fitzgerald (Carnegie Mellon University), Collin Bachert (Carnegie Mellon University), John L. Woolford (Carnegie Mellon University)
Abstract:
Ribosome assembly depends upon the stepwise folding of rRNA into its three-dimensional structure. Near-atomic resolution cryo-EM structures of ribosome assembly intermediates have uncovered the order in which domains of rRNA form their mature structures, yet the mechanisms underlying rRNA folding are not well understood. Assembly factors (AFs) and ribosomal proteins can reconfigure rRNA structures or stabilize rRNA conformations. A class of AF, called RNA helicases, actively remodels rRNA substrates and mediates irreversible transitions in pre-rRNA folding, critical for powering assembly forward. Nineteen phylogenetically conserved RNA helicases have been implicated in ribosome biogenesis in Saccharomyces cerevisiae (yeast). These proteins contain a structurally conserved catalytic core that recognizes RNA in a sequence-independent manner. This raises the question: how are these RNA helicases recruited to their specific rRNA substrates? Our molecular genetic, biochemical, and structural data indicate that the RNA helicase Drs1 is required for structural maturation of rRNA domain III during nucleolar stages of large subunit (60S) assembly in yeast. However, how Drs1 is recruited to the pre-60S is not clear. Here, we have identified a patch of residues on the AF Erb1 that is critical for recruiting Drs1 onto the pre-ribosome. Using a combination of genetic and biochemical approaches, we demonstrate that mutating these residues on Erb1 significantly diminishes the presence of Drs1 on the pre-60S, impairs direct interactions between Erb1 and Drs1, and results in a slow-growth phenotype. We have also examined the role of other AFs implicated in Drs1 recruitment based on protein-protein crosslinking data. Revealing the mechanism by which Drs1 acts is pivotal for linking how RNA helicases locate their substrates during ribosome biogenesis.
Keywords: RNA helicase, ribosome assembly
Friday 02:00-02:15pm: Solution scattering as a structural tool for RNA and RNA:RNA complexes
Aldrex Munsayac (Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA), Wellington C. Leite (Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA), Jesse B. Hopkins (Biophysics Collaborative Access Team, Argonne National Laboratory, Lemont, IL, 60439, USA), Ian Hall (Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA), Hugh M. ONeill (Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA), Sarah C. Keane (Department of Chemistry and Biophysics Program, University of Michigan, Ann Arbor, MI, 48109, USA)
Abstract:
The formation of RNA:RNA complexes represents a critical feature in many biological processes, from gene regulation to viral replication. Structural knowledge of these RNA:RNA complexes remains central to understanding the molecular mechanisms governing their function. Despite their importance, high-resolution structural studies of RNA:RNA complexes remains challenging, accounting for less than 0.002% of the known structures in the PDB, highlighting the need for alternative approaches. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to determine the global architecture of individual RNAs within an RNA:RNA complex. To develop this method, we studied a model RNA:RNA complex, based on the dimerization initiation signal (DIS) from the human immunodeficiency virus. Using SAXS, we measured the solution structures of the individual RNAs in their free state and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof-of-principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.
Keywords: Small-angle scattering, RNARNA complexes, RNA structure modeling
Friday 02:15-02:30pm: Substrate Recognition by Two 3' to 5' RNA Polymerases in Dictyostelium discoideum
Grace Johnecheck (Department of Chemistry and Biochemistry, Center for RNA Biology, and OSBP, The Ohio State University), Yicheng Long (Department of Chemistry and Biochemistry, Center for RNA Biology, and OSBP, The Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry, Center for RNA Biology, and OSBP, The Ohio State University)
Abstract:
Unlike most polymerases that act in the 5' to 3' direction, tRNAHis guanylyltransferase (Thg1) synthesizes RNA 3' to 5'. Thg1 catalyzes an essential reaction during tRNAHis processing by adding a G nucleotide on the 5' end of the tRNA in most eukaryotes, forming an identity element for tRNA aminoacylation. Recent characterization of Thg1 homologs with alternative specificities, known as Thg1-like proteins (TLPs), raises questions about how distinct substrates are recognized by different members of this highly conserved enzyme family. In the slime mold Dictyostelium discoideum (Ddi), DdiThg1 adds G to the 5' end of cytosolic (cy-) tRNAHis, while DdiTLP2 catalyzes the same reaction, but only with mitochondrial (mt-) tRNAHis substrates. In vivo and in vitro, these two enzymes exhibit strict specificity for their respective tRNA substrates. Moreover, unlike Thg1, DdiTLP2 does not depend on the GUG anticodon for tRNAHis recognition, suggesting different mechanisms for tRNA recognition by these two enzymes. We aimed to determine the molecular basis for the distinct RNA substrate specificities of DdiThg1 and DdiTLP2, and thus to provide insight into the general mechanisms of RNA recognition utilized by distinct 3'-5' RNA polymerases. Electrophoretic mobility shift assays (EMSA) revealed no difference in either enzyme's ability to bind to different tRNAs, requiring additional catalytic factors to explain the selective in vitro activities of DdiThg1 and DdiTLP2. Sequence comparison was used to identify a unique residue in DdiTLP2 (R187) that is different from an absolutely conserved D/E residue at the analogous position in the rest of Thg1/TLP family members, including in DdiThg1. The DdiThg1 D150R variant lost the ability to catalyze G-1 addition with cy-tRNAHis and instead gained the ability to act on DdiTLP2’s mt-tRNAHis substrate. Kinetic and conservative amino acid replacement studies suggest that the DdiThg1 D150 residue controls RNA substrate specificity at the adenylation step of the reaction by providing a checkpoint for correct setup of the active site with incoming GTP to be added. Biochemical studies and computational structure prediction suggest that the role of DdiThg1 D150 is H-bonding with the 3'-OH nucleophile on the incoming GTP, which then positions ATP into the correct location for adenylation.
Keywords: RNA polymerase, tRNAHis, tRNA processing
Friday 02:30-02:45pm: Changes in the non-coding RNAs of Glucose-Challenged Human Glomerular Epithelial Cells May Give Insights into the Progression of Diabetic Kidney Disease
Nik Tsotakos (Dept. of Biological Sciences, Penn State Harrisburg), Ryan Castaneira (Dept. of Biological Sciences, Penn State Harrisburg), Daniel Morris (Dept. of Biological Sciences, Penn State Harrisburg)
Abstract:
Diabetic kidney disease is a complication of diabetes that takes years, sometimes decades, to develop. Our previous work on a podocyte-like cell line showed that phenotypic changes following exposure to high glucose, such as downregulation of podocalyxin and nephrin, are gradual and reversible. We also identified changes in the expression of long non-coding RNAs (lncRNA) MEG3, MEG8, and H19 in human glomerular epithelial cells (HGEC) chronically cultured in high glucose levels. In the present work, we identify changes to lncRNAs that are dysregulated following exposure of cells to high glucose. Through a qPCR array, we identified approximately 20 upregulated and 20 downregulated lncRNA in HGEC that were exposed to high glucose for 2 weeks. These changes precede the terminal loss of podocyte markers and may provide insights to novel biomarkers for early-stage diabetic nephropathy. Additionally, ectopic expression of MEG3, a lncRNA upregulated in HGEC chronically treated with high glucose, modulates autophagy by inducing changes in p62/SQSTM1 protein levels.
Keywords: diabetic kidney, MEG3, H19
Friday 03:15-03:30pm: Repression of Ago1 by Ago2 via let-7 microRNAs facilitates embryonic stem cell differentiation
Gabrielle M. Schuh (Biochemistry and molecular biology, Mayo Clinic), Katharine R. Maschhoff (Biochemistry and molecular biology, Mayo Clinic), Annastasia Minor (Biochemistry and molecular biology, Mayo Clinic), Wenqian Hu (Biochemistry and molecular biology, Mayo Clinic)
Abstract:
Argonaute (AGO) proteins are critical regulators of gene expression. Of the four AGOs in mammals, AGO1 and AGO2 are expressed in mouse embryonic stem cells (mESCs). These two proteins have opposing functions in controlling the fate decisions between pluripotency and differentiation in mESCs. AGO2 promotes differentiation predominantly via the let-7 microRNAs, whereas AGO1 maintains pluripotency via modulating protein folding independent of small RNAs. These recent findings raise the question of whether and how these two AGOs are mutually regulated in mESCs. Here, using loss-of-function and gain-of-function approaches, we show that AGO2 represses the expression of Ago1 mRNA via a conserved let-7-microRNA-binding site in its 3’UTR. Mutating this binding site at the endogenous locus abolishes the AGO2-mediated repression of Ago1 mRNA and compromises the exit pluripotency of mESCs. These results reveal that the post-transcriptional regulation of Ago1 by Ago2 and let-7 microRNAs is important for proper stem cell fate decisions.
References:
Qiuying Liu, Xiaoli Chen, Mariah K Novak, Shaojie Zhang, Wenqian Hu (2021) Repressing Ago2 mRNA translation by Trim71 maintains pluripotency through inhibiting let-7 microRNAs. eLife 10:e66288.
Qiuying Liu, Rachel M Pepin, Mariah K Novak, Katharine R Maschhoff, Kailey Worner, Wenqian Hu (2024) AGO1 controls protein folding in mouse embryonic stem cell fate decisions. Developmental Cell 10.1016
Keywords: let-7 microRNA, Argonaute proteins, embryonic stem cell differentiation
Friday 03:30-03:45pm: Substrate Selection and Functional Outcomes by the mRNA modifying enzyme PUS7
Kira Holton (Department of Biological Chemistry, University of Michigan), Brittany Bowman (Department of Biological Chemistry, University of Michigan), Kristin Koutmou (Department of Chemistry, University of Michigan), Chase Weidmann (Department of Biological Chemistry, University of Michigan)
Abstract:
Human RNAs undergo a variety of post-transcriptional modifications, and the extent and effect of most of these modifications is currently unknown. The enzyme PUS7 is responsible for a large fraction of one such RNA modification, pseudouridylation, where uridine bases are isomerized to pseudouridine (Ψ). Altered PUS7 activity is implicated in several diseases, but the mechanisms of PUS7-dependent substrate modification and how Ψ contributes to disease pathogenesis are unclear. While PUS7 can modify almost any UNUAR sequence when reconstituted in solution, only a tiny fraction of these sites (< 3 %) are modified inside the cell. Without understanding the cellular contexts that direct PUS7 target selection, we cannot predict the functional consequences of PUS7-dependent Ψ in RNA. We hypothesize that distinct RNA structural contexts and protein-RNA interactions drive selection of Ψ sites by PUS7 in cells. We are employing live-cell chemical probing and sequencing technologies to identify these cellular contexts. Protein interaction network probing (RNP-MaP) in human cells finds that Ψ modification occurs in RNA regions with limited protein binding, when compared to unmodified UNUAR sites. We are profiling protein engagement at these sites in cells lacking PUS7 to determine whether protein occupancy inhibits Ψ modification or alternatively if Ψ modification limits protein binding. We are similarly probing whether RNA structural motifs are conserved at PUS7-modified sites (by SHAPE-MaP). Additionally, we are concurrently developing a cellular luciferase-based reporter assay to measure mRNA expression, processing, and stability in the presence and absence of Ψ. Preliminary data suggests that Ψ-dependent regulation is mRNA-specific. We anticipate that the knowledge generated from this research will allow us to predict novel PUS7-dependent Ψ sites and may be suggestive of a novel gene regulatory mechanism based on RNA modifications.
Keywords: RNA Modification, PUS7, RNA-Protein Interactions
Friday 03:45-04:00pm: RNase H1 phosphorylation promotes RPA interaction to safeguard genome integrity from R-loop-associated instability
Victor M. Corral (Molecular Pharmacology and Therapeutics Graduate Program, Department of Pharmacology, University of Minnesota, Minneapolis MN), Wannasiri Chiraphapphaiboon (The Masonic Cancer Center, University of Minnesota, Minneapolis MN), Niraja A. Soman (Molecular Pharmacology and Therapeutics Graduate Program, Department of Pharmacology, University of Minnesota, Minneapolis MN), Hai Dang Nguyen (Department of Pharmacology; and The Masonic Cancer Center, University of Minnesota, Minneapolis MN)
Abstract:
Genomic instability arises from a variety of cellular processes, including DNA replication and transcription. R loops are transcription intermediates resulting from the formation of stable RNA:DNA hybrids and a displaced single-stranded DNA (ssDNA). Although R loops have important physiological roles under normal conditions, aberrant accumulation and/or distribution of R loops in the genome become a source of genomic instability and are associated with cancers. R loops must be tightly regulated to prevent R-loop-associated genomic instability. RNase H1 is an enzyme that degrades the RNA moiety within RNA:DNA hybrids of R loops. We previously showed that Replication Protein A (RPA), a ssDNA-binding protein that coats the displaced ssDNA at R loops, is a sensor of R loops and interacts with RNase H1 to promote R-loop resolution(1). How RPA:RNase H1 interaction is regulated is not known. Here, we identified RNase H1 is phosphorylated at Ser76 and Ser233 in human cells. Interestingly, Ser233 phosphorylation is independent of Ser76 phosphorylation, suggesting potentially two independent phosphorylation signaling cascades. Since RNase H1S76 residue is adjacent to its RPA interacting motif, we hypothesized that RNase H1S76 phosphorylation plays a role in resolving R loops through its interaction with RPA. We generated a phospho-specific antibody and detected endogenous RNase H1 phosphorylation at Ser76. Moreover, phosphorylated RNase H1S76 (pRNase H1S76) is associated with R loops and depends on its ability to bind to R loops, but not RPA interaction. Conversely, RNase H1S76A phospho-mutant disrupted RPA interaction to a similar extent as the previously reported RPA-defective binding mutant RNase H1R57A. As a consequence, the RNase H1S76A phospho-mutant failed to suppress PARP inhibitor-induced R-loops and R-loop-associated genomic instability. Lastly, pharmacologic inhibition of DNA checkpoint kinases, ATM/ATR, or cyclin-dependent kinases, CDKs, did not suppress pRNase H1S76, suggesting an unknown kinase involved in R-loop regulation. Our results demonstrate a mechanistic detail in RPA:RNase H1 interaction mediated by RNase H1 phosphorylation at Ser76 to promote R-loop resolution.
References:
1) Nguyen, H. D., et al. (2017). Molecular Cell.
Keywords: R loops, RNase H1, Phosphorylation
Friday 04:00-04:15pm: PUMILIO post-transcriptional regulation of nuclear encoded mitochondrial genes in human cells.
Misbah Khan (The Ohio State University), Ronghao Chen (The Ohio State University), Swetha Rajasakaran (The Ohio State University), Jalal Siddiqui (The Ohio State University), Ganesh Koshre,Rebecca Lisi (The Ohio State University), Wayne Miles (The Ohio State University)
Abstract:
Mitochondria are important energy generating organelles within the cell. Although Mitochondria do contain a small genome, the vast majority of (>95%) proteins that function within the Mitochondria are nuclear encoded (Nuclear Encoded Mitochondrial Genes, NEMG). Mitochondria are specialized environments that enable metabolic enzymatic reactions to occur for efficient energy and macromolecule production. To facilitate this, the pH of the mitochondria is different from the cytoplasm, requiring many of the proteins that fuel mitochondrial activity to be synthesized into protein, either on the surface or inside the mitochondrial to be functional. This requires: 1) the RNA to be transported to the mitochondria AND 2) not to be translated into protein until it completes its journey across the cytoplasm. Errors in this process, diminish mitochondria output and organismal fitness. The PUMILIO family of RNA binding proteins is evolutionarily conserved and bind to defined RNA sequences within the 3’UTR of their substrates. In human cells, PUM1 and PUM2 bind to UGUAHAUA motifs. While profiling PUM1-/-, PUM2-/- and PUM1/2-/- human cells, we found widespread alterations in metabolic and mitochondrial function. Using eCLIP, Ribosome profiling and RNA-sequencing, we identified around 25% of all NEMGs are PUM substrates in human cells and loss of PUM regulation resulted in increased Ribosome occupancy. These RNAs were still mostly localized to the mitochondria correctly. To determine how NEMG that are PUM substrates are transported, we computational mapped motifs and identified a recurrent structures in NEMG vs. non-NEMG PUM substrates. Using this structure as bait, we found LRRPRC binds and in cooperation with PUM, facilitates NEMG RNA transport.
Keywords: PUMILIO, Nuclear Encoded Mitochondrial Genes
Friday 04:15-04:30pm: Ribosomal release factors Hbs1 and Pelota regulate translation of coding regions following upstream open reading frames
Katherine Querry (Department of Cell Biology, University of Pittsburgh School of Medicine ), Narayanan Nampoothiri, Chris Garbark, Nina Gralewski-Goel, Abigail Carney (Department of Cell Biology, University of Pittsburgh School of Medicine ), Mykola Roiuk, Aurelio Teleman (Faculty of Biosciences, Heidelberg University, Heidelberg, Germany. ), Deepika Vasudevan (Department of Cell Biology, University of Pittsburgh School of Medicine )
Abstract:
Over 40% of mammalian mRNAs contain upstream open reading frames (uORFs), which function to inhibit translation of downstream coding regions1. The best studied of these mRNAs encodes for the transcription factor ATF4, a master regulator of the Integrated Stress Response (ISR) pathway. Across phyla, the 5’ leader of ATF4 mRNA contains two uORFs that regulate translation of ATF4, depending on initiator methionyl tRNA (Met-tRNAiMet) levels. Under homeostasis, after termination at the first uORF, the ribosome can continue scanning and reinitiate at the second uORF, which overlaps with the start codon of ATF4, thus blocking ATF4 synthesis. ISR activation results in reduced Met-tRNAiMet availability, which results in a delay in reinitiation, thus permitting synthesis of ATF4 rather than the inhibitory second uORF. We performed a Drosophila RNAi screen using an ATF4 reporter as bait to identify ribosomal factors that may be involved in the regulation of reinitiation. This screen identified Hbs1, a GTPase which aids in post-termination ribosomal recycling when in complex with Pelota. We hypothesize Hbs1 and Pelota act on the stop codon of the first uORF to release the 60s subunit and the polypeptide chain so that the 40s subunit can reinitiate at the ATF4 start codon. Consistently, we see that depletion of Hbs1 and Pelota results in decreased expression of an ATF4 5’ leader reporter. To test the physiological relevance of this regulatory mechanism, we used a Drosophila retinitis pigmentosa disease model where we have previously shown ATF4 to be required in the eye to delay degeneration. Using electroretinograms to measure transduction in the visual system, we observe that loss of Hbs1 and Pelota show retinal degradation similar to ATF4 mutants. Intriguingly, human patients with deletions in Hbs1 have also been shown to have retinitis pigmentosa2, lending further credibility to our results. We are currently pursuing genome-wide ribosome profiling approaches to identify other uORF-containing mRNAs that are regulated by Hbs1 and Pelota and extending our analyses to other known ribosome release factors as well.
References:
1. Vattem KM, Wek RC. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):11269-74. doi: 10.1073/pnas.0400541101.
2. O'Connell AE, Gerashchenko MV, O'Donohue MF, Rosen SM, Huntzinger E, Gleeson D, Galli A, Ryder E, Cao S, Murphy Q, Kazerounian S, Morton SU, Schmitz-Abe K, Gladyshev VN, Gleizes PE, Séraphin B, Agrawal PB. Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation. PLoS Genet. 2019 Feb 1;15(2):e1007917. doi: 10.1371/journal.pgen.1007917.
Keywords: ribosome recycling factors, uORFs, ATF4
Friday 04:30-04:45pm: Translation of human papillomavirus E6 protein from mRNAs with extremely short 5′-UTRs
Ritam Neupane (Department of Biological Chemistry, University of Michigan), Wenzhao Dong (Department of Biological Chemistry, Life Sciences Institute, University of Michigan), Andrew Shurer (Department of Biological Chemistry, University of Michigan), Jay B. Querido (Department of Biological Chemistry, Life Sciences Institute, University of Michigan), Rachel O. Niederer (Department of Biological Chemistry, University of Michigan)
Abstract:
Human Papillomaviruses (HPVs) are responsible for almost 5% of all cancers worldwide. They cause nearly all cervical cancer cases and are estimated to cause the deaths of 342,000 people every year. It was recently shown that a high-risk strain of HPV (HPV-18) uses mRNA with extremely short (≤5 nt) 5′-untranslated regions (5′-UTRs) to make the oncogenic protein E61. While the 5′-cap and the translation initiation proteins eIF4E and eIF4AI were shown to be involved in translation of the E6 mRNA, no known pathway of translation initiation can explain how ribosomes initiate on mRNAs with such extremely short 5′- UTRs. Using a series of biochemical, high throughput, and structural tools, we investigate the mechanism behind this pathway. Our data suggests involvement of an RNA helicase (DDX5) and a potential role of the poly (A) tail of the mRNA in the translation of mRNAs with extremely short 5’-UTRs. We have observed that translation of reporter mRNAs is stimulated by a poly (A) tail even when a cap is lacking. Increasing the length of the poly (A) tail decreases translation from reporter mRNAs. Our lab uses Direct Assessment of Ribosome Targeting (DART) to precisely identify and characterize 5′-UTR elements responsible for translational regulation. A DART assay to study thousands of systematic mutations to functionally characterize sequence and structural elements involved in translation of mRNAs with such extremely short 5′-UTRs is underway. Insights form our work will potentially illuminate a previously undescribed pathway of translation initiation used by viruses.
References:
1.García, A. et al. High-risk human papillomavirus-18 uses an mrna sequence to synthesize oncoprotein E6 in tumors. Proceedings of the National Academy of Sciences 118, (2021).
Keywords: translation initiation, HPV, DART
Friday 05:00-06:00pm: Keynote lecture: Understanding RNA folding in vivo by two complementary approaches
Philip C. Bevilacqua (Departments of Chemistry and of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Penn State University), McCauley Meyer (Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Penn State University), Saehyun Choi, Christine D. Keating (Department of Chemistry, Penn State University), Jacob P. Sieg (Department of Chemistry, Center for RNA Molecular Biology, Penn State University), Sarah M. Assmann (Department of Biology, Center for RNA Molecular Biology, Penn State University), Ryota Yamagami (Department of Chemistry, Center for RNA Molecular Biology, Penn State University)
Abstract:
RNA has an exceptionally diverse and important array of functions, which are driven by RNA folding in the cell. There is thus great interest in understanding how RNA folds in the cell. One approach is chemical probing of RNA in vivo and transcriptome-wide. Another approach is preparing in vivo-like conditions and measuring RNA folding. I will describe the advantages and limitations of each approach and lay out some of the studies from our lab on chemical probing of RNA in vivo over the last decade.
References:
1. Meyer, M. O., Choi, S., Keating, C. D., Bevilacqua, P. C. & Yamagami, R. (2023). Structure-seq of tRNAs and other short RNAs in droplets and in vivo. Methods Enzymol 691, 81-126.
2. Yamagami, R., Sieg, J. P., Assmann, S. M. & Bevilacqua, P. C. (2022). Genome-wide analysis of the in vivo tRNA structurome reveals RNA structural and modification dynamics under heat stress. Proc Natl Acad Sci U S A 119, e2201237119.
3. Meyer, M. O., Yamagami, R., Choi, S., Keating, C. D. & Bevilacqua, P. C. (2023). RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life. Sci Adv 9, eadh5152.
Keywords: tRNA, structure-seq, in vivo
Saturday 08:30-08:45am: Bacterial Ribonucleoprotein bodies (BR-bodies) organize mRNA decay and coordinate other steps of RNA metabolism in bacteria
Vidhyadhar Nandana (Departments of Chemistry and Biological Sciences, Wayne State University), Luis A. Ortiz Rodrguez, Julie S Biteen (Department of Chemistry, Wayne State University), Ali Hatami, Kaveendya S Mallikaarachchi, Yingxi Elaine Zhu (Department of Chemical Engineering, Departments of Chemistry and Biological Sciences, Wayne State University), Katherine Lopez (Phillips Exeter Academy), Seth W Childers (Department of Chemistry, University of Pittsburgh), Jared M Schrader (Departments of Chemistry and Biological Sciences, Wayne State University)
Abstract:
Biomolecular condensates are membraneless assemblies of proteins and RNAs that compartmentalize various biochemical processes in all forms of life. Biomolecular condensates can accelerate enzymatic reactions, impart specificity to the reactions, or serve as storage sites for proteins and RNAs1. Bacterial ribonucleoprotein bodies (BR-bodies) are a type of biomolecular condensates found in bacteria, containing the RNA degradosome and mRNAs. Disrupting BR-bodies in the α-proteobacterium Caulobacter crescentus resulted in a 3-4 fold slowdown in global mRNA decay2. Based on this, we hypothesized that the phase separation of mRNA with components of the RNA degradosome, RNase E, PNPase, and the DEAD Box RNA helicase RhlB may enhance the degradation rate of bacterial mRNAs. Using in-vitro reconstituted minimal BR-bodies and in vivo reconstitutions, we demonstrate that BR-bodies significantly enhance the endonucleolytic activity of RNase E, the exonucleolytic activity of PNPase, and the ATPase activity of DEAD box RNA helicase RhlB, suggesting an acceleration of enzyme kinetics within the condensed state. In line with the role of accelerated decay, in vivo mRNA tracking experiments showed that mRNAs are rapidly degraded when colocalized with BR-bodies. While BR-bodies appear to accelerate enzymatic RNA decay activities during exponential growth, when cells enter stationary phase growth arrests and the cytoplasm becomes more compact and less dynamic3. Single-molecule microscopy revealed that BR-bodies transition from a dynamic, liquid-like state during exponential growth to a more static, arrested state with diminished internal dynamics in stationary phase. mRNA tracking experiments revealed that the reduced dynamics correlated with a switch of internal decay, to mRNA storage, where mRNAs remain colocalized with BR-bodies for extended durations. Finally, by aging in vitro reconstituted BR-bodies, we find that aging reduces internal recovery by FRAP which leads to a strong reduction in RNase E cleavage. Taken together, this suggests that BR-bodies switch from mRNA decay BR-bodies in actively growing cells to mRNA storage bodies in stationary phase.
References:
1. Banani, S.F., Lee, H.O., Hyman, A.A., and Rosen, M.K. (2017). Biomolecular condensates: organizers of cellular biochemistry. Nature Reviews Molecular Cell Biology 18, 285–298. https://doi.org/10.1038/nrm.2017.7.
2. Al-Husini, N., Tomares, D.T., Pfaffenberger, Z.J., Muthunayake, N.S., Samad, M.A., Zuo, T., Bitar, O., Aretakis, J.R., Bharmal, M.-H.M., Gega, A., et al. (2020). BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates. Molecular Cell 78, 670-682.e8. https://doi.org/10.1016/j.molcel.2020.04.001.
3. Parry, B.R., Surovtsev, I.V., Cabeen, M.T., O’Hern, C.S., Dufresne, E.R., and Jacobs-Wagner, C. (2014). The Bacterial Cytoplasm Has Glass-like Properties and Is Fluidized by Metabolic Activity. Cell 156, 183–194. https://doi.org/10.1016/j.cell.2013.11.028.
Keywords: Ribonucleoprotein complexes, biomolecular condensates, RNA decay
Saturday 08:45-09:00am: Data-driven maps of RNA polymerase III transcription and macromolecular interactions identifies new gene targets and regulatory mechanisms
Rajendra KC (Center of Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA), Ruiying Cheng (Cell Developmental Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA), Sihang Zhou (Cell Developmental Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA), Simon Lizarazo (Department of Molecular and Integrative Physiology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA), Duncan Smith (Department of Biology, New York University, New York, NY), Kevin Van Bortle (Cell Developmental Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA)
Abstract:
RNA polymerase III (Pol III) is responsible for transcribing a diverse array of small non-coding RNAs (ncRNAs), including tRNA, 5S rRNA, 7SL, BC200, and others. Pol III also exhibits dynamic activity across various tissues and cancer cells. Despite its crucial role, our understanding of the full spectrum of Pol III transcription and the mechanisms underlying its context-specific activities remains limited, highlighting the need for a comprehensive study of Pol III transcription and its macromolecular interactions. Recent findings have revealed Pol III's involvement in miRNA transcription and its co-transcription with Pol II of certain ncRNAs (e.g., RPPH1, U6, 7SK), adding complexity to the Pol III transcriptome. This underscores the necessity for a meta-study integrating Pol III activity with other RNA polymerases, a challenge compounded by limited genomics data. To address this, we developed a unified framework for quantifying polymerase occupancy using ChIP-seq data across various tissues and conditions. By intersecting this occupancy map with a comprehensive multi-class promoter set—including protein-coding genes (PCGs), non-coding genes, and repetitive elements— we identified widespread Pol III occupancy across thousands of PCGs. Our innovative quantification of Pol III termination using the T4score revealed the production of small nascent RNA transcripts at these PCGs, ending with a run of four thymidines. Additionally, we discovered hundreds of unannotated genomic regions exhibiting high Pol III activity and sensitivity to SSB knockdown, many of which may represent potential new Pol III target genes. In parallel, limited proteomics data have constrained efforts to elucidate Pol III's macromolecular interaction partners. To overcome this, we constructed an extensive protein-protein interaction (PPI) network from the BIOGRID database, modeled as an electrical circuit network. By applying effective conductance as a measure of protein proximity, we identified several new Pol III interaction partners, uncovering additional potential regulatory roles for Pol III.
Keywords: metamap, RNA, polymerase
Saturday 09:00-09:15am: Human CCR4-NOT is a global regulator of gene expression and is a novel silencer of retrotransposon activation
Shardul Kulkarni (Center for Eukaryotic Gene Regulation and Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA), Alexis Morrissey, Courtney Smith, Oluwasegun T. Akinniyi, Shaun Mahony (Center for Eukaryotic Gene Regulation and Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA), Aswathy Sebastian, Istvan Albert (Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, USA), Cheryl A. Keller, Belinda Giardine, Istvan Albert (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 C. Reese (Center for Eukaryotic Gene Regulation and Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA)
Abstract:
CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation, and translation. It has been well studied in budding yeast; however, relatively less is known about its regulation and functions in mammals. To characterize the functions of the human CCR4-NOT complex, we developed a rapid auxin-induced degron system to deplete CNOT1 (the scaffold of the complex) and CNOT4 (E3 ubiquitin ligase) in cell culture. Transcriptome-wide measurements of gene-expression revealed that depleting CNOT1 changed several thousand transcripts, wherein most mRNAs were increased and resulted in a global decrease in mRNA decay rates. In contrast to what was observed in CNOT1-depleted cells, CNOT4 depletion only modestly changed RNA steady-state levels and, surprisingly, led to a global acceleration in mRNA decay. To further investigate the role of CCR4-NOT in transcription, we used transient transcriptome sequencing (TT-seq) to measure ongoing RNA synthesis. Depletion of either subunit resulted in increased RNA synthesis of several thousand genes. In contrast to most of the genome, a rapid reduction in the synthesis of KRAB-Zinc-Finger-proteins (KZNFs) genes, especially those clustered on chromosome 19, was observed. KZNFs are transcriptional repressors of retro-transposable elements (rTEs), and consistent with the decreased KZNFs expression, we observed a significant and rapid activation of rTEs, mainly Long interspersed Nuclear Elements (LINEs). Our data reveal that CCR4-NOT regulates gene expression and silences retrotransposons across the genome by maintaining KZNF expression. These data establish CCR4-NOT as a global regulator of gene expression, and we have identified a novel mammalian-specific function of the complex, the suppression of rTEs.
Keywords: CCR4-NOT, Retrotranspsons, KRAB Zinc finger proteins (KZFPs)
Saturday 09:15-09:30am: Characterization of the UPF1 protein interactome in yeast provides mechanistic insight into mRNP transitions during nonsense-mediated mRNA decay
Sarah LH Nock (Department of Genetics and Genome Sciences, Case Western Reserve University), Kristian E Baker (Department of Genetics and Genome Sciences, Case Western Reserve University)
Abstract:
Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular RNA quality control process recognizing aberrant RNAs and targeting them to rapid degradation. Typical substrates of the NMD pathway include transcripts harboring nonsense mutations which invoke premature translation termination and the production of potentially deleterious, C-terminally truncated polypeptides. NMD is mediated through interactions between the mRNA substrate, the prematurely terminating ribosome, and the NMD machinery – consisting of core proteins UPF1, UPF2, and UPF3. The central factor, UPF1, exhibits essential RNA binding and ATPase activities and is thought to coordinate interaction between the NMD and translation machineries, subsequently promoting accelerated decay of substrates. Despite considerable investigation into the molecular events underlying NMD, how UPF1 mediates its function on terminating ribosomes and promotes substrate degradation, and the precise roles for UPF2 and UPF3 in this process remain unknown.
To identify UPF1 protein interaction partners key for mediating NMD in vivo, we have applied proximity-based protein labeling (employing a fusion of UPF1 to the catalytically-enhanced biotin ligase, TurboID) in a yeast strain engineered for removal of abundant naturally-occurring biotinylated proteins from our samples. In addition to monitoring functional UPF1, we have analyzed a number of NMD-deficient UPF1 mutants that stall NMD at various stages of the pathway, thereby enabling us to monitor protein dynamics throughout the NMD process. Data demonstrating enhanced sensitivity in our assay and the detection of novel UPF1 protein partners will be presented.
Keywords: UPF1, proximity labeling, nonsense-mediated mRNA decay (NMD)
Saturday 09:30-09:45am: Mapping the transcriptional effects of the ER stress sensor IRE1 with optogenetic oligomerization and long-read sequencing
Jacob W. Smith (Department of Chemistry and Biochemistry, The Ohio State University), Vladislav Belyy (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
The Unfolded Protein Response (UPR) is a eukaryotic stress response pathway that can switch between promoting cellular homeostasis and regulated cell death in response to imbalances in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) is one of three known ER membrane-resident stress sensors, and its activity in response to stress is thought to promote the cytoprotective aspects of the UPR. IRE1 responds to unfolded proteins in the ER lumen by forming homo-oligomers that enable trans-autophosphorylation and activation of the cytosolic RNase domain, which then facilitates non-canonical splicing of the mRNA of transcription factor XBP1 into an active isoform that induces a sweeping transcriptional program. IRE1 has been shown to play an important role in diseases where cells are faced with chronic ER stress, such as neurodegeneration and diabetes. A lack of direct experimental means of activation has made it difficult to study IRE1 signaling independently of the other UPR sensors, leading us to engineer an IRE1 construct with an added light-inducible oligomerizing domain to cluster and subsequently activate the cytosolic RNase domain. We used long-read sequencing to analyze the transcriptome of human cells expressing this IRE1 construct and subjected to varying exposures of light or chemical stress. Our data have indicated potential new players in the IRE1 signaling pathway and support the hypothesis that IRE1’s RNase domain can degrade a broad set of mRNAs in addition to XBP1, its canonical target. This method has also enabled us to investigate alternative splicing patterns and novel splicing substrates of IRE1. By using a specific and direct method to investigate the role of IRE1 signaling in the complex network of the UPR, our data provide insights into the role of IRE1 in the human cell’s ability to adapt and respond to severe ER stress and may guide the development of future therapeutics.
Keywords: Transcriptomics, Splicing, Stress
Saturday 09:45-10:00am: GCN2 monitors mRNA translation termination
Katharine R. Maschhoff (Department of Biochemistry and Molecular Biology, Mayo Clinic), Kailey Worner (Department of Biochemistry and Molecular Biology, Mayo Clinic), Gabrielle M. Schuh (Department of Biochemistry and Molecular Biology, Mayo Clinic), Wenqian Hu (Department of Biochemistry and Molecular Biology, Mayo Clinic)
Abstract:
Controlling mRNA translation is critical for proper protein production. Although translation initiation and elongation regulations are becoming increasingly clear, whether and how translation termination is monitored remains poorly understood. Using an acute protein degradation system coupled with phenotypic rescue via ectopic expression, here we show that the impaired translation termination reaction leads to rapid activation of GCN2, resulting in eIF2a phosphorylation and inhibition of translation initiation, which occurs prior to ribosome collisions. Ribosome profiling analyses reveal that GCN2 monitors terminating ribosomes and prevents ribosome collisions and mitigates translation readthrough when translation termination is compromised. This rapid activation of GCN2 by compromised translation termination occurs in both stem and somatic cells, and in mouse and human cells. These results reveal a conserved surveillance mechanism of translation termination.
Keywords: GCN2, Translation Termination, Ribosome Collision
Saturday 10:30-10:45am: Predictive computational method development for T-box riboswitch drug discovery
Emily A. Fairchild (Department of Chemistry & Biochemistry, Ohio University & Department of Chemistry, Leipzig University), Destini McCartney (Department of Chemistry & Biochemistry, Ohio University), Danushika Hearth (Molecular & Cellular Biology Program, Ohio University & Department of Chemistry & Biochemistry, Ohio University), Sebastian Schmutzler (Department of Chemistry, Leipzig University), Ralf Hoffmann (Department of Chemistry, Leipzig University), Jennifer V. Hines (Molecular & Cellular Biology Program, Ohio University & Department of Chemistry & Biochemistry, Ohio University)
Abstract:
The T- Box Riboswitch is a novel antibiotic target for Gram positive bacteria found at the 5´- end of crucial genes for cell survival and uses charged and uncharged tRNA as a ligand molecule.1 The antiterminator of the T-Box Riboswitch binds to the acceptor end of tRNA and has a highly conserved sequence and structure.1 To develop a computational workflow to map possible antiterminator-ligand interactions, we chose to investigate deca-peptides due to ease of synthesis and diversity of functional groups. Our goal is to develop an automatable workflow (utilizing Schrödinger software) with broad applicability to RNA targets and diverse ligands. We selected 16 deca-peptides to synthesize based on our existing established docking protocols.2,3 We then used data from primary screening specificity assays to inform the refinement of the computational method and the subsequent data analysis. Analysis of the docking data focused on the location of the peptide and the interactions it made with the antiterminator. We used statistical methods (PCA and k-medoids clustering analysis) to group compounds based on distance and scoring function data. The training set of peptides was also tested in an in vitro T-Box riboswitch transcription readthrough assay4 for effectiveness on the riboswitch function. The correlation between the computational and experimental results will be discussed.
References:
(1) Zhang, J.; Ferré-D’Amaré, A. R. Structure and Mechanism of the T-Box Riboswitches. Wiley Interdiscip. Rev. RNA 2015, 6 (4), 419–433. https://doi.org/10.1002/wrna.1285.
(2) Orac, C. M.; et.al. Synthesis and Stereospecificity of 4,5-Disubstituted Oxazolidinone Ligands Binding to T-Box Riboswitch RNA. J. Med. Chem. 2011, 54 (19), 6786–6795. https://doi.org/10.1021/jm2006904.
(3) Armstrong, I.; et.al. RNA Drug Discovery: Conformational Restriction Enhances Specific Modulation of the T-Box Riboswitch Function. Bioorg. Med. Chem. 2020, 28 (20), 115696. https://doi.org/10.1016/j.bmc.2020.115696.
(4) Zeng, C.; et.al. Factors That Influence T Box Riboswitch Efficacy and tRNA Affinity. Bioorg. Med. Chem. 2015, 23 (17), 5702–5708. https://doi.org/10.1016/j.bmc.2015.07.018.
Keywords: RNA Drug Design, T-Box Riboswitch, Computational Drug Design
Saturday 10:45-11:00am: Construction of RNA four-way junction to co-deliver RNAi and chemical drugs for efficient treatment of colon cancer lung metastasis with undetectable toxicity
Kai Jin (Division of Pharmaceutics & Pharmacology, College of Pharmacy; Center for RNA Nanotechnology and Nanomedicine; The Ohio State University, Columbus, OH, United States.), Xin Li (Division of Pharmaceutics & Pharmacology, College of Pharmacy; Center for RNA Nanotechnology and Nanomedicine; The Ohio State University, Columbus, OH, United States.), Peixuan Guo (Division of Pharmaceutics & Pharmacology, College of Pharmacy; Center for RNA Nanotechnology and Nanomedicine; The Ohio State University, Columbus, OH, United States.)
Abstract:
RNA therapeutics emerge as the third milestone in pharmaceutical drugs. Here is to report the in vitro and in vivo assessments of the pathology and safety aspects of various RNA nanoparticles, encompassing RNA three-way junction (3WJ) containing 2-F modified pyrimidine, folic acid, and survivin siRNA, as well as RNA four-way junction (4WJ) with 2-F modified pyrimidine and 24 copies of SN38. The investigation involved animal models and patient serum. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo examinations include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis, serum profiling, and organ weight analysis. The extensive safety evaluations revealed that no significant toxicity, side effects, or immune responses were observed for RNA nanoparticles. The physicochemical foundation guiding the strategic construction of a branched RNA four-way junction (4WJ) nanoparticle was also investigated. The use of the 4WJ leads to enhanced high thermostability and drug payload for cancer therapy, particularly targeting metastatic tumors in the lung. The therapeutic effect of the 4WJ nanostructure functionalized with the anti-cancer chemical drug SN38 and tested in two distinct cancer models: colorectal cancer xenograft and orthotopic lung metastasis of colon cancer. The 4WJ RNA drug complex exhibited effective and spontaneous cancer targeting, demonstrating cancer inhibition. The 4WJ showed swift renal excretion, rapid body clearance, and minimal organ accumulation, with no detectable toxicity or immunogenicity. These findings underscore RNA nanoparticles as effective and safe drug delivery vehicles poised for future clinical translations.
References:
1. Kai Jin, Mitch A. Phelps, et al Yuan-Soon Ho, Peixuan Guo. In Vitro and In Vivo Evaluation of the Pathology and Safety Aspects of Three- and Four-Way Junction RNA Nanoparticles. Molecular Pharmaceutics. 2024, https://doi.org/10.1021/acs.molpharmaceut.3c00845
2. Xin Li, Kai Jin, Mitch A. Phelps, et al Yuan Soon Ho, Peixuan Guo. RNA four-way junction (4WJ) for spontaneous cancer-targeting, effective tumor-regression, metastasis suppression, fast renal excretion and undetectable toxicity. Biomaterials, 2024: 305, 122432, https://doi.org/10.1016/j.biomaterials.2023.122432.
Keywords: RNA Based Therapeutics, RNA Interactions, RNA Drug Conjugation
Saturday 11:00-11:15am: CASC15 lncRNA as a Mediator of Vascular Senescence in the Renin-Angiotensin System
Warlley Rosa Cunha (Center for Molecular Medicine and Genetics Wayne State University, Detroit, MI), Maria Martin de la Vega (Center for Molecular Medicine and Genetics,Wayne State University, Detroit, MI), Anika Kulkarni (Michigan State University, East Lansing, MI), Delphine Gomez (University of Pittsburgh. Pittsburgh. PA), Cristina Espinosa-Diez (Center for Molecular Medicine and Genetics,Wayne State University, Detroit, MI)
Abstract:
The renin-angiotensin system regulates vascular function and maintains blood pressure homeostasis. Angiotensin-II (Ang-II) and angiotensin-1-7 (Ang-1-7) have opposing effects on vascular health. Ang-II promotes vascular senescence and pathophysiological remodeling by inducing oxidative stress, inflammation, vascular hypertrophy, and endothelial dysfunction. In contrast, Ang-1-7, known for its vasodilatory, anti-inflammatory, and antioxidative properties, counteracts these detrimental effects. Our lab has characterized CASC15, a lncRNA enriched in vascular smooth muscle and endothelial cells, which has been shown to influence cellular responses to stress. Previous findings demonstrated that Ang-II reduces CASC15 expression in vascular smooth muscle cells (SMCs), promoting senescence. However, the ability of Ang 1-7 to reverse this effect by restoring CASC15 expression requires further investigation. We treated mouse SMCs with Ang-II, and other cellular stressors such as hydrogen peroxide (H2O2) and doxorubicin to assess CASC15 expression through RT-qPCR. CASC15 expression decreased significantly following Ang-II treatment, while co-treatment with Ang 1-7 restored CASC15 expression beyond control levels. H2O2 and doxorubicin also reduced CASC15 expression. To examine the impact of CASC15 loss, SMCs were transfected with an antisense oligonucleotide to suppress CASC15 expression, and DNA damage was evaluated, resulting in increased γH2AX foci, indicating elevated DNA damage. Ang 1-7 prevented DNA damage in both control and CASC15-inhibited cells. In conclusion, CASC15 is a crucial SMC regulator, and its loss promotes vascular senescence, hypertrophy, and DNA damage, effects countered by Ang-1-7. Targeting CASC15 could offer new strategies for mitigating vascular aging and improving vascular health, particularly chronic kidney disease. Future work will focus on the role of CASC15 responses in hypertensive remodeling in vivo.
Keywords: LncRNAs, senescence, Angiotensin
Saturday 11:15-11:30am: Title not available online - please see the booklet.
Tiffany Barwell (University of North Carolina at Charlotte )
Abstract not available online - please check the booklet.
Saturday 11:30-11:45am: Purine mRNA modifications impact translation speed
Rachel Giles (Chemistry, University of Michigan), Tyler Smith (Chemistry, University of Michigan), Kristin Koutmou (Chemistry, University of Michigan)
Abstract:
Cells chemically modify all three major classes of biomolecules (DNA, RNA and protein) to control their structure, function and stability. In RNAs, nucleoside modifications are incorporated either enzymatically or as the result of RNA damage. Regardless of how they are added, the insertion of chemical modifications into mRNA coding sequences can influence translation elongation and termination. Here we investigate the consequences of recently discovered guanosine mRNA modifications, N1-methyl guanosine (m1G) and N2-methyl guanosine (m2G), and a well-established adenosine mRNA modification, inosine (I), on protein synthesis. Non naturally occurring purine modifications, 2,6 – diaminopurine (DAP) and 2-aminopurine (2AP), were also examined. All of these modifications alter the hydrogen bonding potential between codon and anticodon nucleotides as well potential stereochemical interactions between codons and their cognate amino-acyl tRNAs. We find that inclusion of modifications around varied positions of the guanosine base in mRNA codons (GUG, CGU, UGA and UAG) impact translation elongation and termination in a fully reconstituted E. coli translation system. Our findings indicate that these modifications alter the rates of translation elongation and termination in a context dependent manner, with modifications in the first and second positions of the codon slowing elongation the most. These data support the growing body of evidence indicating that mRNA modifications can alter protein production by the ribosome.
Keywords: Translation, RNA modifications, Kinetics
Saturday 11:45-12:00pm: Selective Small Molecule Targeting of Homologous RNA-Binding Proteins
Johann M. Roque (Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame IN), Julia A. Haas (Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame IN), Beth P. Anderson (Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame IN), Ariel J. Thelander (Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame IN), Blanton S. Tolbert (Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA), Brittany S. Morgan (Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame IN)
Abstract:
RNA-Binding Proteins (RBPs) are exceptional therapeutics targets in a multitude of cancers and neurological diseases; yet despite their therapeutic promise, few chemical probes and/or drugs have been developed for RBPs. One of the major challenges is the abundance and exceptionally high structural conservation of RNA-binding domains (RBDs), where the sites of greatest diversity are in “unligandable” dynamic loops and linkers. Herein, we describe the selective targeting of conserved cysteine residues located in the dynamic loops of two homologous RBPs: heterogenous nuclear RiboNucleoProteins (hnRNPs) H and F. Specifically, the ligands target quasi-RNA Recognition Motifs 1 and 2 (qRRMs1,2), which share a highly conserved tertiary structure, greater than 85% sequence similarity, yet have unique molecular motions and conformational ensembles. We hypothesized that the slower protein motions in hnRNP H qRRMs1,2 would lead to longer-lived, pocket-like structures that could be exploited for selective ligand targeting. Indeed, covalent fragments were identified for qRRMs1,2 and optimized to have ten-fold selectivity for hnRNP H. Furthermore, traditional structure-affinity relationships were elucidated for the lead ligand, which targets a cysteine residue in a dynamic loop substructure. Our current efforts are focused on improving the potency and selectivity of the lead ligand through fragment building strategies and utilizing biomolecular NMR and molecular dynamics to explore the basis of ligand recognition and selectivity. The guiding principles discovered will be first-in-kind, describing how molecular motions in dynamic loops can be harnessed for ligand selectivity. The principles will be key for developing the first small molecules for hnRNP H into proteome-wide, selective chemical probes and for identifying selective small molecules for an array of other RBPs. The selective targeting of RBDs and RBPs will revolutionize our understanding of RBP structure, function, and therapeutic potential, and it will also establish a precedent of exploiting protein loops and dynamics for selective ligand targeting in chemical biology and drug discovery
Keywords: