Talk Abstracts

Friday 01:15-01:30pm: RNA binding proteins, upstream open reading frames, and translational control of human gene expression

Cassia Williams-Rogers (Department of Biological Sciences, Carnegie Mellon University), Matthew Agar-Johnson (Department of Biological Sciences, Carnegie Mellon University), Gemma May (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Translation is an integral part of the conversion of DNA’s genetic information into functional proteins. Translation is controlled at the initiation step by the scanning of the preinitiation complex (PIC) along the 5’ transcript leader until the start codon is recognized. Upstream open reading frames (uORFs) that present potential start sites for translation in the 5’ transcript leader generally suppress translation of downstream genes, but the mechanisms by which uORF usage is regulated are not well defined. Previous work in Drosophila has proposed a translational control mechanism in which the RNA binding protein (RBP) Sex-lethal increases uORF usage in msl-2 and other transcripts by binding immediately downstream of uORF start codons (Medenbach, et al., 2011). However, whether uORF regulation in humans occurs by the same mechanism remains unknown. To address this, we performed large scale computational analyses of human transcript annotations and RBP site conservation and frequency data from GENCODE and the ENCODE project (Van Nostrand, et al., 2020) to investigate the potential role of RBPs in human uORF regulation. We found 5,963 genes with highly conserved AUG uORFs that are enriched for functions and processes related to control of transcription and neuronal / neurotransmitter activity. Furthermore, 281 of these genes have both highly conserved AUG uORFs and downstream RBP binding sites and have even greater enrichment in RNA polymerase II related activities, serine / threonine kinase activity, and chromatin modification functions (P < 0.05). Most notably, using publicly available ribosome profiling data we found that uORFs with downstream RBP binding sites have ~1.5-fold higher ribosome occupancy (p = 3.31e-7) than uORFs without such sites. We will discuss ongoing experiments to test human RBPs regulation of uORF activity using tissue culture reporter assays. Overall, these results suggest novel roles for human RBPs in regulating hundreds of uORFs from critical genes.

Keywords: RNA binding proteins (RBPs), upstream open reading frames (uORFs)

Friday 01:30-01:45pm: Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation by 5′ UTR elements

Rachel O. Niederer (Molecular Biophysics and Biochemistry, Yale University), Maria F. Rojas-Duran (Molecular Biophysics and Biochemistry, Yale University), Boris Zinshteyn (Panorama Medicine), Wendy V. Gilbert (Molecular Biophysics and Biochemistry, Yale University)

Abstract:
Translational control of gene expression plays an essential role in a wide range of cellular processes, ranging from stress responses to immune regulation. The protein output per mRNA is ultimately governed by a combination of cis elements and trans factors. However, the key mRNA features that distinguish efficiently translated from poorly translated mRNAs remain largely unknown. To elucidate the varied mechanisms by which translational control is achieved, we developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5′ untranslated regions (5′ UTRs). We find that 5′ UTRs confer thousand-fold differences in ribosome recruitment in vitro. Using DART, we identified novel translational enhancers and silencers and determined a functional role for most alternative 5′ UTR isoforms expressed in yeast. Our analysis revealed both anticipated and novel trends in the data. For example, engineered stems are generally inhibitory to recruitment at levels proportional to their folding strength. However, the effects are highly context-dependent with some strong stems apparently promoting recruitment in specific positions. Strikingly, we observed a strong global anticorrelation between %C and ribosome recruitment (Spearman R = -0.544) and found C-rich motifs over-represented among poorly recruiting TLs. The identified C-rich motifs are necessary and sufficient to repress translation both in vitro and in vivo. We also observe differential expression of 5′ UTR isoforms containing C-rich motifs in response to glucose starvation. This suggests these novel elements could be used to regulate translation of specific messages under different growth conditions. DART enables both the discovery of novel elements as well as systematic assessment of the translational regulatory potential of 5′ UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems.

Keywords: Translational control, RNA structure, Translation initiation

Friday 01:45-02:00pm: Effectors of stress-responsive translational repression in C. neoformans

Corey M. Knowles (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, 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 undergo the rapid transition from surviving in its environmental niche, to surviving the harsh environment inside of the human lung. Here, it is subject to the sudden temperature shift to the human core temperature of 37°C, and oxidative stress from resident lung macrophages, among other stressors. In a wild type (WT) strain, exposure to these stressors results in ribosome collision accompanied by a repression in translation, and rapid decay of abundant homeostatic mRNAs, many of which code for ribosomal proteins. This response results in a translatome reprogramming, that promotes translation of mRNAs encoding stress response effectors. Our previous work has identified Ccr4-dependent mRNA decay to be a requirement for translatome reprogramming in response to stress in C. neoformans, and as a result, a ccr4Δ mutant is broadly stress sensitive. This observation has led us to investigate additional paths to translational repression, testing the hypothesis that stress responsive translatome reprogramming will require regulation of translation at the ribosome. Gcn2, the sole kinase of eIF2α in C. neoformans, is required for translational repression and subsequent RP mRNA decay during oxidative stress from H₂O₂, but is completely dispensable for adaptation to host temperature stress of 37°C. Additionally, a gcn2Δ mutant is sensitive to oxidative stress, and exhibits persistent disome accumulation. These results point to deadenylation-dependent decay as a convergence point for translatome reprogramming in C. neoformans, and suggest that individual stressors and their magnitude contribute to the translational response to stress in this important pathogen through different ribosome associated pathways. Future work will determine which components of the ribosome quality control machinery are necessary for recognizing and resolving these changes in translation in response to environmental stressors relevant to host adaptation.

Keywords: translation regulation, ribosome collision, cellular stress

Friday 02:00-02:15pm: Pnrc2-dependent mRNA decay and translational control mechanisms promote oscillatory gene expression during vertebrate segmentation

Monica Mannings (The Ohio State University Molecular Genetics Department), Thomas Gallagher (The Ohio State University Molecular Genetics Department), Sharon Amacher (The Ohio State University Molecular Genetics Department)

Abstract:
During early vertebrate embryogenesis, muscle and skeletal stem cells are grouped into reiterated segments, called somites, in a process called somitogenesis. Sequential somite formation is established by a genetic oscillator called the segmentation clock, comprised of a network of genes expressed rhythmically in the presomitic mesoderm. Precise control of segmentation clock oscillations is driven by robust temporal regulation of mRNA production, translation, and mRNA decay, and our work explores post-transcriptional mechanisms that regulate oscillatory expression. Previously, we demonstrated that Proline-rich nuclear receptor coactivator 2 (Pnrc2) regulates oscillatory mRNA decay in zebrafish and loss of pnrc2 results in stabilization and accumulation of clock transcripts. Surprisingly, pnrc2 mutant embryos exhibit normal protein expression and somite patterning is not disrupted. We are currently addressing this molecular phenotype by analyzing the translation status of clock transcripts in wild-type and pnrc2 mutant embryos using polysome profiling. We found segmentation clock transcripts her1, her7, dlc, and rhov are enriched in ribosome-unbound fractions in pnrc2 mutants, compared to wild type, which indicates that stabilized transcripts are translationally repressed. To further dissect the mechanism of clock transcript regulation, we performed in vivo reporter analyses that revealed that disruption of two RNA binding protein motifs, a Pumilio Response Element (PRE) and AU-rich Element (ARE), within the her1 3’UTR markedly increased stability and polysome association of reporter mRNAs, suggesting one or both elements are important for translational regulation of clock transcripts. Our work investigating the role of Pumilio and ARE-binding proteins in regulating segmentation clock mRNA stability and translation will demonstrate how essential, vertebrate patterning processes are robustly maintained through multiple layers of post-transcriptional regulation.

Keywords: decay, translation, oscillations

Friday 02:15-02:30pm: The Musashi family of stem cell factors is critical for the survival and function of terminally differentiated photoreceptor neurons.

Fatimah Matalkah (Biochemistry/WVU), Bohye Jeong (Biochemistry/WVU), Visvanathan Ramamurthy (Biochemistry/WVU), Peter Stoilov (Biochemistry/WVU)

Abstract:
The Musashi (MSI) proteins are RNA binding proteins consisting of two paralogs MSI1/2 that are highly conserved across species. The MSI proteins are known for their role in maintaining undifferentiated state of stem cells. In the context of mature retina, we show that MSI proteins maintain high expression level with a distinct switch occurring between MSI1 and MSI2 expression during postnatal development. Our observations reveal a new role for the MSI proteins in terminally differentiated cells. To understand the role of the MSI in mature photoreceptors we utilized an inducible mouse knockout model that allows for the timed deletion of MSI at postnatal day 30. Ablation of the MSI proteins in the fully developed photoreceptors caused progressive loss of vision. The results demonstrate that the MSI are required for the maintenance of the differentiated photoreceptors and functionally can compensate for each other. To investigate the molecular mechanism, we used CLIP-Seq to identify MSI1 targets and the binding sites in the retinal transcriptome. CLIP-Seq revealed MSI binding to the 3’-UTR of large number of transcripts. Among the identified targets are proteins that are involved in the phototransduction pathway or are critical for the maintenance of photoreceptors outer segment structure. Proteomics analysis showed deletion of the MSI results in a significant reduction in proteins involved in the phototransduction pathway including RHO, GNB1, and GNAT1 with a significant increase in GFAP and STAT3. Interestingly, the results also detected on protein level a switch in the alternative splicing that we previously detected by RNA-Seq. Closer examination of the protein levels of MSI targets revealed that the MSI function as translational activator. Thus, we hypothesize that the high expression of MSI proteins in mature retina is evolutionary meant to keep up with the high demand of protein synthesis that is required to maintain the constant regeneration of the light receptive organelle of the photoreceptors. Importantly, the protein expression from a significant proportion of the MSI bound mRNA is not affected by the loss of MSI. This observation implies that MSI is either acting in cooperation with additional factors, or there is a built-in redundancy in the translational regulation of many of the MSI bound transcripts.

Keywords: Musashi, RNA binding proteins, Photoreceptors

Friday 03:00-03:15pm: Probing the structure and function of the Bacillus subtilis thrS T-box riboswitch

Alexander T. Runyon (Microbiology Department, The Ohio State University), Tina M. Henkin (Microbiology Department, The Ohio State University)

Abstract:
Many amino acid-related genes in Gram-positive bacteria are regulated at the level of transcription attenuation by cis-acting RNA structures known as T-box riboswitches. A specific uncharged tRNA that corresponds to the amino acid specificity of the downstream gene is used as the regulatory ligand. ~95% of T-box riboswitches are predicted to fold into the canonical pattern, which includes a set of conserved sequence and structural elements. Current biochemical understanding of T-box riboswitch function derives from noncanonical models that lack one or more of these conserved elements. The Bacillus subtilis thrS riboswitch is located upstream of the threonyl-tRNA synthetase coding sequence and contains all of the major conserved elements, making it a canonical riboswitch. Unlike other canonical T-box RNAs, tRNA-dependent antitermination in vitro has been demonstrated with thrS, which allows comparisons with biochemical data obtained from non-canonical riboswitches. Point mutations have been generated in conserved elements in thrS and in vitro transcription, tRNA binding assays, and RT-qPCR indicate that the conserved elements in thrS are critical for tRNA-dependent antitermination activity, a subset of which are critical for tRNA binding. Structural studies performed with SHAPE confirm the overall structural pattern and mutational disruption of conserved elements. In addition, some elements of thrS have been modified to resemble the corresponding elements of the non-canonical ileS US riboswitch found in Actinobacteria. These mutations reduce thrS function, although not as severely as point mutations that disrupt key elements. Analysis of the thrS system provide a better understanding of the function of canonical T-box RNAs, which represent the major class of these elements found in nature.

References:
Kreuzer KD, Henkin TM. 2018. The T-Box riboswitch: tRNA as an effector to modulate gene regulation. Microbiol Spectrum 6(4).
Grundy FJ, Henkin TM. 1993. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74:475–482.
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.
Suddala KC, Zhang J. 2019. High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nat Struct Mol Biol 26:1114–1122.
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

Friday 03:15-03:30pm: Protein cofactors and substrate dictate Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Ila A. Marathe (Department of Chemistry and Biochemistry, Ohio State University), Walter J. Zahurancik (Department of Chemistry and Biochemistry, Ohio State University), Stella M. Lai (Department of Chemistry and Biochemistry, Ohio State University), Khan L. Cox, Michael G. Poirier (Department of Physics, Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, Ohio State University)

Abstract:
The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ~250-500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ~10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

Keywords: RNase P, Conformational dynamics, FRET

Friday 03:30-03:45pm: Sarecycline interferes with tRNA accommodation and tethers mRNA to the 70S ribosome in a context dependent manner

Zahra Batool (Department of Biolgical Sciences, University of Illinois at Chicago), Ivan B. Lomakin (Department of Molecular Biophysics and Biochemistry, Yale University), Yury S. Polikanov (Department of Biolgical Sciences, University of Illinois at Chicago), Christopher G. Bunick (Department of Molecular Biophysics and Biochemistry, Yale University)

Abstract:
Sarecycline is a new narrow-spectrum tetracycline-class antibiotic approved for the treatment of acne vulgaris. Tetracyclines share a common four-ring naphthacene core and inhibit protein synthesis by interacting with the 70S bacterial ribosome. Sarecycline is distinguished chemically from other tetracyclines because it has a 7-[[methoxy(methyl)amino]methyl] group attached at the C7 position of ring D. To investigate the functional role of this C7 moiety, we determined the X-ray crystal structure of sarecycline bound to the Thermus thermophilus 70S ribosome. Our 2.8-Å resolution structure revealed that sarecycline binds at the canonical tetracycline binding site located in the decoding center of the small ribosomal subunit. Importantly, unlike other tetracyclines, the unique C7 extension of sarecycline extends into the messenger RNA (mRNA) channel to form a direct interaction with the A-site codon to possibly interfere with mRNA movement through the channel and/or disrupt A-site codon–anticodon interaction. Based on our biochemical studies, sarecycline appears to be a more potent initiation inhibitor compared to other tetracyclines, possibly due to drug interactions with the mRNA, thereby blocking accommodation of the first aminoacyl transfer RNA (tRNA) into the A site. Following up on these observations, ribosome profiling analysis also showed strong context dependent inhibition at the start codon. Overall, our structural and biochemical findings rationalize the role of the unique C7 moiety of sarecycline in antibiotic action.

References:
Batool Z., Sarecycline interferes with tRNA accommodation and tethers mRNA to the 70S ribosome, PNAS, 2020

Keywords: sarecycline, ribosome, antibiotic

Friday 03:45-04:00pm: Crystal Structure of the RNA Cleaving 10-23 DNAzyme Captured in a Pre-Catalytic State

Evan Cramer (Department of Biochemistry, West Virginia University), Sarah Starcovic (Department of Biochemistry, West Virginia University), Aaron Robart (Department of Biochemistry, West Virginia University)

Abstract:
Deoxyribozymes (DNAzymes) are short single-stranded DNA sequences capable of catalyzing chemical reactions such as RNA cleavage, thymine excision, RNA/DNA ligation, and thymine dimer resolution. Among these, RNA cleaving DNAzymes have emerged as the most prominent due to their applications in medicine as potential protein expression knockdown therapeutics and antiviral agents; however, the lack of a structural insight into catalysis hinders further optimization. Here, we report a structure of the RNA cleaving 10-23 DNAzyme, which requires magnesium as a cofactor. In crystallo, the DNAzyme forms a dimer mediated primarily through a palindromic sequence within the catalytic core. The substrate adopts a sharp kink at the cleavage site that exposes the scissile phosphate with magnesium ions coordinated. This structure reveals that the 10-23 DNAzyme appears to function similarly to RNA cleaving ribozymes such as Group II Introns and functions by inducing a strained substrate conformation that allows the cofactor to interact with the consensus site.

Keywords: DNAzyme, X-Ray Crystallography, RNA Cleavage

Friday 04:00-04:15pm: Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures

Meredith K. Purchal (Program in Chemical Biology, University of Michigan), Daniel E. Eyler, Mehmet Tardu, Megan M. Korn (Department of Chemistry, University of Michigan), Monika K. Franco, Taslima Khan (Program in Chemical Biology, University of Michigan), Ryan McNassor, Hari Sharma, Leena Malik (Department of Chemistry, University of Michigan), Markos Koutmos (Program in Chemical Biology, Department of Chemistry, Department of Biophysics, University of Michigan), Kristin S. Koutmou (Program in Chemical Biology, Department of Chemistry, University of Michigan)

Abstract:
Pseudouridine is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of non-coding and protein coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal mRNA modifying Pus enzymes. Pus7 catalyzes pseudouridine formation into a diverse set of mRNA sequences and is distinct among the Pus proteins because it shares minimal sequence homology with other pseudouridine synthase family members. We solved the first crystal structure of Saccharomyces cerevisiae Pus7, revealing the architecture of the eukaryotic specific insertion domains thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified amino acids and an insertion in the protein important for substrate binding and modification. Our data demonstrate that Pus7 preferentially binds substrates > 25 nucleotides in length possessing the previously identified UGUAR (R = purine) consensus sequence. Furthermore, we observed that RNA secondary structure is not a strong requirement for Pus7 binding. In contrast, the rate constants for pseudouridine incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less structured contexts more quickly. Although less structured substrates were preferred, Pus7 fully modified every tRNA, mRNA and non-natural RNAs containing the consensus recognition sequence we tested. These data suggest that Pus7 is a promiscuous enzyme, and lead us to propose a model for Pus7 substrate selection wherein the enzyme targets its substrates largely based upon the accessibility of a modifiable sequence.

Keywords: RNA modifications, Pseudouridine synthase, crystallography

Friday 04:15-04:30pm: High-Resolution Profiling of Telomerase RNA Structure Dynamics in the Eukaryotic Pathogen Trypanosoma brucei

Kaitlin E. Klotz (Department of Biological Sciences-University of North Carolina at Charlotte), Abhishek Dey, Anais Monroy-Eklund (Department of Biology-University of North Carolina at Chapel Hill), Justin Davis (Department of Biological Sciences-University of North Carolina at Charlotte), Arpita Saha, Bibo Li (Department of Biological, Geological and Environmental Sciences-Cleveland State University), Alain Laederach (Department of Biology-University of North Carolina at Chapel Hill), Kausik Chakrabarti (Department of Biological Sciences-University of North Carolina at Charlotte)

Abstract:
Telomerase is a ribonucleoprotein (RNP) comprised of a reverse transcriptase (TERT) and an RNA template (TR) for extending linear chromosomes to preserve genomic integrity and address the end replication problem within eukaryotic cells. The TR catalytic core, which is responsible for executing the reactions to add repeats to the ends of linear chromosomes, resides near the 5’ end of the telomerase RNP and it is functionally conserved amongst eukaryotes. Until recently, studying the conformation of the cellular telomerase RNP has been difficult due to its long, folded structure and low abundance in vivo. To address this problem and directly analyze the in-cell TbTR architecture, we employed a novel approach which coupled structure-specific in vivo chemical modification with mutational profiling, followed by next-generation sequencing to elucidate the conformation of the TbTR catalytic core. We investigated whether proper assembly of the catalytic core components of telomerase is a requirement for RNA folding and function through affinity purification of the telomerase RNP complex from T. brucei, then we probed the native RNA structure using mutational profiling. This immunoprecipitated telomerase demonstrated telomere repeat addition activity in a telomerase assay, suggesting that this RNA is a vital part of the active telomerase complex. Interestingly, we observed that T. brucei TR exists in two distinctly different folding states in discrete developmental stages within both the insect and mammalian host. Taken together, the described work provides the first detailed analysis of the in vivo folding architecture of telomerase RNA at nucleotide resolution. Because telomerase is the key mechanism employed by the parasite to preserve telomere integrity and thus, maintenance of the sub-telomeric virulence genes, we anticipate that developing a greater understanding of TR dynamics in telomerase function will provide crucial insights into T. brucei telomere synthesis, sub-telomeric virulence gene regulation and the ability of the parasite to survive.

Keywords: Telomerase RNA, Structure Dynamics, Trypanosoma brucei

Saturday 08:45-09:00am: NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain

Bing Wang (Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Dmitri Svetlov (Svetlov Scientific Software, Pasadena, CA 91106, USA), Irina Artsimovitch (Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA)

Abstract:
The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.

Keywords: NMPylation, nsp9, RdRp

Saturday 09:00-09:15am: Understanding the role of CLP1 in mammalian mRNA transcription and cleavage in neurodegeneration

Geneva LaForce (Department of Genetics and Genome Sciences, Case Western Reserve University), Jordan Farr, Cydni Akesson (Department of Genetics and Genome Sciences, Case Western Reserve University), Otis Pinkard, Thomas Sweet (Center for RNA Science and Therapeutics, Case Western Reserve University), Jeff Coller (Department of Molecular Biology and Genetics, Johns Hopkins University), Ping Ji, Eric Wagner (Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston), Ashleigh Schaffer (Department of Genetics and Genome Sciences, Case Western Reserve University)

Abstract not available online - please check the booklet.

Saturday 09:15-09:30am: pre-piRNA trimming and 2′-O-methylation protect piRNAs from 3’ tailing and degradation in C. elegans

Benjamin Pastore (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, Ohio State Biochemistry Program, The Ohio State University ), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University ), Ian F. Price (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, Ohio State Biochemistry Program, The Ohio State University ), Wen Tang (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University )

Abstract:
The Piwi/piRNA pathway suppresses transposable elements and promotes fertility in diverse organisms. Maturation of piRNAs involves pre-piRNA trimming followed by 2′-O-methylation at their 3’ termini. Here we report that 3’ termini of C. elegans piRNAs are subject to nontemplated nucleotide addition and piRNAs with 3’ addition exhibit extensive base-pairing interaction with their target RNAs. Animals deficient for PARN-1 (pre-piRNA trimmer) and HENN-1 (2′-O-methyltransferase) accumulate piRNAs with 3’ nontemplated nucleotides. In henn-1 mutants piRNAs are shortened prior to 3’ addition whereas long isoforms of untrimmed piRNAs are preferentially modified in parn-1 mutant animals. Loss of either PARN-1 or HENN-1 results in modest reduction in steady-state levels of piRNAs. Deletion of both enzymes leads to depletion of piRNAs, desilencing of piRNA targets, and impaired fecundity. Together, our findings suggest that pre-piRNA trimming and 2′-O-methylation act collaboratively to protect piRNAs from tailing and degradation.

References:
Ozata, D.M., Gainetdinov, I., Zoch, A., O'Carroll, D., and Zamore, P.D. (2019). PIWI-interacting RNAs: small RNAs with big functions. Nature reviews Genetics 20, 89-108.

Fuchs Wightman, F., Giono, L.E., Fededa, J.P., and de la Mata, M. (2018). Target RNAs Strike Back on MicroRNAs. Frontiers in genetics 9, 435.

Keywords: piRNA, nontemplated nucleotide addition, degradation

Saturday 09:30-09:45am: Export, accumulation and degradation of 5’ end-extended, spliced tRNAIleUAU in yeast

William A Marshall (Molecular Genetics, Ohio State University), Kunal Chatterjee (Biology, Wittenberg University), Anita K Hopper (Molecular Genetics, Ohio State University)

Abstract:
The export of tRNA from the nucleus to the cytoplasm is an essential process that is integrally involved in cellular metabolism, aging, response to stress, and more. As transcribed, pre-tRNA contains extra leader and trailer sequences at the 5’ and 3’ ends, respectively. Normally, removal of the leader and trailer sequences occurs before tRNA is exported from the nucleus to the cytoplasm. However, 5’ end-extended spliced tRNA (aberrant tRNA) has been detected in yeast, indicating that tRNA was prematurely exported, since splicing of tRNA occurs in the cytoplasm at the surface of the mitochondria in S. cerevisiae. Aberrant tRNA cannot participate in translation, as the 3’ trailer prevents CCA addition and amino acid charging. We report that Mex67, but not the primary tRNA exportin Los1 or the newly bona fide tRNA exporter Crm1, exports the aberrant tRNA precursor. We also learned that two members of the 3’ to 5’ RNA exosome, Dis3 and Rrp6, function in reducing pools of the aberrant tRNA. While Dis3 is active in both the cytoplasm and the nucleus, Rrp6 is restricted to the nucleus, so aberrant tRNA must be reimported from the cytoplasm to be turned over in an Rrp6-dependent fashion, underscoring the role of the tRNA retrograde pathway in tRNA quality control. Also, members of the 5’ to 3’ rapid tRNA decay (RTD) pathway, Rat1 and Xrn1, do not function in turnover of this 5’ extended aberrant tRNA, perhaps because the 5’ extension on aberrant tRNA contains a cap or triphosphate that protects the tRNA from the RTD. Further studies are needed to determine whether 5’ extended tRNA that co-purifies with Mex67-Mtr2 is capped, as well as how 3’ trailer sequences or modifications at individual nucleotides could impact export, accumulation, and degradation of tRNA.

References:
Kilchert C, Wittmann S, Vasiljeva L. The regulation and functions of the nuclear RNA exosome complex. Nat Rev Mol Cell Biol. 2016 Apr;17(4):227-39. doi: 10.1038/nrm.2015.15. Epub 2016 Jan 4. PMID: 26726035.

Kramer EB, Hopper AK. Retrograde transfer RNA nuclear import provides a new level of tRNA quality control in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2013 Dec 24;110(52):21042-7. doi: 10.1073/pnas.1316579110. Epub 2013 Dec 2. PMID: 24297920; PMCID: PMC3876269.

Ohira T, Suzuki T. Precursors of tRNAs are stabilized by methylguanosine cap structures. Nat Chem Biol. 2016 Aug;12(8):648-55. doi: 10.1038/nchembio.2117. Epub 2016 Jun 27. PMID: 27348091.

Keywords: tRNA, Export, turn over

Saturday 09:45-10:00am: Understanding the biological significance of multiple zebrafish Trm10 homologs

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 a subset of tRNAs. Trm10 enzymes are ubiquitous throughout Eukarya and Archaea, with eukaryotes encoding up to three homologs of Trm10. Humans, for example, encode the three homologs TRMT10A, TRMT10B and TRMT10C. Previously, we showed that human TRMT10A and TRMT10B have distinct, non-redundant biochemical activities. Human TRMT10A, like yeast Trm10, catalyzes m1G9 formation on multiple tRNA species whereas TRMT10B forms m1A9 specifically on tRNAAsp. To further probe the significance of these distinct enzyme activities and test their generality across multiple vertebrate models, we are using Danio rerio (zebrafish), which also encodes two cytosolic homologs, Trmt10a and Trmt10b. Like human TRMT10A, zebrafish Trmt10a rescues the trm10∆ phenotype in yeast 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. Intriguingly, however, we discovered that human and zebrafish TRMT10B homologs differ significantly in their in vitro activities. In contrast to the human enzymes, zebrafish Trmt10a and Trmt10b do not show the same pattern of unique in vitro substrate specificities, although they do modify some tRNAs with different catalytic rates. These studies reveal that determinants of substrate specificity of individual Trm10 homologs are complex, and often can not be fully recapitulated by in vitro analysis. In order to fully elucidate the biological functions of these enzymes, we are using mutant zebrafish lines to analyze the specific roles of each enzyme in tRNA modification in vivo.

Keywords: tRNA modification, Trm10, m1R9

Saturday 10:00-10:15am: Genetic variation and microRNA targeting of A-to-I RNA editing fine tune human tissue transcriptomes

Eddie Park (Center for Computational and Genomic Medicine, The Childrens Hospital of Philadelphia), Yan Jiang (State Key Laboratory of Molecular Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology), Lili Hao (National Genomics Data Center & CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics), Jingyi Hui (State Key Laboratory of Molecular Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology), Yi Xing (Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, University of Pennsylvania)

Abstract:
Background
A-to-I RNA editing diversifies the transcriptome and has multiple downstream functional effects. Genetic variation contributes to RNA editing variability between individuals and has the potential to impact phenotypic variability.

Results
We analyze matched genetic and transcriptomic data in 49 tissues across 437 individuals to identify RNA editing events that are associated with genetic variation. Using an RNA editing quantitative trait loci (edQTL) mapping approach, we identify 3117 unique RNA editing events associated with a cis genetic polymorphism. Fourteen percent of these edQTL events are also associated with genetic variation in their gene expression. A subset of these events are associated with genome-wide association study signals of complex traits or diseases. We determine that tissue-specific levels of ADAR and ADARB1 are able to explain a subset of tissue-specific edQTL events. We find that certain microRNAs are able to differentiate between the edited and unedited isoforms of their targets. Furthermore, microRNAs can generate an expression quantitative trait loci (eQTL) signal from an edQTL locus by microRNA-mediated transcript degradation in an editing-specific manner. By integrative analyses of edQTL, eQTL, and microRNA expression profiles, we computationally discover and experimentally validate edQTL-microRNA pairs for which the microRNA may generate an eQTL signal from an edQTL locus in a tissue-specific manner.

Conclusions
Our work suggests a mechanism in which RNA editing variability can influence the phenotypes of complex traits and diseases by altering the stability and steady-state level of critical RNA molecules.

Keywords: RNA editing, microRNA, RNA-seq

Saturday 10:15-10:30am: Global RNA binding analysis defines the hierarchy of RNA-UPF protein interactions during early stages of NMD

Savannah Mills (Department of Biochemistry, Case Western Reserve University), DaJuan Whiteside (Department of Genetics and Genome Sciences, Case Western Reserve University), Kristian E Baker (Department of Genetics and Genome Sciences, Case Western Reserve University)

Abstract not available online - please check the booklet.

Saturday 11:00-11:15am: Metabolic starvation in tumors regulates Alternative Splicing to potentiate tumorigenicity

Safiya Khurshid (Abigail Wexner Research Center, Nationwide Childrens Hospital ), Ruoning Wang (Abigail Wexner Research Center, Nationwide Childrens Hospital ), Dawn Chandler (Abigail Wexner Research Center, Nationwide Childrens Hospital )

Abstract not available online - please check the booklet.

Saturday 11:15-11:30am: Vaccinia virus K7 protein inhibits biochemical activities and liquid-liquid phase separation of the DEAD-box RNA helicase DDX3X

Sarah Venus (Center for RNA Science and Therapeutics, Case Western Reserve University), Kaba Tandjigora (Center for RNA Science and Therapeutics, Case Western Reserve University), Eckhard Jankowsky (Center for RNA Science and Therapeutics, Case Western Reserve University)

Abstract:
The DEAD-box RNA helicase DDX3X, which functions in translation initiation and cellular signaling, is targeted by proteins from diverse viruses, including the vaccinia virus K7 protein. K7 binds the N-terminus of DDX3X, a region that facilitates DDX3X association with translation initiation factors and stress granules. While interactions between K7 and DDX3X are thought to disrupt immune signaling pathways, the impact of K7 on DDX3X’s biochemical activities and roles in RNA metabolism is unknown. Here we show that K7 inhibits the ATP-dependent RNA unwinding and ATP hydrolysis activities of DDX3X. We further show that K7 impairs the formation of recombinant DDX3X phase-separated droplets in vitro, as well as DDX3X-containing stress granules within human cells. Our data reveal that the targeting of the intrinsically disordered N-terminus of DDX3X is an effective way to interfere with both of the disparate functions of DDX3X, its ability to resolve RNA structure and its association with stress granules. Our findings provide new mechanistic insight into strategies by which viruses target DDX3X to alter RNA metabolism in the host.

Keywords: DDX3X, stress granule, vaccinia

Saturday 11:30-11:45am: Title not available online - please see the booklet.

Ullas V. Chembazhi (Department of Biochemistry, University of Illinois, Urbana-Champaign, Illinois, USA), Chaitali Misra (Department of Biochemistry, University of Illinois, Urbana-Champaign, Illinois, USA), Sushant Bangru (Department of Biochemistry, University of Illinois, Urbana-Champaign, Illinois, USA), Auinash Kalsotra (Department of Biochemistry, University of Illinois, Urbana-Champaign, Illinois, USA)

Abstract not available online - please check the booklet.

Saturday 11:45-12:00pm: Title not available online - please see the booklet.

Mariah K. Novak (Biochemistry and Molecular Biology, Mayo Clinic), Qiuying Liu, Rachel Pepin, Wenqian Hu (Biochemstry and Molecular Biology, Mayo Clinic)

Abstract not available online - please check the booklet.

Saturday 12:00-12:15pm: microRNA-mediated regulation of microRNA machinery controls cell fate decisions

Rachel M. Pepin (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA), Qiuying Liu (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA), Mariah K. Novak (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA), Taylor Eich (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA), Wenqian Hu (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905, USA)

Abstract:
microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. Here, using mouse embryonic stem cell (mESC) fate decisions between pluripotency and differentiation as a model, we found that Ago2 is the major developmentally regulated Argonaute protein in mESCs. In pluripotency, microRNA-182/microRNA-183 repress Ago2. Specific inhibition of this repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.

Keywords: microRNA, Ago2, pluripotency

Saturday 12:15-12:35pm: Structural investigation of the oncomiR-1 RNA

Sarah C. Keane (Departments of Biophysics and Chemistry, University of Michigan)

Abstract not available online - please check the booklet.

Saturday 12:35-12:55pm: FMRP inhibits translation elongation independent of RNA G-quadruplexes

MaKenzie R. Scarpitti (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, 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:
Loss of expression of 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. Notably, FMRP is thought to inhibit translation elongation after being recruited to target transcripts via binding RNA G-quadruplexes (G4) within the coding sequence. Here we directly test this model and report that FMRP inhibits translation elongation independent of 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 enables FMRP to bind mRNA and all four polymeric RNA sequences. FMRP only inhibits translation when bound to mRNA through this ncRBD. Consistent with stalling elongation and accumulating slowed ribosomes, FMRP inhibited transcripts co-sediment with heavier polysomes compared to transcripts bound by FMRP deleted of the ncRBD. Together, this work shifts our understanding of how FMRP inhibits translation elongation and supports a model where repression is driven by local FMRP and mRNA concentration rather than target mRNA sequence.

Keywords: ribosome, translational control, fragile X