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Friday 01:15-01:30pm: Linking local RNA structural dynamics to ribosome binding in the prfA RNA thermosensor
David Beier (Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA), Elizabeth Duran (Biophysics Program, University of Michigan, Ann Arbor, Michigan 48109, USA), Nils Walter (Department of Biological Chemistry and Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA), Sarah Keane (Biophysics Program and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA)
Abstract:
The 5′-untranslated region (UTR) of the Listeria monocytogenes prfA messenger (m) RNA controls the translation of the PrfA transcriptional regulator in a temperature dependent manner1. PrfA is an important transcription factor that controls the expression of a cluster of virulence genes involved in pathogenesis2. At low temperature (4-30 oC) the prfA mRNA transcript is detected, however translation of PrfA does not occur. Concurrent with a temperature shift to 37 oC upon successful infection of a human host, translation of PrfA occurs. Since mRNA levels of prfA remain similar as protein levels change, the 5′-UTR of prfA is characterized as an RNA thermosensor (RNAT). Thorough analysis of the prfA 5′-UTR is essential to better understand how RNA-mediated processes in L. monocytogenes initiate and prolong infection.
The Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) assay uses transient fluorescent oligonucleotides to probe the accessibility of specific regions within an RNA structure3. Monitoring binding events to a specific region of an RNA allows for the determination of RNA structure accessibility, and ultimately the conformational dynamics of the RNA. This analysis can be used to identify regions of the prfA 5′-UTR that unfold with increasing temperature to expose elements required for translation initiation. Our SiM-KARTS data reveals that the initial destabilization of the local RNA structure occurs between 30 and 34 oC. This destabilization occurs proximal to the apical loop of the RNAT, upstream of the Shine-Dalgarno (SD) sequence. Unfolding near the SD sequence occurs between 34 and 37 oC. We do not observe destabilization of the helical region containing the AUG start codon at the temperatures probed. Fluorescent ribosomal toeprinting suggests that the interaction of the prfA RNAT and 70S ribosome is temperature dependent, as truncated cDNA products are first observed at 37 oC, but not 30 or 34 oC. Together, these results indicate that the stability of the local structure at and upstream of the SD sequence, but not the AUG start codon, is essential for ribosome binding.
References:
(1) Johansson, J.; Mandin, P.; Renzoni, A.; Chiaruttini, C.; Springer, M.; Cossart, P. An RNA Thermosensor Controls Expression of Virulence Genes in Listeria Monocytogenes. Cell 2002, 110 (5), 551–561. https://doi.org/10.1016/S0092-8674(02)00905-4.
(2) Zhang, H.; Hall, I.; Nissley, A. J.; Abdallah, K.; Keane, S. C. A Tale of Two Transitions: The Unfolding Mechanism of the PrfA RNA Thermosensor. Biochemistry 2020, 59 (48), 4533–4545. https://doi.org/10.1021/ACS.BIOCHEM.0C00588/SUPPL_FILE/BI0C00588_SI_001.PDF.
(3) Chauvier, A.; Cabello-Villegas, J.; Walter, N. G. Probing RNA Structure and Interaction Dynamics at the Single Molecule Level. Methods 2019, 162–163, 3–11. https://doi.org/10.1016/J.YMETH.2019.04.002.
Keywords: Thermosensor, Single-Molecule, Structural Dynamics
Friday 01:30-01:45pm: Title not available online - please see the booklet.
Huaqun Zhang (Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US), Audrey Kehling (Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US), GeunYoung Sim (Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US), Andrew Savidge (Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US), Jackson Secor (Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US), Kotaro Nakanishi (Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, US)
Abstract not available online - please check the booklet.
Friday 01:45-02:00pm: A study of non-templated polymerization for understanding the emergence of RNA from prebiotic mixtures
Xiwen Jia (Department of Chemistry and Chemical Biology, Harvard University), Stephanie J. Zhang (Department of Chemistry and Chemical Biology, Harvard University), Lijun Zhou (Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital), Jack W. Szostak (Department of Chemistry and Chemical Biology, Harvard University; Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital; HHMI)
Abstract:
Prebiotically plausible synthesis of ribonucleotides (RNA) shares similar reaction pathway as arabino- (ANA) and threo-nucleotides (TNA). We investigated the non-templated primer extension using a mixture of activated ribo-, arabino- and threo-nucleotides in the light of understanding the process of emergence of RNA from prebiotic mixtures. We found that the mechanism of non-templated primer extension occurs primarily through bridged dinucleotides. In non-templated primer extension with pre-activated monomers, RNA and ANA shares similar extension profile including rate and yield, while TNA barely has any extension. For competition experiments, we used a prebiotically plausible spontaneous air dry process to speed up non-templated primer extension. We observed that TNA is slightly filtered out whereas ANA has similar incorporation compared to RNA depending on the initial abundance of nucleotides. We concluded that the selection bias based on identities of nucleotides in the non-templated polymerization is close to non-existent. However, the later templated copying step exerts strong incorporation preference towards ribonucleotides, thus generating mostly RNA oligomers.
Keywords: Genetics, Oligomers, Monomers
Friday 02:00-02:15pm: SHAPE probing of the HIV-1 Reverse Transcriptase Initiation Complex reveals RNA flexibility changes adjacent to the Primer-Binding Site
Chathuri Pathirage (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University ), William Cantara (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University), Steven Tuske (Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ), Eddy Arnold (Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ), Karin Musier-Forsyth (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
HIV-1 uses host tRNALys3 as the primer for reverse transcription (RT). The 18 nucleotides (nt) at the 3’ end of tRNALys3 base pair with a complementary primer-binding site (PBS) sequence in the HIV-1 5’ UTR to initiate RT. Extended base-pairing interactions are necessary for efficient RT initiation. Based on recent cryo-EM data using a truncated PBS construct and crosslinking approach to stabilize the complex, tRNALys3 is restructured into an extended helix with a base paired anticodon loop in the reverse transcriptase initiation complex (RTIC). Here, we performed UV crosslinking coupled with selective 2’- hydroxyl acylation analyzed by primer extension (XL-SHAPE) on the complete 5’UTR to probe conformational changes upon tRNA annealing and reverse transcriptase (RTase) binding. Nt flexibility was decreased in the 4 nt downstream of the PBS upon unmodified tRNA annealing to the 5’UTR, suggesting extended primer-template base pairing. We also observed increased flexibility in the primer activation signal (PAS) sequence of the 5’UTR upon tRNA annealing, suggesting dynamics in this region. SHAPE probing of tRNALys3 heat-annealed to the 5’UTR showed that the anticodon loop of the tRNA has high SHAPE reactivity, suggesting that it maintains canonical structural features. UV crosslinking of RTase to the primer-template complex identified crosslinking sites in the PBS and SL2 regions of the 5’UTR packaging signal. The SHAPE reactivity profile of the 5’UTR when RTase is bound to the heat-annealed primer-template complex showed increased nt flexibility in the region upstream of the PBS, suggesting a destabilizing effect. Overall, our results indicate that 5’UTR regions adjacent to the PBS undergo significant nt flexibility changes upon both primer annealing and RTase binding, while tRNALys3 maintains canonical structural features in the anticodon and D domains. Studies aimed at establishing the role of tRNA modifications in RT initiation, are underway.
Keywords: HIV-1 RNA, reverse transcription initiation, tRNA primer annealing
Friday 02:15-02:30pm: Fluorogenic U-rich internal loop (FLURIL) tagging with bPNA enables intracellular RNA and DNA tracking
Yufeng Liang (Department of Chemistry and Biochemistry, The Ohio State University), Sydney Willey (Department of Biological Chemistry and Pharmacology, The Ohio State University), Yu-Chieh Chung (Department of Biological Chemistry and Pharmacology, The Ohio State University), Yi-Meng Lo, Shiqin Miao, Sarah Rundell (Department of Chemistry and Biochemistry, The Ohio State University), Li-Chun Tu (Department of Biological Chemistry and Pharmacology, The Ohio State University), Dennis Bong (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
RNA and DNA are highly dynamic and provide complex, essential regulation to cells. Fluorescent methods for intracellular RNA imaging and tracking in living cells are crucial for understanding RNA and DNA function and organization. While many useful fluorescent tools exist, they often require multiple binding sites and large proteins to reach a sufficient signal-to-noise ratio, which can potentially impact the structure and function of the target of interest. In this research, we developed a novel method for intracellular RNA and DNA tracking: fluorogenic U-rich internal loop (FLURIL) tagging. Fluorophore-labeled bifacial peptide nucleic acids (fbPNAs) fluorescently brighten when forming base-triple hybrid stems specifically within genetically encoded U4xU4 U-rich internal loops in RNA. The cell-permeable fbPNA (~1 kD) binds with minimal structural modifications, and does not require RNA-binding proteins or fluorescent proteins. Using FLURIL tagging of the bacteriophage MS2 hairpin in fixed HEK-293 cells, we successfully visualized its binding with MS2 coat protein (MCP). Next, we visualized a native mammalian ribonucleoprotein (RNP) complex by FLURIL tagging of RNA containing UG repeats which bind to transactive response DNA binding protein (TDP-43). In addition, we tested FLURIL live-cell tracking by targeting guide RNA (gRNA) in a CRISPR-based imaging system to visualize and track a genomic locus in live U2OS cells. Compared with CRISPR-Sirius, which uses MS2/MCP-HaloTag labeled gRNA for live-cell genomic loci tracking, FLURIL had a similar brightness while adding significantly less steric bulk to the target locus. Overall, FLURIL tagging effectively tracks RNA, RNPs, and genomic loci and can be applied to living cells in real time. By minimally altering the native structure of RNA, FLURIL tagging allows accurate tracking of the structure and function of RNA which could improve studies in diseased cells and potentially lead to the development of new treatments.
Keywords: RNA-based fluorescent tagging, Imaging RNA and DNA tracking, live-cell imaging
Friday 02:30-02:45pm: Temperature-sensitive RNAs: flanking sequence of a known RNA thermometer (RNAT) and identification of novel RNATs genome-wide
Elizabeth A. Jolley (Department of Chemistry, Pennsylvania State University), Kathryn M. Bormes (Department of Chemistry, Pennsylvania State University, current affiliation Sidney Kimmel Medical College, Thomas Jefferson University), Helen Yahknin (Department of Biology and Molecular Biology, Pennsylvania State University), David C. Tack, Paul Babitzke (Department of Biology and Molecular Biology, Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry, Pennsylvania State University)
Abstract:
RNA structure is known to regulate bacterial gene expression by several distinct mechanisms. These riboregulators respond to a variety of environmental and cellular stimuli, one of which is temperature. RNA elements that change structure with temperature are termed RNA thermometers (RNATs). We conducted two studies on the effects of heat on bacterial RNAs: one on elements upstream of an RNAT and one on genome-wide effects of heat on RNA folding. Often found upstream of heat shock protein coding sequences, RNATs have a thermolabile RNA hairpin that sequesters the Shine-Dalgarno (SD) sequence and sometimes have additional hairpins in the sequence further upstream. While many RNATs have been studied to confirm that they exist or to determine their function, few studies have examined the role that upstream hairpins may play. We examined this often-overlooked region and determined that the upstream hairpins likely act as folding guides for the final RNAT structure.1
During a heat shock, cells utilize heat shock proteins to acclimate to this new environmental condition. However, there could be other genes that make use of similar temperature-response mechanisms to adjust to growth at different temperatures. We conducted a genome-wide study of RNA structure in Bacillus subtilis at four temperatures (23°C, 30°C, 37°C, 42°C). This study revealed many RNAs with significant reactivity changes with increasing temperature. Two of these RNAs, glpF (glycerol permease) and glpT (glycerol-3-phosphate permease), were found to increase expression in an RNAT-controlled manner.2 Together, these studies1,2 exemplify the many structural changes that RNA undergoes to alter cellular physiology in response to changing environmental conditions.
References:
1Jolley, EA; Bormes, KA; Bevilacqua, PC. (2022) J. Mol. Biol., in press
2Jolley, EA; Yahknin, H; Tack, DC; Babitzke, P; Bevilacqua, PC. In preparation
Keywords: flanking sequence, RNAT, In vivo probing
Friday 03:15-03:30pm: MicroRNAs Have Macro Impacts on the Regulation of Alternative Splicing
Whitney D. Jimenez (Center for Childhood Cancer and Blood Disorders, Nationwide Childrens Hospital), Hannah R. Ackerman (Center for Childhood Cancer and Blood Disorders, Nationwide Childrens Hospital), Matias Montes (Stanford University), Dawn Chandler (Center for Childhood Cancer and Blood Disorders, Nationwide Childrens Hospital)
Abstract not available online - please check the booklet.
Friday 03:30-03:45pm: tRNA introns: A novel class of small non-coding regulatory RNAs
Regina Nostramo (Department of Molecular Genetics, The Ohio State University), Anita K. Hopper (Department of Molecular Genetics, The Ohio State University)
Abstract:
From archaea to humans, a subset of tRNA encoding genes possess introns. These introns are spliced out and rapidly degraded as part of the tRNA maturation process. tRNA intron splicing is essential in all studied eukaryotes because for at least one tRNA family, all reiterated tRNA genes contain an intron. Thus, the genome cannot be fully decoded without tRNA intron removal. This raises the intriguing question of why tRNA introns have been evolutionarily conserved. One possibility is that like many other components of tRNA biology that multitask with pathways for other RNAs (Hopper & Nostramo, 2019), the released introns may serve biological roles, functioning as small non-coding regulatory RNAs by interacting with mRNAs through sequence complementarity. To test this hypothesis, we first generated a strain lacking the tRNAIleUAU introns and assessed changes in mRNA levels of 32 ORFs with long stretches of complementarity to the intron, relative to wild-type cells. Absence of the tRNAIle introns elicited significant increases in mRNA levels for genes with intron complementarity as compared to ORFs without. Conversely, modest overexpression of the free tRNAIle intron elicited decreases in mRNA levels for genes with complementarity. These data suggest that tRNAIle introns function as inhibitors of sequence-specific gene expression/mRNA turnover. Extension of our analyses to the tRNATrpCCA intron supports these findings. Marked H2O2-induced increases in the tRNATrp intron elicited a decrease in ATG5 mRNA levels. However, this effect was abolished when complementarity between the intron and ATG5 mRNA was disrupted. Interestingly, evidence of an additional mechanism for the function of tRNA introns was also observed, in which tRNA introns can act as enhancers of translation. Overall, our data support the exciting possibility that tRNA introns are the newest member of non-coding regulatory RNAs and can function as both positive and negative regulators of gene expression.
References:
Hopper AK and Nostramo R. (2019) tRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs. Front Genet. 10:96.
Keywords: tRNA, introns, gene expression
Friday 03:45-04:00pm: Arginine methylation of Puf4 drives diverse protein functions
Murat C. Kalem (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, NY, USA & University of California, San Francisco, CA ), Sean Duffy (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA ), Shichen Shen (Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14214, USA ), Jan Naseer Kaur (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA ), Jun Qu (Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14214, USA ), John C. Panepinto (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA )
Abstract not available online - please check the booklet.
Friday 04:00-04:15pm: Quantitative and systematic definitions of inherent specificities of RNA binding proteins
Soon Yi (Department of Biochemistry and Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH), Xuan Ye (Department of Biochemistry and Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH), Eckhard Jankowsky (Department of Biochemistry and Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH)
Abstract not available online - please check the booklet.
Friday 04:15-04:30pm: Metabolites can bind and regulate human 5′UTRs
Jeffrey T. Morgan (Howard Hughes Medical Institute, Department of Biochemistry, University of Utah), Kevin G. Hicks (Department of Biochemistry, University of Utah), Jared Rutter (Howard Hughes Medical Institute, Department of Biochemistry, University of Utah)
Abstract not available online - please check the booklet.
Saturday 08:45-09:00am: RESC8 and RESC14 cooperate to mediate RESC function and dynamics during trypanosome RNA editing
Katherine Sortino (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Brianna Tylec (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Natalie M. McAdams (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Runpu Chen (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Yijun Sun (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Laurie K. Read (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.)
Abstract:
Uridine insertion/deletion RNA editing is unique to kinetoplastid organisms and essential for their survival and virulence. This intricate process requires the action of several multiprotein complexes and small trans-acting RNAs called guide RNAs. The RNA editing holoenzyme comprises three dynamically interacting complexes: RECC (RNA Editing Catalytic Complex), RESC (RNA Editing Substrate Binding Complex), and REH2C (RNA Editing Helicase 2 Complex). Previous studies in Trypanosoma brucei showed that RESC components, RESC14 and RESC8, are critical in maintaining normal protein-protein and protein-RNA interactions within the editing holoenzyme. To further clarify the role of these proteins and the RESC complex in RNA editing, we performed high-throughput sequencing of ATP synthase subunit 6 (A6) mRNA in RESC14, RESC8, and RESC13 RNAi cells, and compared the phenotypes of these cell lines. We measured a highly significant overlap between exacerbated pause sites arising on A6 mRNA in cells depleted of RESC14 and RESC8, suggesting an overlapping function between these proteins during editing progression. In contrast, RESC13 depleted cells exhibited different A6 mRNA pause sites, indicative of a function distinct from that of RESC14/8. To probe the biochemical basis of the RESC14/8 interdependence, we performed Native PAGE analysis and showed that RESC8 is not incorporated into large RNA-containing editing complexes in the absence of RESC14. We also found through Native PAGE analysis that RESC14 is needed for incorporation of other RESC factors into large RNA-containing complexes, but is dispensable for RECC, KREH1 and REH2C complex formation. To better understand the interaction of RESC14 and RESC8, co-immunoprecipitation assays were performed and identified an RNA inhibited interaction between RESC14 and RESC8. We synthesize our findings into a model showing how RESC14 and RESC8 cooperate to mediate necessary protein and RNA rearrangements during editing progression. Together, our findings suggest that RESC14 and RESC8 rely on each other for necessary protein and RNA rearrangements during editing progression.
Keywords: Trypanosome, RNA editing, Holoenzyme
Saturday 09:00-09:15am: Identification of a tRNA-specific function for the tRNA methyltransferase Trm10 in Saccharomyces cerevisiae
Isobel Bowles (Ohio State Biochemistry Program), Aiswarya Krishnamohan, Abi Hubacher, Jane Jackman (Chemistry and Biochemistry, OSU)
Abstract:
tRNA methyltransferase 10 (Trm10) methylates N1 of guanosine at the 9th position of tRNA molecules using methyl donor S-adenosyl methionine (SAM). Upon deletion of trm10, Saccharomyces cerevisiae strains exhibit growth defects in the presence of antitumor drug 5-fluorouracil (5FU). We hypothesized that tRNA stability decreases with the lack of the m1G9 modification in trm10Δ strains and that certain tRNA species may be more reliant upon the methylated G9 nucleotide. We showed that when Trm10 substrate tRNATrp is overexpressed in trm10Δ strains, growth hypersensitivity to 5FU is rescued, while overexpression of 37 other tRNA species in S. cerevisiae does not rescue growth in the presence of the drug. We then demonstrated that levels of tRNATrp decrease in trm10Δ strains, and that these levels decrease further in the presence of 5FU, but another Trm10 substrate (tRNAGly) remains at a similar level in all conditions. These data indicate that the loss of m1G9 from tRNATrp is specifically responsible for the trm10Δ growth defect, even though Trm10 modifies 13 other tRNA substrates in S. cerevisiae. To identify the specific pathway associated with hypomodified tRNATrp quality control, pairwise deletion strains were created. These studies revealed that pairwise deletion of trm10 in combination with a known enzyme associated with multiple tRNA quality control pathways can rescue the 5FU-dependent growth defect. The effects of the m1G9 modification on tRNA structure and its interaction with Trm10 are also being determined with chemical footprinting methods, including SHAPE. Trm10 interactions with the tRNA at sites distant from the m1G9 modification were revealed that may play a role in the sensitivity of hypomodified tRNATrp. Together, these studies provide insight into the biological impact of loss of this highly conserved modification.
Keywords: tRNA modification, tRNA surveillance, SHAPE
Saturday 09:15-09:30am: Cleavage and Polyadenylation Specificity Factor (CPSF30) involved in plant immunity system
Lichun Zhou (Department of Plant and Soil Sciences, University of Kentucky, Lexington KY 40546, USA), Huazhen Liu (Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA), Pradeep Kachroo (Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky, Lexington KY 40546, USA)
Abstract:
In plants, Systemic Acquired Resistance (SAR) is a form of systemic immunity that protects uninfected parts of a pathogen-inoculated plant against further disease. In a previous study, it was shown that a novel plant polyadenylation factor subunit, CPSF30, is required for resistance against a bacterial pathogen [1]. The Arabidopsis thaliana ortholog of the 30-kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor(CPSF30) gene encodes two proteins, CPSF30S and CPSF30L. To better understand how CPSF30 contributes to resistance to plant pathogens, we treated a set of mutant and complemented Arabidopsis lines with an avirulent strain of Pseudomonas syringae to induce SAR. The set of lines included a mutant (oxt6) that does not express CPSF30 as well as lines that express either CPSF30S, CPSF30L, or both proteins in the oxt6 background. The results show that lines that express CPSF30S can establish an SAR, but lines that do not express CPSF30S are deficient in SAR. All of the wild type, mutant, and transgenic plants have similar transcriptional responses in the inoculated leaf to the avirulent pathogen. However, they differ in the (immune) distal leaves, and the differences relate to the well-established transcriptional response in SAR. We conclude that CPSF30S is acting downstream from the initial challenge by the avirulent pathogen and outside of the inoculated leaf. In a recent study, it has shown that the non-coding RNA TAS3a is a precursor for tasi-RNAs D7 and D8, which are early mobile signals for SAR [2]. TAS3a transcripts undergo alternative polyadenylation, and poly(A) site choice affects the ability to generate the D7 and D8 tasi-RNAs. We observed TAS3a poly(A) site usage shift from distal to proximal in plants that do not make CPSF30S. We propose that CPSF30S-dependent usage of the distal TAS3a poly(A) site is the mechanism that links CPSF30 with SAR. Taken together, these studies provide new insights into the connections between mRNA polyadenylation and the plant immune system.
References:
1. Bruggeman, Q., et al., The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis. Plant Physiol, 2014. 165(2): p. 732-746.
2. Shine et al., Phased small RNA–mediated systemic signaling in plants. Science Advance, 2022.
Keywords: Plant RNA polyadenylation, Systemic Acquired Resistance, tasi-RNAs
Saturday 09:30-09:45am: Dynamic Phosphorylation Of RNA Helicase eIF4A Mediates Translational Responses To Cell Cycle Transitions And Nutrients Starvation
Ansuman Sahoo (Biological Sciences Department, University at Buffalo), Qian He, Marium Ashraf, Samantha Nelson, Gerald Koudelka (Biological Sciences Department, University at Buffalo), Shichen Shen, Jun Qu (Pharmaceutical Sciences Department, University at Buffalo), Robert A. Zollo, Joseph Barbi (Roswell Park Comprehensive Cancer Center), Sarah E. Walker (Biological Sciences Department, University at Buffalo)
Abstract:
The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate cell cycle stages and found that three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed to complete cell division. In addition to these repressive events, we observed reciprocal phosphorylation of S177, a site immediately adjacent to the DEAD-box, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A enhanced normally reduced eIF4A•eIF4G-interaction during G2/M and increased polysomes, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. S177 also showed reduced phosphorylation upon removal of glucose, coinciding with global translation initiation shut-down. Mutation to S177D mimicking constitutive phosphorylation decreased P body levels upon glucose removal, while phosphodeficient S177A led to decreased growth and translation rates and increased P body levels, suggesting S177 phosphorylation mediates changes in eIF4A activity needed for altering translation and RNA turnover in response to changing cellular states. We also observed enhanced phosphorylation of T146 and other sites, events likely to evict eIF4A from RNA and eIF4G upon glucose removal. Together, these data suggest that parallel phosphorylation and dephosphorylation of alternative sites in the essential translation initiation factor eIF4A stimulates translation during periods of robust growth and holds the capacity to rapidly shut down protein synthesis by impaired eIF4A binding to eIF4G and RNA during unfavorable conditions.
Keywords: Translation, Helicase, Phosphorylation
Saturday 09:45-10:00am: PYM1 controls of Exon Junction Complex occupancy at canonical and non-canonical positions and can thereby modulate NMD
Manu Sanjeev (Molecular Genetics, The Ohio State University), Lauren Woodward (Molecular Genetics, The Ohio State University), Robert Patton (Physics, The Ohio State University), Ralf Bundschuh (Physics, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University)
Abstract:
RNA binding proteins are critical for RNA processes in the cell. The Exon Junction Complex (EJC) is an important RNA binding protein complex present in all vertebrate cells. EJCs are deposited ~24 nucleotides upstream of exon-exon junctions during pre-mRNA splicing. Once deposited, EJCs remain stably bound to mRNA and modulates mRNA fate at multiple post-transcriptional steps. A critical EJC function is to aid the Nonsense Mediated mRNA Decay (NMD) pathway to identify and decay transcripts that undergo premature translation termination. Since there are more exon junctions than EJCs at steady state, EJC subunits are continuously disassembled and recycled. However, the molecular understanding of this process is incomplete. Here, we tested the roles of two activities implicated in EJC disassembly, (i) the elongating ribosome and (ii) a ribosome-associated factor called PYM1, which binds the Y14/MAGOH heterodimer of the EJC core. Using an EJC footprinting assay in HEK293 cells, we find that unlike inhibition of translation elongation, blocking EJC-PYM1 interaction shows no defect in EJC disassembly. Thus, PYM1 does not function as an EJC disassembly factor. Surprisingly, we find that the loss of EJC-PYM1 interaction leads to decreased EJC footprints at the -24 position (canonical EJC binding site) and increased EJC footprints away from exon junctions (non-canonical EJC binding sites). Among transcripts with increased non-canonical EJC binding are intronless mRNAs that do not normally contain the EJC. These observations suggest a role of PYM1 in which it aids proper EJC recycling and suppresses EJC deposition at non-canonical sites. Consistent with this idea, we find that PYM1 depletion upregulates canonical EJC dependent NMD targets and downregulates of transcripts that accumulate non-canonical EJCs. In summary, our work reveals that PYM1 is dispensable for EJC disassembly and suggests a model where PYM1 prevents splicing-independent deposition of EJC onto RNAs.
References:
[1] L. A. Woodward, J. W. Mabin, P. Gangras, and G. Singh, “The exon junction complex: a lifelong guardian of mRNA fate: EJC: assembly, structure, and function,” WIREs RNA, vol. 8, no. 3, p. e1411, May 2017, doi: 10.1002/wrna.1411.
[2] N. H. Gehring, S. Lamprinaki, A. E. Kulozik, and M. W. Hentze, “Disassembly of Exon Junction Complexes by PYM,” Cell, vol. 137, no. 3, pp. 536–548, May 2009, doi: 10.1016/j.cell.2009.02.042.
[3] S. Ghosh, A. Obrdlik, V. Marchand, and A. Ephrussi, “The EJC Binding and Dissociating Activity of PYM Is Regulated in Drosophila,” PLoS Genet, vol. 10, no. 6, p. e1004455, Jun. 2014, doi: 10.1371/journal.pgen.1004455.
Keywords: EJC, PYM1, NMD
Saturday 10:00-10:15am: The role of KPAP1 methylation in moderating mitochondrial mRNA 3’ tail addition in Trypanosoma brucei
Clara M. Smoniewski (Dept. of Biomedical Sciences, U of MN Medical School, Duluth campus), Poorya Mirzavand Borujeni (Institute of Parasitology,McGill University), Marshall Hampton (Department of Mathematics and Statistics, University of Minnesota Duluth), Reza Salavati (Institute of Parasitology,McGill University), Sara L. Zimmer (Dept. of Biomedical Sciences, U of MN Medical School, Duluth campus)
Abstract:
Trypanosoma brucei is a protozoan parasite with unique molecular mechanisms to control expression of its nuclear and mitochondrial (mt) genomes. Its mt mRNAs have regulatory non-templated heterogenous adenine/uridine tails added post-transcriptionally to their 3’ ends. We previously found population level differences in tail characteristics, such as length and composition, between transcripts. The mechanism for imparting these differences is unknown. Tail adenines are added by kinetoplast poly(A) polymerase I (KPAP1), possessing two previously identified arginine methylation sites. Methylation of protein arginine residues is a known mechanism for altering protein:protein and protein:nucleic acid associations. We have mutated KPAP1 at its arginine methylation sites to mimic constant methylation (methyl-mimic) or no methylation (methyl-mutant). One of these mutated versions of KPAP1 or a wildtype version were inducibly expressed in procyclic T. brucei along with simultaneous silencing of native KPAP1. The three cell lines were compared using circTAILseq, which combines enzymatic circularization of RNA, RT-PCR, and Illumina sequencing. We found that tail length and composition are impacted by mutation of KPAP1 arginine methylation sites. Addition of adenine to pre-edited mRNA tails seems more robust but less processive under activity of methyl-mimic KPAP1; the reverse can sometimes be observed for never-edited mRNA. This suggests a role for methylation in KPAP1 regulation. Surprisingly, some transcripts yielded circTAILseq libraries when the ligase responsible for RNA circularization was omitted, although the size of the library’s molecules was smaller. These libraries consist of cDNAs possessing many 3’ and 5’ UTRs ligated together, sometimes possessing a short tail between the two UTRs. Further experiments suggest that these products reflect a minor sub-population of mt mRNAs that are intracellularly circularized. To our knowledge, this is the first circRNA to be identified in a Kinetoplastid species.
References:
Aphasizheva, I. & Aphasizhev, R. (2010). Mol Cell Bio 30, 1555-1567.; Etheridge, R.D. et al. (2008). EMBO J 27, 1596–1608.; Fisk, J.C. et al. (2013). Mol Cell Proteomics 12, 302–311.; Gazestani, V.H. et al. (2016). RNA 22, 477-86.; Gazestani, V.H. et al. (2018). Int J Parasitol 48, 179-189.; Hampton, M. et al., (2021). PLOS ONE 16: e0244858.; Zhang, L. et al. (2017). EMBO J 36, 2435–2454.
Keywords: Trypanosomes, Mitochondria, mRNA 3 tails
Saturday 10:15-10:30am: Title not available online - please see the booklet.
Benjamin Pastore (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University), Jillian A. Wagner (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University), Ian F. Price (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University), Wen Tang (Department of Biological Chemistry and Pharmacology, The Center for RNA Biology, The Ohio State University)
Abstract not available online - please check the booklet.
Saturday 11:00-11:15am: Phase separation of demethylase LSD1 enriches lncRNA TERRA on telomeres to promote R-loop formation for telomere maintenance in ALT cancer cells
meng xu (Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University), Dulmi Senanayaka (Klingler College of Arts and Sciences, Department of Chemistry, Marquette University, Milwaukee, WI 53233), David Chenoweth (Department of chemistry, University of Pennsylvania ), Nicholas J Reiter (Klingler College of Arts and Sciences, Department of Chemistry, Marquette University, Milwaukee, WI 53233), Huaiying Zhang (Department of Biology, Carnegie Mellon University, Pittsburgh, PA 15213)
Abstract not available online - please check the booklet.
Saturday 11:15-11:30am: The mRNA regulatory function of Brat is essential for development and neurogenesis
Robert P. Connacher (Dept. Biochemistry, Molecular Biology, & Biophysics, University of Minnesota), Yichao Hu (Dept. Molecular Genetics, University of Toronto & Institute of Genetics, Zhejiang University), Michael B. OConnor (Dept. Genetics, Cell Biology, and Development, University of Minnesota), Xiaohang Yang (Institute of Genetics, Zhejiang University), Howard D. Lipshitz (Dept. Molecular Genetics, University of Toronto), Richard T. Roden, Aaron C. Goldstrohm (Dept. Biochemistry, Molecular Biology, & Biophysics, University of Minnesota)
Abstract not available online - please check the booklet.
Saturday 11:30-11:45am: Myotonic Dystrophy Type 1 Adversely Alters affects Liver Function and Liver Lipid Synthesis
Zac Dewald (Biochemistry, University of Illinois), Auinash Kalsotra (Biochemistry, University of Illinois)
Abstract:
Myotonic Dystrophy type 1 (DM1) is multi-systemic muscular dystrophy, affecting 1 in 3000 people. DM1 is caused by a (CTG)n repeat expansion in the ubiquitously expressed gene DMPK. The (CUG)n containing RNAs resulting from the transcription of diseased DMPK sequester several RBPs, many of which regulate juvenile-to-adult development of many tissues. Studies have shown that in addition to muscle pathologies, DM1 patients exhibit increased susceptibility toward glucose intolerance, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome. Furthermore, DM1 patients are abnormally sensitive to various analgesics and anesthetics, with complications ranging from prolonged anesthesia recovery to pulmonary dysfunction. These findings suggest a predisposition for liver dysfunction in DM1 patients.
To understand the effects of DM1 in the liver, we generated a DM1 mouse model which expresses CUG repeat-containing RNA specifically within the liver. Through these mice, we show that the expression of toxic CUG RNA in hepatocytes sequesters MBNL proteins, causing a reduction in mature hepatocellular activity. We have shown these transcriptomic changes driven by DM1 lead to changes in liver morphology, injury, and increased lipid accumulation. We show that DM1 sensitizes the liver to diet induced NAFLD, increasing the likelihood of NAFLD development when patients consume high fat, high sugar diets. Our data suggest this is due to misregulation of fatty acid metabolism and homeostasis. Specifically, the 28th exon of acetyl-CoA carboxylase 1 (ACC1), the rate-limiting enzyme in fatty acid synthesis, shows increased inclusion in the livers of the DM1 mice. This exon is implicated in affecting ACC1 phosphorylation and activity, and we have shown that ACC1 levels decrease in the DM1 afflicted mice. We further demonstrate that DM1 mice livers are defective in drug metabolism and clearance, with marked impairment against zoxazolamine-induced paralysis and acetaminophen-induced hepatotoxicity. These results reveal that expression of CUG repeat-containing RNA disrupts normal hepatic functions and predisposes the liver to injury, fatty liver disease, and drug clearance pathologies which jeopardize the health of DM1 patients and complicate the treatment of DM1.
Keywords: Myotonic Dystrophy Type 1, NAFLD, Alternative Splicing
Saturday 11:45-12:00pm: Title not available online - please see the booklet.
Eng-Soon Khor (Aab Cardiovascular Research Institute , University of Rochester), Feng Jiang (Aab Cardiovascular Research Institute , University of Rochester), Omar M. Hedaya (Aab Cardiovascular Research Institute , University of Rochester), Waihong Wilson Tang (Department of Cardiovascular Medicine, Cleveland Clinic), Peng Yao (Aab Cardiovascular Research Institute , University of Rochester)
Abstract not available online - please check the booklet.
Saturday 12:00-12:20pm: Repeat G4 RNA structures preferentially interact with the Lysine Specific Demethylase-1-CoREST complex, mask nucleosome recognition, and trigger phase separation.
Dulmi Senanayaka (Department of Chemistry, Marquette University), Meng Xu (Department of Biological Sciences, Carnegie Mellon University), William J Martin (Department of Biochemistry, Vanderbilt University School of Medicine), Owen Schneider (Marquette University), Manuel Ascano, Jr (Department of Biochemistry, Vanderbilt University School of Medicine), Nick Reiter (Department of Chemistry, Marquette University)
Abstract not available online - please check the booklet.
Saturday 12:20-12:40pm: Tracking single chromosome localization, orientation, and movement in living cells by fluorescent guide RNAs
Yu-Chieh Chung (Department of Biological Chemistry and Pharmacology, The Ohio State University), Madhoolika Bisht (Department of Molecular Genetics, The Ohio State University), Li-Chun Tu (Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract not available online - please check the booklet.