Talk abstracts
Friday 01:00-01:15pm: Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA
GeunYoung Sim (Molecular, Cellular and Developmental Biology and Center for RNA Biology, The Ohio State University), Mi Seul Park (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Audrey C. Kehling (Department of Chemistry and Biochemistry, The Ohio State University), Kotaro Nakanishi (Department of Chemistry and Biochemistry, Molecular, Cellular and Developmental Biology and Center for RNA Biology, The Ohio State University)
Abstract:
RNA interfering is a eukaryote-specific gene silencing by 20~23 nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3′ end-shortened variants of let-7a, miR-27a, and specific miR-17-92 families that make AGO3 an extremely competent slicer, increasing target cleavage up to ~ 82-fold in some instances. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.
References:
1. Posted on bioRxiv, doi: https://doi.org/10.1101/2020.07.16.207720
2. Park MS, Araya-Secchi R, Brackbill JA, et al. Multidomain Convergence of Argonaute during RISC Assembly Correlates with the Formation of Internal Water Clusters. Mol Cell. 2019;75(4):725-740.e6. doi:10.1016/j.molcel.2019.06.011
Keywords: Argonaute, small RNAs, RNA cleavage
Friday 01:15-01:30pm: Protein Arginine Methylation Regulates Long Non-coding RNAs in Cryptococcus neoformans
Murat C. Kalem (Department of Microbiology and Immunology - SUNY University at Buffalo), Harini Subbiah (Department of Microbiology and Immunology - SUNY University at Buffalo), John C. Panepinto (Department of Microbiology and Immunology - SUNY University at Buffalo)
Abstract not available online - please check the printed booklet.
Friday 01:30-01:45pm: The role of Musashi proteins in the maintenance of mature photoreceptor cells
Fatimah Matalkah (Biochemistry/WVU), Bohye Jeong (Biochemistry/WVU), Visvanathan Ramamurthy (Biochemistry/WVU), Peter Stoilov (Biochemistry/WVU)
Abstract not available online - please check the printed booklet.
Friday 01:45-02:00pm: Nucleotide-level resolution of RNA folding interactions within peptide-based complex coacervates
McCauley Meyer (Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802), Saehyun Choi, Fatma Pir Cakmak, Philip C. Bevilacqua, Christine D. Keating (Department of Chemistry, Pennsylvania State University, University Park, PA 16802)
Abstract:
The RNA World Hypothesis states that RNA or an RNA-like polymer may have acted as both the initial genetic material and the catalyst for the reactions of life. In the 1980s, the first ribozymes were discovered, demonstrating that RNA could act as a catalyst. Since then, it has become apparent that RNA folding is integral to function in a way similar to protein enzyme folding. Because of this, it’s important to try to understand RNA folding under prebiotically relevant conditions.
On the early Earth, a problem that would have been faced by the first enzymes was the scarcity of organic material. To overcome this issue, organic material would need to be localized and concentrated on either a mineral surface or in some type of compartment, like a protocell. An ideal protocell candidate should partition molecules required for catalysis such as Mg2+, nucleotides, RNAs, amino acids, and peptides. A model protocell able to do this is one made of complex coacervates.
Herein, I describe turbidity measurements, FRET, local pH, and RNA folding studies within complex coacervate droplets made out of (Lys)n-(Asp)n, and (Lys)10-ATP1. tRNAphe from S. cerevisiae, was used for these RNA folding studies. tRNAphe was subjected to in-line probing (ILP) under the following conditions (0.5 mM Mg2+, 15 mM KCl, and 10 mM Tris, pH 8.3) initially to determine its native fold. Then, tRNAphe was placed inside of (Lys)n-(Asp)n and (Lys)10-ATP coacervates where we found that under all of these coacervate conditions, the tRNA had lost its tertiary contacts and the acceptor stem was unfolded. Upon changing Mg2+ conditions and charge-ratio of polyanions to polycations, more native folding of tRNAphe was observed. Future studies will focus on evaluating if under the same conditions ribozymes fold and function well. These experiments provide one of the first detailed views of RNA folding in protocells and pave the way for combining Next-Generation sequencing to study RNA folding within protocells.
References:
1.Pir Cakmak, F.; Choi, S.†; Meyer, M. O.†; Bevilacqua, P. C.; and Keating, C. D. Prebiotically-relevant low polyion multivalency can improve functionality of membraneless compartments. Submitted and uploaded to BioRxiv: https://www.biorxiv.org/content/10.1101/2020.02.23.961920v1
Keywords: RNA folding, complex coacervates, liquid-liquid phase separation
Friday 02:00-02:15pm: Not just a phase: understanding ribosome biogenesis using liquid-liquid phase biology
Amber J. LaPeruta (Carnegie Mellon University Department of Biological Sciences), Jelena Micic (Carnegie Mellon University Department of Biological Sciences), Daniel M. Wilson (Carnegie Mellon University Department of Biological Sciences), Fiona Fitzgerald (Carnegie Mellon University Department of Biological Sciences), John L. Woolford Jr. (Carnegie Mellon University Department of Biological Sciences)
Abstract:
The ribosome is an extremely complex structure composed of 79 ribosomal proteins (RPs) and 4 ribosomal RNAs (rRNAs) (25S, 18S, 5.8S and 5S), and is synthesized with the assistance of >200 assembly factors (AFs). The initial stages of ribosome biogenesis take place in the nucleolus, a membraneless organelle within the nucleus that is assembled via liquid-liquid phase separation. The interrelation between ribosome assembly and the nucleolus has been noted for decades. Nucleolar function in response to environmental signals regulates ribosome biogenesis, and inhibition of or defects in ribosome biogenesis alter nucleolar structure. This raises numerous questions. How does a nucleolus form as ribosomes assemble? What qualities of pre-ribosomes retain them in the nucleolus, and what changes must occur in order for them to be released? To answer these questions, we analyzed cryo-EM structures of pre-ribosomes within and outside of the nucleolus from the perspective of liquid-liquid phase biology. A major precept in phase biology is that highly valent molecules and complexes, which are capable of undergoing multiple simultaneous interactions, have greater phase separation potential. We hypothesize that the valency of pre-ribosomes decreases as they mature because the number of heterogeneous trans-interacting RNA and protein domains, which cannot be visualized by cryo-EM, decreases. To explore these ideas, we determined the abundance of predicted trans-interacting RNA and protein structures not accounted for in the cryo-EM structures of nascent large ribosomal subunits undergoing nucleolar and non-nucleolar stages of assembly. Using these data, we have identified qualities of pre-ribosomes that support nucleolar retention. Furthermore, we created a general model for how rRNA, RPs, and AFs function during ribosome biogenesis to ultimately decrease the valency of pre-ribosomes and facilitate their release from the nucleolus.
Keywords: ribosome, nucleolus, liquid-liquid phase
Friday 02:15-02:30pm: Utilization of high-pressure NMR for the study of an intrinsically disordered domain of hnRNP A1
Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University), Jake Peterson (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University), Julien Roche (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)
Abstract:
The hnRNP A1 protein is involved in numerous processes of nascent RNA transcripts. These roles include, but are not limited to, translational control, splicing regulation, and mRNA stabilization. The protein performs its functions by first binding the RNA at a high affinity site before recruiting other proteins to aid in its RNA processing activities.
hnRNP A1 is composed of three domains, two structurally identical RRMs that form the nucleic acid binding protein UP1, and a disordered, glycine rich C-terminal domain (G-CTD). As the more structured domain, UP1 has been studied extensively. Its RNA binding properties have been examined through numerous biochemical and biophysical methods. Its structure, both free and bound, has been determined through NMR, X-ray crystallography, and small-angle X-ray scattering (SAXS).
In contrast to UP1, the G-CTD has not been very well characterized. However, its roles in hnRNP A1 activity requires that it be studied with just as much detail as UP1. The G-CTD is responsible for assembling the multi-protein complexes the protein utilizes to carry out its various functions. It has been shown to be a low complexity domain (LCD) capable of mediating liquid-liquid phase separation (LLPS). This partitioning leads to the formation of stress granules, cytosolic bodies which are believed to be formed by mRNAs stalled in translation.
The utilization of SAXS and high-pressure NMR has revealed the LC domain, at standard conditions, adopts a disordered, but compact conformation that is stabilized by polar and electrostatic interactions. The application of pressure increases solvent density, resulting in a disruption of the intra-protein interactions and the compact nature of the domain. The application of high pressure might also stabilize helical formation in the domain. The extended high-pressure conformation potentially reveals attributes of the domain which play a role in phase separation.
Keywords: RRM, IDP, high-pressure NMR
Friday 03:00-03:15pm: uORF control of cardiac gene expression: Lessons learned from a single nucleotide that regulates cardiomyocyte biology
Omar M. Hedaya (Biochemistry and Biophysics, University of Rochester ), Feng Jiang (Biochemistry and Biophysics, University of Rochester ), Venkata Kadiam (Cardiovascular Research Institute, University of Rochester ), Jiangbin Wu (Cardiovascular Research Institute, University of Rochester ), Darren Khorr (Cardiovascular Research Institute, University of Rochester ), Peng Yao (Cardiovascular Research Institute, University of Rochester )
Abstract not available online - please check the printed booklet.
Friday 03:15-03:30pm: Pumilio-mediated post-transcriptional regulation of DICER1 protein in Dicer1 syndrome
Swetha Rajasekaran (Department of Molecular Genetics, The Ohio State University), Wayne Miles (Cancer Biology and Genetics, The James Comprehensive Cancer Center)
Abstract not available online - please check the printed booklet.
Friday 03:30-03:45pm: Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During The Cell Cycle
Ansuman Sahoo (Department of Biological Sciences, University at Buffalo), Marium Ashraf (Department of Biological Sciences, University at Buffalo), Sarah E. Walker (Department of Biological Sciences, University at Buffalo)
Abstract:
The eukaryotic translation initiation factor 4A (eIF4A) supports mRNA recruitment to the ribosomal preinitiation complex, thus controlling cell-wide protein synthesis. A conserved Threonine residue of eIF4A (T146 in yeast) near the catalytically-important DEAD motif is present in a consensus CDK1/CDKA/Cdc28 motif and found to be phosphorylated in large-scale studies (Soulard et al., 2010). In plants, eIF4A is phosphorylated at this site by CDKA (a key regulator of the cell cycle) and this event decreased eIF4A activity (Bush et al., 2016). To further dissect the molecular function of eIF4A phosphorylation, we analyzed specific in vitro and in vivo changes that take place because of phosphorylation of eIF4A at different stages of the cell cycle. We mutated several phosphosites in eIF4A to phosphodeficient (Ala) and phosphomimetic (Asp/Glu) forms and analyzed the effects of these mutations on yeast growth and translation. Our data show that besides T146, multiple residues in eIF4A were phosphorylated at the G2 to M transition, and versions of eIF4A that could not be phosphorylated or mimicked constitutive phosphorylation were lethal, suggesting dynamic phosphorylation of eIF4A is essential. A phosphomimetic version of eIF4A (T146D) abolished RNA binding, suggesting that translation of mRNAs that occurs during mitosis (when eIF4A is phosphorylated) is independent of the eIF4F cap-binding complex. The activity of the essential Glc7/PP1 complex promoted dephosphorylation of eIF4A and cell division. Together these data suggest that eIF4A acts as a switch to couple production of specific proteins to phases of the cell cycle. Phosphorylation arrests canonical translation during mitosis, while dephosphorylation of eIF4A is needed to derepress cap dependent translation to complete cell division.
References:
1. Soulard, Alexandre, et al. "The rapamycin-sensitive phosphoproteome reveals that TOR controls protein kinase A toward some but not all substrates." Molecular biology of the cell 21.19 (2010): 3475-3486.
2. Bush, Maxwell S., et al. "eIF4A RNA helicase associates with cyclin-dependent protein kinase A in proliferating cells and is modulated by phosphorylation." Plant physiology 172.1 (2016): 128-140.
Keywords: eIF4A, Phosphorylation, Cell cycle
Friday 03:45-04:00pm: 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), Kiel Tietz (The Ohio State University Molecular Genetics Department), Sharon Amacher (The Ohio State University Molecular Genetics Department)
Abstract:
During early vertebrate embryogenesis, muscle and skeletal precursors are sequentially 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 called cyclic genes or segmentation clock genes, that are reiteratively expressed in the unsegmented mesoderm with defined periodicity. Precise regulation of mRNA production, translation, and decay drive the pace and amplitude of segmentation clock oscillations and our work explores post-transcriptional mechanisms that regulate oscillatory expression. Previous work in the Amacher lab showed that Proline-rich nuclear receptor coactivator 2 (Pnrc2) promotes cyclic gene mRNA decay in zebrafish embryos and that loss of pnrc2 results in accumulation of cyclic gene mRNAs. Despite increased levels of cyclic gene mRNAs, pnrc2 mutants display normal protein oscillations and segment boundary formation. Using polysome profiling to probe the translation status of cyclic gene mRNAs in pnrc2 mutant embryos, we show here that the accumulated cyclic gene mRNAs her1, her7, dlc, and rhov are disengaged from translational machinery. Cyclic gene mRNA 3’UTR analysis identified two cis-regulatory elements within the 3’UTRs of her1 and dlc, a Pumilio Response Element (PRE) and AU-rich Element (ARE), that promote reporter transcript decay. Both Pumilio and ARE-binding proteins are well-known regulators of mRNA decay and translation. We are investigating their role in regulating cyclic gene mRNA stability and translation, in wild-type and pnrc2 mutant backgrounds, to understand mechanistically how vertebrate patterning is robustly maintained using multiple layers of post-transcriptional regulation.
Keywords: decay, translation, oscillations
Friday 04:00-04:15pm: Human UPF3-dependent nonsense-mediated mRNA decay is regulated by a specific exon junction complex composition
Zhongxia Yi (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University)
Abstract not available online - please check the printed booklet.
Friday 04:15-04:30pm: Unraveling the roles of 5’ transcript leaders in gene regulation
Christina Akirtava (Carnegie Mellon University, Biological Sciences), Hunter Kready (Carnegie Mellon University, Biological Sciences), Lauren Nazzaro (Carnegie Mellon University, Biological Sciences), Matt Agar-Johnson (Carnegie Mellon University, Biological Sciences), Gemma E May (Carnegie Mellon University, Biological Sciences), C. Joel McManus (Carnegie Mellon University, Biological Sciences)
Abstract:
Translation initiation is regulated by sequences surrounding the start codon, the “Kozak context” (1), cis.-acting sequences and structures in the 5’ transcript leader (TL), and corresponding trans.-acting factors. Previous work evaluating the in vitro. translation efficiency of 96 native yeast TLs showed that changes of 50-200nt in TL lengths varied translation up to 100-fold (2). Larger-scale in vivo. reporter studies of fixed-length synthetic TLs identified upstream AUGs as major repressors of gene expression (3-5). However, the in vivo. regulation of translation by native yeast TLs has not been systematically studied. To investigate cis.-regulatory translational control by native TLs in vivo., we assayed gene expression from ~11,000 endogenous TLs of S. cerevisiae. and S. paradoxus. using Fluorescence-Activated Cell Sorting and high-throughput sequencing (FACS-seq) (6). Additionally, we tested all Kozak variants surrounding AUG start codons in S. cerevisiae. We find Kozak context influences expression over a ~20-fold range, with the expected strong preference for -3 A. Our results show that alternative transcription start sites that change leader length by as little as 10 nucleotides can impact gene expression as much as ~16 fold. Finally, we trained a machine learning model on native 5’ TLs to identify regulatory features that increase sequence-based predictions of translation. Although Kozak strengths explain much of the variance in expression, our model quantitates the influence of upstream open reading frames, mRNA folding around the 5’ cap, and other structures that regulate the rate of translation initiation in vivo.. Thus, our results identify the range and relative regulatory impacts of Kozak context and other cis.-acting sequences and structures on translation from native yeast TLs in vivo.
References:
1)Kozak, M. (1984). Compilation and analysis of sequences upstream… NAR, 12(2), 857–872.
2)Rojas-duran, M. F., & Gilbert, W. V. (2012). Alternative transcription start site selection leads to large differences in translation activity in yeast, 2299–2305.
3)Cuperus et al., (2017). Deep Learning Of The Regulatory Grammar Of Yeast 5′ Untranslated Regions From 500,000 Random Sequences. BioRxiv, 163(2), 1–10.
4)Dvir et al.,(2013). Deciphering the rules by which 5’-UTR sequences… PNAS USA, 110(30), E2792–E2801
5)Sample et al., (2019). Human 5′ UTR design and variant effect prediction from a massively parallel translation assay. Nat. Bio., 37(7), 803–809.
6)Noderer et al., (2014). Quantitative analysis of mammalian translation initiation sites by FACS-seq. Molecular Systems Bio., 10(8), 748–748.
Keywords: translation, modeling, UTR
Saturday 09:30-09:55am: Targeting −1 programmed ribosomal frameshiting of SARS-CoV-2
Yu Sun (Department of Neuroscience, Yale University), Laura Abriola (Yale Center for Molecular Discovery, Yale University), Yulia Surovtseva (Yale Center for Molecular Discovery, Yale University), Brett Lindenbach (Department of Microbial Pathogenesis, Yale University), Junjie Guo (Department of Neuroscience, Yale University)
Abstract not available online - please check the printed booklet.
Saturday 10:00-10:30am: RNA Therapeutics: Informational drugs as a pandemic response tool
Anastasia Khvorova (University of Massachusetts Medical School)
Abstract:
Keywords:
Saturday 10:30-11:00am: Accelerated 3D RNA structure determination and the SARS-CoV-2 genome
Zhang, K., Zheludev, I. N. (Biochemistry Department, Stanford University), Hagey, R. J., Wu, M. T.-P., Haslecker, R. (Biochemistry Department, Stanford University), Hou, Y. J., Kretsch, R., Pintilie, G. D., Rangan, R. (Biochemistry Department, Stanford University), Kladwang, W., Li, S., Pham, E. A., Bernardin-Souibgui, C. (Biochemistry Department, Stanford University), Baric, R. S., Sheahan, T. P., D Souza, V., Glenn, J. S., Chiu, W. (Biochemistry Department, Stanford University), Das, R. (Biochemistry Department, Stanford University)
Abstract:
The discovery and design of biologically important RNA molecules has typically outpaced three-dimensional structural characterization. This talk will describe Ribosolve, a hybrid method integrating cryo-EM. biochemistry, and computer modeling, and its application to conserved segments of the SARS-CoV-2 RNA genome. The virus's frameshifting element is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date and illustrates how rapid structure determination can lead to functional insights with implications for biomedical targeting of RNA.
Keywords:
Saturday 01:00-01:15pm: Structure and assembly of human Multi-tRNA Synthetase Complex reveals novel mechanism of disease association
Krishnendu Khan (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, USA), Camelia B. Gogonea (Department of Chemistry, Cleveland State University, USA), Belinda Willard (Lerner Research Institute Proteomics and Metabolomics Core, Cleveland Clinic, USA), Valentin Gogonea (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, USA), Paul L. Fox (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, USA)
Abstract not available online - please check the printed booklet.
Saturday 01:15-01:30pm: A missense variant impairing TRMT1 function in tRNA modification is linked to intellectual disability
Kejia Zhang (Department of Biology, Center for RNA Biology, University of Rochester), Dragony Fu (Department of Biology, Center for RNA Biology, University of Rochester)
Abstract:
Transfer RNAs (tRNAs) are subject to numerous post-transcriptional modifications. In mammalian cells, tRNA methyltransferase 1 (TRMT1) is a tRNA methyltransferases that catalyzes the formation of the dimethylguanosine (m2,2G) modification in more than half of tRNA species. Frameshift mutations in the TRMT1 gene have been shown to cause autosomal-recessive intellectual disability (ID) in the human population but additional TRMT1 variants remain to be characterized. Here, we describe a homozygous missense variant in TRMT1 in a patient displaying developmental delay, ID, and epilepsy. The missense variant changes a conserved arginine residue to a cysteine (R323C) within the methyltransferase domain of TRMT1 and is expected to perturb protein folding. Patient cells expressing the TRMT1-R323C variant exhibit a severe deficiency in m2,2G modifications within tRNAs, indicating that the mutation causes loss-of-function. Notably, the TRMT1 R323C mutant retains the ability to bind tRNA but is unable to rescue m2,2G formation in TRMT1-deficient human cells. Our results identify a pathogenic point mutation in TRMT1 that severely perturbs tRNA modification activity, and provide the first demonstration that m2,2G modifications are disrupted in patients with TRMT1-associated ID disorders. These findings underscore the key relationship between human neurodevelopment and tRNA modifications.
References:
Dewe, J. M., Fuller, B. L., Lentini, J. M., Kellner, S. M., & Fu, D. (2017). TRMT1-Catalyzed tRNA Modifications Are Required for Redox Homeostasis To Ensure Proper Cellular Proliferation and Oxidative Stress Survival. Mol Cell Biol, 37(21).doi:10.1128/MCB.00214-17
Najmabadi, H., Hu, H., Garshasbi, M., Zemojtel, T., Abedini, S., Chen, W.,... Ropers, H. (2011).Deep sequencing reveals 50 novel genes for recessive cognitive disorders. Nature, 478(7367), 57-63. doi:10.1038/nature10423
Keywords: tRNA modification, TRMT1, neurodevelopment
Saturday 01:30-01:45pm: ADAR3 alters the MAVS/NF-κB signaling pathway in glioblastoma
Reshma Raghava Kurup (Genome, Cell and Developmental Biology Program, Indiana University), Emilie Oakes (Genome, Cell and Developmental Biology Program, Indiana University), Aidan Manning (Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN 47405), Pranathi Vadlamani (Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN 47405), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN 47405)
Abstract not available online - please check the printed booklet.
Saturday 01:45-02:00pm: Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei
Joseph T. Smith Jr. (Department of Microbiology and Immunology, University at Buffalo), Eva Dolezelova, Alena Zikova (Institute of Parasitology, Biology Centre Czech Academy of Science), Brianna Tylec (Department of Microbiology and Immunology, University at Buffalo), Jonathan Bard (Genomics and Bioinformatics Core, University at Buffalo), Runpu Chen, Yijun Sun (Department of Computer Science and Engineering, University at Buffalo), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo)
Abstract not available online - please check the printed booklet.
Saturday 02:00-02:15pm: Role of unique C-terminal domain in a plant aminoacyl-tRNA trans-editing protein
Jun-Kyu Byun (Department of Chemistry and Biochemistry, The Ohio State University), John Vu (Department of Chemistry and Biochemistry, The Ohio State University), William A. Cantara (Department of Chemistry and Biochemistry, The Ohio State University), Jawad Abid (Department of Chemistry and Biochemistry, The Ohio State University), Jyan-Chyun Jang (Department of Horticulture and Crop Science, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 02:15-02:30pm: Disease-associated point mutations in a bifunctional aminoacyl-tRNA synthetase elicit the integrated stress response
Danni Jin (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210), Nathan Kudlapur (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210), Ronald Wek (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, 46202), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210)
Abstract not available online - please check the printed booklet.
Saturday 02:45-03:00pm: Chemistry and catalysis join forces in prebiotic RNA ligation
Saurja DasGupta (Molecular Biology, Massachusetts General Hospital), Travis Walton (Biological Chemistry & Molecular Pharmacology, Harvard Medical School), Daniel Duzdevich (Molecular Biology, Massachusetts General Hospital), Seung Soo Oh (Materials Science and Engineering, Pohang University of Science and Technology POSTECH), Jack Szostak (Molecular Biology, Massachusetts General Hospital)
Abstract:
The ability of RNA to function as the carrier of heritable information as well as enzymes (ribozymes) have made it central to the emergence of life on earth. Modern biology uses protein polymerases to assemble RNA building blocks that are activated by triphosphate groups (NTPs); however, these building blocks are not sufficiently reactive for non-enzymatic RNA assembly. Non-enzymatic polymerization/ligation of monomers/oligomers activated by intrinsically reactive moieties like prebiotically-relevant 2-aminoimidazoles (2AI) can generate short RNA sequences, but these processes are inefficient (1). The appearance of ribozymes that catalyze RNA assembly was therefore a vital transition in the chemical evolution of life. Ribozymes that use triphosphate-activated monomers or oligomers as substrates for RNA assembly have been identified through in vitro selection/evolution experiments (2, 3). The evolutionary connection between chemical assembly of reactive, prebiotic building blocks and the enzymatic assembly of building blocks activated with triphosphates would be provided by ribozymes that catalyze RNA assembly using prebiotic 2AI-activated substrates.
We used in vitro selection to identify ligase ribozymes that utilize 2AI-activated RNA as substrates (4). Ligation was found to be dependent on a specific sequence at the 3' end of the substrate, which made ligation of shorter substrates inefficient. We rebooted selection to identify sequences that catalyze the ligation of RNA oligomers that lack the specific 3' sequence. After 10 rounds, we have identified RNA pools that join RNA pieces as short as 4 nt. These sequences will provide starting points for in vitro evolution to select polymerase ribozymes that use 2AI-activated monomers as substrates.
We have also identified ligase ribozymes that function at sub-millimolar concentrations of Mg2+. The low Mg2+ requirement presents an opportunity to constitute RNA-catalyzed RNA assembly inside prebiotic compartments made of fatty acids, as these vesicles are unstable at higher concentrations of Mg2+ (5). Achieving RNA-catalyzed RNA assembly within model protocells will bring us one step closer to assembling a self-replicating chemical system, capable of exhibiting Darwinian evolution.
References:
1. Li, L., Prywes, N., Tam, C. P., O'Flaherty, D. K., Lelyveld, V. S., Izgu, E. C., Pal, A., Szostak, J. W. J. Am. Chem. Soc., 2017, 139, 1810−1813.
2. Bartel, D. P., Szostak, J. W. Science, 1993, 261, 1411-1418.
3. Tjhung, K. F., Shokhirev, M. N., Horning, D. P., Joyce, G.F. Proc. Natl. Acad. Sci., 2020, 117, 2906-2913.
4. Walton, T., DasGupta, S., Duzdevich, D., Oh, S. S., Szostak, J. W. Proc. Natl. Acad. Sci., 2020, 117, 5741-5748.
Keywords: Ribozyme, Origin of Life, In vitro selection
Saturday 03:00-03:15pm: Structural basis of sequestration of the Anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome
Zakkary A. McNutt (Department of Microbiology & Center for RNA Biology & The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA), Bappaditya Roy, Bethany L. Boleratz, Dean E. Watkins (Department of Microbiology & Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Vikash Jha, Dushyant Jahagirdar, Kaustuv Basu (Department of Anatomy and Cell Biology & Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada), Elan A. Shatoff, Ralf Bundschuh (Department of Physics & Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Joaquin Ortega (Department of Anatomy and Cell Biology & Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada), Kurt Fredrick (Department of Microbiology & Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA)
Abstract not available online - please check the printed booklet.
Saturday 03:15-03:30pm: Structure-function relationship in biogenesis of microRNA-31
Anita Kotar (Biophysics program, University of Michigan), Sarah Keane (Biophysics program, Department of Chemistry, University of Michigan)
Abstract not available online - please check the printed booklet.
Saturday 03:30-03:45pm: Sequence and structure control assembly of RNA condensates
Raghav Poudyal (Chemistry, Pennsylvania State University), Jacob Sieg (Chemistry, Pennsylvania State University), Bede Portz (School of Medicine, University of Pennsylvania), Christine Keating (Chemistry, Pennsylvania State University), Philip Bevilacqua (Chemistry, Pennsylvania State University)
Abstract:
Intracellular condensates formed through liquid-liquid phase (LLPS) primarily contain proteins and RNA among other biomolecules. Recent evidences point to tremendous contributions of RNA self-assembly in formation of intracellular condensates. As a majority of previous studies on LLPS have focused on protein biochemistry, how biological RNAs affect LLPS remain unexplored. In this study, we explore the effects of crowding, metal ions, and RNA structure towards formation of condensates that are largely composed of RNAs. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by the formation of RNA droplets in the presence of polyethylene glycol. In the absence of crowders, elevated Mg2+ concentrations promoted assembly of specific riboswitches. In silico prediction identified key structural and sequence elements that potentiate the formation of condensates; these predictions were corroborated by experimental observations. We implement structure-guided designs to generate condensates with novel functions. Finally, we show that RNA condensates protect RNAs from nucleases, suggesting potential biological roles for such higher-order RNA assemblies. Overall, our work provides mechanistic insights on contributions of both intrinsic RNA properties and extrinsic environmental conditions that promote the formation and regulation of condensates composed of RNAs.
Keywords: RNA structure, phase separation, riboswitch
Saturday 03:45-04:05pm: Translational induction of ATF4 mRNA during stress requires noncanonical initiation factors eIF2D and DENR
Deepika Vasudevan (Cell Biology, NYU School of Medicine), Sarah D. Neuman (Dept. of Pharmaceutical Sciences, University of Wisconsin-Madison), Amy Yang (Cell Biology, NYU School of Medicine), Arash Bashirullah (Dept. of Pharmaceutical Sciences, University of Wisconsin-Madison), Hyung Don Ryoo (Cell Biology, NYU School of Medicine)
Abstract:
Certain conditions of cellular stress impose restrictions on mRNA translation by activating stress response kinases that phospho-inactivate the α-subunit of the initiator methionine-carrying complex, eIF2. Such restrictive translation conditions paradoxically stimulate the synthesis of the transcription factor ATF4 to induce a stress responsive gene expression program. In a Drosophila RNAi screen, we discovered eIF2D as a regulator of ATF4, and subsequently validated this observation with two independent eIF2D mutant alleles. Based on domain analysis, we found that the eIF2D homolog complex, DENR-MCTS1, also exerted similar effects on ATF4. While deletion of eIF2D or DENR had little effect on ATF4 mRNA levels, it resulted in marked decrease of an ATF4 5’UTR-dsRed reporter in response to amino acid deprivation or ER stress. Disrupting the tRNA-binding activity of eIF2D also resulted in decreased expression of the ATF4 5’UTR-dsRed reporter. Taken together, these data suggest that eIF2D and DENR-MCTS1 are non-canonical initiation factors required for the translational induction of ATF4. Consistently, loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by ER stress, and developmental defects similar to ATF4 mutants. eIF2D and DENR deficient human cells also show impaired ATF4 protein induction in response to ER stress, indicating that such regulation is conserved in higher organisms.
Keywords: Integrated Stress Response, reinitiation, eIF2D
Saturday 04:05-04:25pm: lncRNA phase separation in nuclear organization.
Huaiying Zhang (Carnegie Mellon University)
Abstract:
Keywords: