Talk abstracts
Friday 01:00-01:15pm: Emergence of ribonuclease activity in ribozymes through intersection of neutral networks
Saurja DasGupta (The University of Chicago), Joseph A. Piccirilli (The University of Chicago)
Abstract:
Ribozymes present the most primitive catalytic systems in Biology, and are thought to have played a central role in jumpstarting life. A major class of ribozymes, referred to as small endonucleolytic ribozymes catalyze site-specific RNA cleavage. While the structural and mechanistic features of these RNAs have been studied extensively, we have little understanding about their evolutionary histories. It is generally thought that enzymes acquire novel functions through adaptive walks along paths virtually neutral to mutational perturbations. This involves gradual acquisition of new functions while conserving its existing ones till the emergence of an ‘optimally evolved’ catalyst through bifunctional intermediates. We explored mutational landscapes in the sequence spaces of two functional RNAs – the VS and hairpin endonucleolytic ribozymes, by rational design, in-silico structure prediction and in-vitro functional assays. We discovered ‘neutral networks’ or mutational paths in the fitness landscapes containing sequences that retained cleavage activity for both ribozymes, and identified a sequence space where neutral networks for both ribozymes intersect. Sequences that fall in this region could potentially adopt secondary structures corresponding to both VS and hairpin folds and are catalytically active toward both VS and hairpin substrates. Catalytic promiscuity in primitive RNA might have driven exploration of sequence space and eventually certain sequences through mutations could’ve acquired a new function on encountering a novel functional space. Intersection of neutral networks therefore provide an evolutionary mechanism for the acquisition of new function in catalytic RNA. Subsequently, the gene for intersection sequences could duplicate and diverge by adaptive evolution to more optimal activities exemplified by wild-type VS and hairpin ribozymes, relieving constraints of dual and therefore decreased activities. This work paves a way for further investigations into the neutral networks of other related ribozymes and might provide insights into their evolutionary origins, enriching our understanding of RNA evolution in general, and in the context of primitive life prevalent in the RNA World.
References:
1. The RNA World, 3rd Edition. Edited by Gesteland, ,R. F., Cech, T. R. and Atkins, J. F. 2006.
2. Lilley, D. M. J. The Origins of RNA Catalysis. TiBS, 28 (9), 2003.
3. Wagner, A. Robustness, evolvability and neutrality. FEBS Lett, 579, 2003.
4. Schultes, E. A. & Bartel, D. P. One sequence, two ribozymes: Implications for the emergence of New Ribozyme folds. Science, 289, 2000.
Keywords: Ribozymes, Evolution, Neutral Networks
Friday 01:15-01:30pm: Dissecting the functional consequences of the A-to-I RNA editing landscape in Glioblastoma to uncover a compendium of prognostic editing events
Seyedsasan Hashemikhabir (Biohealth Informatics, Indiana University), Britya Ghosh (Biochemistry & Molecular Biology, Indiana University), Heather Ann Hundley (Biochemistry & Molecular Biology, Indiana University), Sarath Chandra Janga (Biohealth Informatics, Indiana University)
Abstract not available online - please check the printed booklet.
Friday 01:30-01:45pm: Title not available online - please see the printed booklet.
Daniel L. Kiss (Center for RNA Biology, Department of Biological Chemistry and Pharmacology, The Ohio State University), William D. Baez (Center for RNA Biology, Department of Physics, The Ohio State University), Bernice Agana (Department of Chemistry and Biochemistry, The Ohio State Universit), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State Universit), Ralf Bundschuh (Center for RNA Biology, Department of Physics,and Division of Hematology, The Ohio State University), Daniel R Schoenberg (Center for RNA Biology, Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract not available online - please check the printed booklet.
Friday 01:45-02:00pm: In vivo genome-wide probing of RNA structure in Bacillus subtilis reveals regulatory features
Laura E. Ritchey (Chemistry and Center for RNA Molecular Biology, Penn State University), David C. Tack (Biology, Penn State University), Sarah M. Assmann (Biology and Center for RNA Molecular Biology, Penn State University), Philip C. Bevilacqua (Chemistry, Biochemistry & Molecular Biology, and Center for RNA Molecular Biology, Penn State University), Paul Babitzke (Biochemistry & Molecular Biology, Penn State University)
Abstract not available online - please check the printed booklet.
Friday 02:00-02:15pm: Title not available online - please see the printed booklet.
Angela M Yu (Tri-Institutional Program in Computational Biology and Medicine, Cornell University, Weill Cornell Medicine, MSKCC; Department of Chemical and Biological Engineering, Northwestern University), Paul M. Gaspar (Department of Chemistry and RNA Institute, University at Albany ), Eric J. Strobel (Department of Chemical and Biological Engineering, Northwestern University), Kyle E. Watters (Department of Molecular and Cell Biology, University of California, Berkeley), Alan A. Chen (Department of Chemistry and RNA Institute, University at Albany ), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University)
Abstract not available online - please check the printed booklet.
Friday 02:15-02:30pm: RNA folding nearest neighbor parameter derivation and RNA secondary structure prediction
Hongying Sun (Department of Biochemistry and Biophysics, University of Rochester), Jeffrey Zuber (Department of Biochemistry and Biophysics, University of Rochester), David H. Mathews (Department of Biochemistry and Biophysics, University of Rochester)
Abstract:
The folding stability of RNA secondary structure can be estimated using a nearest neighbor model, and this model is in widespread use to predict RNA secondary structures. Nearest neighbor parameters for predicting RNA folding free energy change at 37°C are based on a database of optical melting measurements on small model systems. This work revises and expands the nearest neighbor model by including the latest experimental results on the melting stability of small model systems. A statistical model called AIC was applied to determine and select nearest neighbor parameters that are significantly important to the stability of loops and to prevent overfitting. Surprisingly, we found that the AU helix-end penalty was removed by AIC model selection for hairpin loops, indicating that the AU end penalty should not be applied to hairpin loops. We also found that the stability of hairpin loops is independent of first mismatch sequence, which was assumed to be important in the previous 2004 model. We did a benchmark on a set of 3856 RNA sequences with known structures by implementing both 2004 and the new nearest neighbor parameters in the RNAstructure software package for RNA secondary structure prediction. Secondary structure prediction identified 1% more of the known pairs using the new model compared to 2004 model, and this improvement is statistically significant. Therefore, the new hairpin loop model predicts RNA secondary structure more accurately. We are implementing the complete new set of nearest neighbor parameters and hypothesize that this will improve the accuracy of RNA secondary structure prediction significantly.
References:
1. Chen JL, Dishler AL, Kennedy SD, et al. Testing the Nearest Neighbor Model for Canonical RNA Base Pairs: Revision of GU Parameters. Biochemistry. 2012;51(16):3508-3522. doi:10.1021/bi3002709.
2. Bellaousov S, Mathews DH. 2010. ProbKnot: Fast prediction of RNA secondary structure including pseudoknots. RNA 16: 1870-1880.
3. Mathews DH. 2004. Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization.
RNA 10: 1178-1190.
4. Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH. 2004. Incorporating chemical modification constraints into a dynamic program-
ming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci USA 101: 7287-7292.
Keywords: RNA Folding, RNA Secondary Structure Prediction, Nearest Neighbor Model
Friday 02:30-02:45pm: Selectivity in substrate binding by the exonuclease Rrp6p
Armend Axhemi (The Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Sukanya Srinivasan (The Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Ulf Peter Guenther (European Molecular Biology Laboratory, Heidelberg, Germany ), Elizabeth V Wasmuth (Structural Biology Program, Sloan Kettering Institute, New York, NY ), Christopher D. Lima (Howard Hughes Medical Institute, Sloan Kettering Institute, New York, NY ), Eckhard Jankowsky (The Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)
Abstract not available online - please check the printed booklet.
Friday 03:15-03:30pm: Dynamic remodeling of mRNA splicing and translation programs drives hepatocyte proliferation during liver regeneration.
Sushant Bangru (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Waqar Arif (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Joseph Seimetz, Amruta Bhate, Jackie Chen, Edrees Rashan (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Russ P. Carstens (Dept. of Medicine and Genetics, Perelman school of medicine, University of Pennsylvania), Sayeepriyadarshini Anakk (Dept. of Molecular and Integrative Physiology, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Dept. of Biochemistry, and Carl R. Woese Institute of genomic biology at University of Illinois, Urbana-Champaign)
Abstract:
During liver regeneration, most new hepatocytes arise from pre-existing ones; yet, the underlying mechanisms that drive these cells from quiescence to proliferation remain poorly defined. By combining high-resolution transcriptome and polysome profiling of purified hepatocytes isolated from quiescent and toxin-injured adult mouse livers, we uncover a pervasive shift in ribosome occupancies for transcripts encoding metabolic and RNA processing factors. This translational remodeling modulates protein levels of a set of splicing factors including Epithelial splicing regulatory protein 2 (ESRP2), which activates an early postnatal splicing program in regenerating hepatocytes. We find that regeneration-regulated exons are enriched within unstructured regions of proteins and frequently harbor amino-acid residues that can be post-translationally modified to rewire protein-protein interactions. Finally, we demonstrate that dynamic regulation of ESRP2 upon toxin-injury tunes hepatocyte proliferation by coordinating alternative splicing of core components of the Hippo signaling pathway. Altogether, our data reveal that after sustaining injury, an early postnatal splicing program is redeployed in adult hepatocytes, which reprograms critical signaling pathways to enable hepatocyte proliferation and proper liver regeneration.
Keywords: alternative splicing , liver regeneration, translational regulation
Friday 03:30-03:45pm: Title not available online - please see the printed booklet.
Rene Arvola (Department of Biological Chemistry, University of Michigan), Chase Weidmann (Department of Chemistry, University of North Carolina Chapel Hill; Department of Biological Chemistry, University of Michigan), Chen Qui, Traci Tanaka Hall (National Institutes of Health, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences ), Jordan Killingsworth (Department of Biological Chemistry, University of Michigan), Tzu-Fang Lou, Zachary Campbell (Department of Biological Sciences, University of Texas at Dallas), Aaron Goldstrohm (Department of Biological Chemistry, University of Michigan; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota)
Abstract not available online - please check the printed booklet.
Friday 03:45-04:00pm: Title not available online - please see the printed booklet.
Anne Witzky (Department of Molecular Genetics, Center for RNA Biology, Ohio State University, Columbus, OH 43210), Katherine R. Hummels (Department of Biology, Indiana University, Bloomington, Indiana 47405), Andrei Rajkovic (Department of Microbiology, Center for RNA Biology, Ohio State University, Columbus, OH 43210), Rodney Tollerson II (Department of Microbiology, Ohio State University, Columbus, OH 43210), Daniel B. Kearns (Department of Biology, Indiana University, Bloomington, Indiana 47405), Michael Ibba (Department of Microbiology, Center for RNA Biology, Ohio State University, Columbus, OH 43210)
Abstract not available online - please check the printed booklet.
Friday 04:00-04:15pm: Proximal 3'UTR introns elicit EJC-dependent NMD to manipulate levels of key developmental regulators
Pooja Gangras (Department of Molecular Genetics), Thomas L. Gallagher (Department of Molecular Genetics), Kiel T Tietz (Department of Molecular Genetics), Natalie C. Deans (Department of Molecular Genetics), Sharon L. Amacher (Department of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics)
Abstract not available online - please check the printed booklet.
Friday 04:15-04:30pm: Pnrc2 Regulates 3’UTR-Mediated Decay Of Cyclic Transcripts During Somitogenesis
Kiel T. Tietz (Department of Molecular Genetics, The Ohio State University), Thomas L. Gallagher (Department of Molecular Genetics, The Ohio State University), Zachary T. Morrow (Department of Molecular Genetics, The Ohio State University), Nicolas L. Derr (Department of Molecular Genetics, The Ohio State University), Sharon L. Amacher (Department of Molecular Genetics, The Ohio State University)
Abstract not available online - please check the printed booklet.
Friday 04:30-04:45pm: A family expands: Characterization of human TRMT10B, a multifunctional tRNA methyltransferase
Nathan Howell (Ohio State University), Jane Jackman (Ohio State University)
Abstract not available online - please check the printed booklet.
Friday 04:45-05:00pm: IF2 and unique features of initiator tRNAfMet help establish the translational reading frame
Bappaditya Roy (Department of Microbiology and Center for RNA Biology, Ohio State University, Columbus, Ohio, 43210, USA), Qi Liu (Department of Microbiology and Center for RNA Biology, Ohio State University, Columbus, Ohio, 43210, USA), Shinichiro Shoji (Department of Microbiology and Center for RNA Biology, Ohio State University, Columbus, Ohio, 43210, USA), Kurt Fredrick (Department of Microbiology and Center for RNA Biology, Ohio State University, Columbus, Ohio, 43210, USA)
Abstract:
Translation begins at AUG, GUG, or UUG codons in bacteria. Start codon recognition occurs in the P site, which may help explain this first-position degeneracy. However, the molecular basis of start codon specificity remains unclear. In this study, we measured the codon dependence of 30S•mRNA•tRNAfMet and 30S•mRNA•tRNAMet complex formation. We found that complex stability varies over a large range with initiator tRNAfMet, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNAMet, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNAfMet is a series of three G-C basepairs in the anticodon stem, which are known to be important for efficient initiation in vivo. A mutation targeting the central of these G-C basepairs causes the mRNA binding specificity pattern to change in a way reminiscent of elongator tRNAMet. Unexpectedly, for certain complexes containing fMet-tRNAfMet, we observed mispositioning of mRNA, such that codon 2 is no longer programmed in the A site. This mRNA mispositioning is exacerbated by the anticodon stem mutation and suppressed by IF2. These findings suggest that both IF2 and the unique anticodon stem of fMet-tRNAfMet help constrain mRNA positioning to set the correct reading frame during initiation.
Keywords: Initiation, ribosome, start codon selection
Saturday 08:30-08:45am: Title not available online - please see the printed booklet.
Seth Lyon (Chemistry and Biochemistry, The Ohio State University), Venkat Gopalan (Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 08:45-09:00am: Sharing the load: Mex67-Mtr2 co-functions with Los1 in primary tRNA nuclear export
Kunal Chatterjee (The Ohio State University Comprehensive Cancer Research Centre, Centre for RNA Biology, Department of Molecular Genetics, The Ohio State University.), Shubhra Majumder, Yao Wan, Vijay Shah, Jungian Wu, Hsiao-Yun Huang, (Department of Molecular Genetics, Centre for RNA Biology, The Ohio State University), Anita K. Hopper (Department of Molecular Genetics, Centre for RNA Biology, The Ohio State University)
Abstract:
tRNAs perform essential role of delivering amino acids to the cytoplasmic protein synthesis machinery. To execute this role in protein production, eukaryotic tRNAs must be escorted out of the nucleus, their site of synthesis, to the cytoplasm, their site of function. Via primary nuclear export, newly transcribed, end matured, partially modified tRNAs are shuttled to the cytoplasm, where, in yeast, the tRNA splicing machinery is located. Genetic and biochemical studies have identified a set of RanGTPase binding proteins, β-importins, implicated in this tRNA movement. Two members of the β-importin family, Los1 (Exportin-t in vertebrates) and Msn5 (Exportin 5) serve overlapping but distinct functions in tRNA export. Although Los1 and Msn5 both participate in tRNA nuclear export, they cannot be the only nuclear exporters for tRNAs in yeast as los1Δ msn5Δ double mutant cells are viable. An unbiased comprehensive screening representing ~90% of the total yeast proteome recently conducted in our laboratory uncovered novel proteins involved in tRNA subcellular dynamics. Two such proteins Mex67 (vertebrate TAP) and Mtr2 (vertebrate p15), which are known to be mRNA nuclear exporters, when inactivated, accumulate end-matured, unspliced tRNAs in yeast nuclei. Remarkably, merely 5-fold over-expression of Mex67-Mtr2 substitutes for Los1 in los1Δ cells. Moreover, in vivo co-immunoprecipitation assays with Mex67 document that the Mex67 binds tRNAs. We also show that tRNA exporters surprisingly exhibit differential tRNA substrate preferences, possibly indicating that the proteome can be regulated at the step of tRNA nuclear export. Thus, our data show that the mRNA export machinery, Mex67-Mtr2, co-functions with Los1, in primary nuclear export for a subset of yeast tRNAs.
Keywords: tRNA subcellular dynamics, tRNA trafficking, Sachharomyces cerevisiae
Saturday 09:00-09:15am: Title not available online - please see the printed booklet.
Qinyu Hao (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL), Susan M. Freier (IONIS Pharmaceuticals, Carlsbad, CA, USA), Supriya G. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL), Sui Huang (Northwestern University, Chicago, IL), Brian McStay (Centre for Chromosome Biology, NUI Galway, Ireland), Kannanganattu V. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL)
Abstract not available online - please check the printed booklet.
Saturday 09:15-09:30am: Only particular guide RNAs activate human Argonaute3 for RNA cleavage
Mi Seul Park (Department of Chemistry and Biochemistry, The Ohio State University), Hong-Duc Phan (Ohio State Biochemistry Program, The Ohio State University), Florian Busch, Samantha H. Hinckley (Department of Chemistry and Biochemistry, The Ohio State University), James A. Brackbill (Department of Chemistry and Biochemistry, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Kotaro Nakanishi (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 09:30-09:45am: CTD phosphatase Ssu72 stimulates read-through of ncRNA at NNS terminated genes
Jose F. Victorino (Department of Biochemistry and Molecular Biology, Center for Computational Biology and Bioinformatics, Indiana University School of Medicine), Whitney R. Smith-Kinnaman (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Neil McCracken (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Rachel A. Chan (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Hongyu Gao, Yunlong Liu, (Center for Computational Biology and Bioinformatics, Department of Medical and Molecular Genetics, Indiana University School of Medicine), Amber L. Mosley (Department of Biochemistry and Molecular Biology, Center for Computational Biology and Bioinformatics, Indiana University School of Medicine)
Abstract not available online - please check the printed booklet.
Saturday 09:45-10:00am: Hypoxia-regulated microRNA-210 expression and role in kidney development
Shelby L. Hemker (Department of Pediatrics, Division of Nephrology, University of Pittsburgh, Pittsburgh, PA), Andrew N. Clugston (Department of Developmetnal Biology, Department of Pediatrics, Division of Nephrology, University of Pittsburgh, Pittsburgh, PA), Yu Leng Phua (Department of Pediatrics, Division of Nephrology, University of Pittsburgh, Pittsburgh, PA), Dennis Kostka (Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA), Jacqueline Ho (Department of Pediatrics, Division of Nephrology, University of Pittsburgh, Pittsburgh, PA)
Abstract not available online - please check the printed booklet.
Saturday 10:00-10:15am: Title not available online - please see the printed booklet.
Alan C. Kessler (Department of Microbiology, The Center for RNA Biology, The Ohio State University ), Gabriel Silveria dAlmeida (Department of Microbiology, The Center for RNA Biology, The Ohio State University ), Sneha S. Kulkarni, Zdenek Paris (Institute of Parasitology, Biology Centre, Czech Academy of Sciences and Faculty of Science), Mellie J. Paulines, Robert L. Ross, Patrick A. Limbach (Department of Chemistry, Rieveschl laboratories for Mass Spectrometry, University of Cincinnati ), Mary Anne T. Rubio (Department of Microbiology, The Center for RNA Biology, The Ohio State University ), Juan D. Alfonzo (Department of Microbiology, The Ohio State Biochemistry Program, The Center for RNA Biology, The Ohio State University )
Abstract not available online - please check the printed booklet.
Saturday 10:45-11:00am: Assessment of thermal stability of phosphorothioate-DNA, DNA, RNA, 2’-F RNA and LNA in the context of Phi29 pRNA 3WJ
Xijun Piao (College of Pharmacy, The Ohio State University), Hongzhi Wang (College of Pharmacy, The Ohio State University), Daniel W. Binzel (College of Pharmacy, The Ohio State University), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy; College of Medicine; Dorothy M. Davis Heart and Lung Research Institute; James Comprehensive Cancer Center, The Ohio State Univ.)
Abstract:
Thermodynamically-stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2’-F nucleotides (2’-F) and locked nucleic acids (LNA) on the thermal and in vivo stability of RNA and the three-way junction (3WJ) of bacteriophage phi29 motor pRNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2’-F < 2’-F/2’-F < 2’-F/LNA < LNA/LNA. This proposition is supported by the studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically-modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures (Tms) from 21.2 oC to over 95 oC. The LNA 3WJ was resistant to boiling temperature denaturation, urea denaturation and 50% serum degradation. Intravenous injection of fluorescent LNA/2’-F hybrid 3WJs into mice revealed its exceptional in vivo stability and the presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2’-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve its pharmacokinetics profile.
References:
(1) Guo P. Nature Nanotechnology. 2010; 5:833.
(2) Shu D, Shu Y, Haque F, Abdelmawla S, Guo P. Nature Nanotechnology 2011; 6(10):658-67.
(3) Binzel DW, Khisamutdinov EF, Guo P. Biochemistry. 2014; 53(14):2221-31.
(4) Piao X, Wang H, Binzel DW, Guo P. in revision, 2017.
Keywords: Three-way junction, thermal stability, in vivo stability
Saturday 11:00-11:15am: tRNA dynamics during antibiotic dependent translation arrest
James Marks (University of Illinois at Chicago), Junhong Choi (Stanford University), Nora Vazquez-Laslop (University of Illinois at Chicago), Joseph Puglisi (Stanford University), Alexander Mankin (University of Illinois at Chicago)
Abstract:
Chloramphenicol and linezolid are inhibitors of bacterial protein synthesis. They bind to the A-site of the peptidyl transferase center in the ribosome, the site occupied by all incoming aminoacyl-tRNAs during translation elongation. Therefore it was thought that chloramphenicol and linezolid inhibit formation of every peptide bond because they interfere with the accommodation of any aminoacyl-tRNA. However, we have recently shown that chloramphenicol and linezolid are not universal inhibitors but, instead, their action depends on the amino acid sequence of the nascent peptide. Specifically, translation is strongly arrested when an alanine is in the penultimate position of the nascent chain. To investigate how antibiotic dependent arrest influences translation dynamics we used single-molecule FRET to discretely monitor ribosome and tRNA conformations in real-time during translation of model templates in the presence or absence of chloramphenicol. We found that, in spite of the presence of chloramphenicol, the ribosome progressed normally when translating the non-arrest template. In contrast, a ribosome translating an arrest peptide in the presence of chloramphenicol binds A-site tRNA but in a non-productive fashion because it remains in a partially accommodated state for an extended period of time. After the non-productive A-site tRNA finally dissociates from the ribosome, another round of non-productive binding can occur. Our findings not only contribute to understanding the mechanism of action of ribosomal antibiotics but also reveal critical aspects tRNA dynamics in the peptidyl transferase center during protein synthesis.
Keywords: Ribosome, Antibiotic, tRNA
Saturday 11:15-11:30am: Versatile RNA Tetra-U Helix Linking Motif as a Toolkit for Nucleic Acid Nanotechnology
Tori Goldsworthy (Department of Chemistry, , Ball State University ), My N. Bui, Zhihai Li, Emil F. Khisamutdinov (Department of Chemistry, Ball State University), M. Brittany Johnson, Angelica N. Martins, Ian Marriott (Department of Biology, University of North Carolina Charlotte ), Mathias Viard (Basic Science Program, Leidos Biomedical Research Inc., RNA Biology Laboratory, Frederik National Laboratory for Cancer Research ), Emily Satterwhite, Kirill Afonin (Department of Chemistry, University of North Carolina Charlotte )
Abstract:
RNA nanotechnology employs ribonucleic acid to engineer addressable nanostructures in one, two, and three dimensions, making it highly attractive for medical applications. Although RNA nanotechnology is an emerging field, the notion of RNA-nanoparticle (NP) production is no longer novel. Despite its advantages, RNA biomaterial is fragile and prone to enzymatic degradation. Herein, we demonstrate a RNA/DNA hybrid approach which uses a computer-assisted de novo RNA tetra-uracil (tetra-U) motif as a toolkit to overcome obstacles in RNA nanotechnology including simplicity, efficiency, versatility, stability, and the production costs of functional RNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stability, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, hexagonal and other polygonal NPs, representing the power of simplicity that is in high demand in RNA nanotechnology.
Keywords: nanoparticle
Saturday 11:30-11:45am: piRNA mediated down regulation of ferritin heavy chain 1 mRNA in Triple Negative Breast Cancer cells
Sumirtha Balaratnam (Kent State University), Soumitra Basu (Kent State University)
Abstract:
The piwi interacting RNAs (piRNAs) are small non-coding RNAs mostly 24-32 nucleotides in length. The piRNAs are defined by their ability to specifically bind to the PIWI proteins, a requirement for their functions. The piRNAs are involved in germline development, transposons control, transcriptional and posttranscriptional gene regulation. However, piRNA mediated post-transciptional gene regulation in somatic cells is not well understood. We discovered a human piRNA (piR-FTH1) which has a complementary sequence in the ferritin heavy chain 1 (FTH1) mRNA. We demonstrate that piR-FTH1 expression is inversely correlated to the expression of FTH1 in most tumor cell lines. piR-FTH1 negatively regulates the FTH1 expression at posttranscriptional level in MDA-MB 231 cell lines. Further we confirmed that transfected piR-FTH1 knocks down the FTH1 mRNA through piR-FTH1-HIWI-RISC pathway by binding to HIWI2 and HILI. FTH1 repression by piR-FTH1 increased the chemotherapuetic agent Doxorubicin’s sensitivity by a remarkable 20 fold compared to the cell line treated with control siRNA sequence. Since most of the current piRNA mediated knockdown of target mRNA were only described in germ line cells, here we report piRNA mediated post transcriptional gene regulation in somatic cells. Thus, it will have a broader impact on treatment of various diseases that are linked to elevated level of specific mRNAs which have a piRNA target.
Keywords: piRNA, post-transcriptional regulation, Ferritin heavy chain 1
Saturday 11:45-12:00pm: Rbm24-mediated post-transcriptional gene regulation is necessary for vertebrate eye development
Soma Dash (Department of Biological Sciences, University of Delaware), Lindy K. Brastorm (Department of Biological Sciences, University of Iowa), C. Anthony Scott (Department of Biological Sciences, University of Iowa), Diane C. Slusarski (Department of Biological Sciences, University of Iowa), Salil A. Lachke (Department of Biological Sciences, University of Delaware)
Abstract:
While transcriptional and signaling regulation are well-defined in eye development, post-transcriptional control (PTC) mediated by RNA binding proteins (RBP) remains less clear. Recently we described three RBPs Tdrd7, Celf1 and Caprin2 required for vertebrate lens development. Here we define the function of another RBP Rbm24 - identified using iSyTE (integrated Systems Tool for Eye gene discovery) – in vertebrate eye development. In zebrafish rbm24a expression begins in the ocular tissue at 1-somite stage, while in mouse it is expressed in lens and retina at embryonic day (E) 9.5. rbm24a morpholino knockdown zebrafish morphants exhibit microphthalmia (abnormally small eye). We generated new targeted germline deletion mouse mutants of Rbm24 (Rbm24-/-), which exhibit 100% penetrant microphthalmia and 50% penetrant anophthalmia (absence of eye). Interestingly, the human anophthalmia-linked gene SOX2 is downregulated at both mRNA and protein levels in Rbm24-/- ocular tissue. RNA immunoprecipitation assay suggests that Rbm24 binds to Sox2 mRNA in E14.5 mouse eye tissue. Further, to characterize Rbm24 mediated control over Sox2 we co-transfected NIH3T3 cells with Rbm24 overexpression vector and luciferase reporter vector wherein Renilla luciferase is fused to 3’UTR of Sox2. Following actinomycin-D treatment, we find the half-life of luciferase mRNA fused to 3’UTR of Sox2 is increased in Rbm24 overexpressing cells compared to control. This suggests that Rbm24 binds to the 3’UTR of Sox2 and stabilizes its mRNA. Further, mutation of the AU-rich elements (ARE) in the 3’UTR of Sox2 in the luciferase reporter vector failed to increase the mRNA half-life of luciferase with Rbm24 overexpression. This suggests that Rbm24-medited Sox2 mRNA stability is mediated by its binding to the ARE sites in 3’UTR of Sox2. These data identify Rbm24 as a new factor controlling vertebrate eye development and define a new RBP-mediated post-transcriptional mechanism to control Sox2 expression.
Keywords: RNA Binding Proteins, Post-transcriptional regulation
Saturday 12:00-12:20pm: Translation repression via modulating the cytoplasmic poly(A) binding protein in inflammatory response
Xu Zhang (Biochemistry and Molecular Biology, Mayo Clinic), Xiaoli Chen (University of Central Florida), Qiuying Liu (Biochemistry and Molecular Biology, Mayo Clinic), Shaojie Zhang (University of Central Florida), Wenqian Hu (Biochemistry and Molecular Biology, Mayo Clinic)
Abstract:
Gene expression is precisely regulated in inflammatory response to control infection and limit detrimental effects of inflammation. Here, we profiled global mRNA translation dynamics in mouse primary macrophage-mediated inflammatory response and identified hundreds of differentially translated mRNAs. These mRNAs’ 3’UTRs have enriched binding motifs of several RNA-binding proteins, implying extensive translational regulatory networks. We characterized one such protein, Zfp36, as a translation repressor. Using primary macrophages from a Zfp36-V5 epitope tag knock-in mouse generated by CRISPR/Cas9-mediated genome editing, we found that the endogenous Zfp36 directly interacts with the cytoplasmic poly(A) binding protein. Importantly, this interaction is required for the translational repression of Zfp36’s target mRNAs in resolving inflammation. Altogether, these results uncovered critical roles of translational regulations in controlling appropriate gene expression during inflammatory responses and revealed a new biologically relevant molecular mechanism of translational repression via modulating the cytoplasmic poly(A) binding protein.
Keywords: translational control , poly(A) binding protein, Zfp36
Saturday 12:20-12:40pm: From the structure and function of ribosome to disease-curing antibiotics
Yury Polikanov (University of Illinois at Chicago)
Abstract:
Antibiotic resistance in the US costs an estimated $20 billion a year, because many pathogenic microorganisms quickly develop resistance to the drugs. This demands a constant search for new antibiotics. Our current research is focused on one of the major targets of antibacterial drugs – the ribosome, which is a very complex molecular machine responsible for protein synthesis in all living organisms. Since ribosome inhibitors are among the most successful antimicrobial drugs in clinic, our ambition is to determine the structures of such inhibitors bound to their target in order to understand how they work and how they could be improved. Recently we established a biochemistry-inspired multifaceted approach, which allowed us not to only directly visualize interactions of various drugs with the ribosome with atomic precision, but also to assess the effects of the drug binding on the ribosome functionality. In my talk I will present our recent advances that enabled us to elucidate the mechanisms of action of novel classes of antibiotics, all of which are not represented among the clinically used drugs and, therefore, hold value for future structure-based rational drug design.
References:
1. Metelev M, et al. (2017) Klebsazolicin inhibits 70S ribosome by obstruction of the peptide exit tunnel. Nature Chemical Biology. In press.
2. Almutairi MM, et al. (2017) Co-produced natural ketolides methymycin and pikromycin inhibit bacterial growth by preventing synthesis of a limited number of proteins. Nucleic Acids Research. In press.
3. Osterman IA, et al. (2017) Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state. Nucleic Acids Research, DOI: 10.1093/nar/gkx413.
Keywords: Ribosome, Structure, Antibiotic