Talk abstracts
Friday 01:00-01:15pm: Title not available online - please see the printed booklet.
Jamie L. Bingaman (Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University), Sixue Zhang (Department of Chemistry, University of Illinois at Urbana-Champaign), David R. Stevens (Department of Chemistry, University of Illinois at Urbana-Champaign), Neela H. Yennawar (Macromolecular X-Ray Facility, The Huck Institutes of the Life Sciences, The Pennsylvania State University), Sharon Hammes-Schiffer (Department of Chemistry, University of Illinois at Urbana-Champaign), Philip C. Bevilacqua (Department of Chemistry, Center for RNA Molecular Biology, and Department of Biochemistry, Microbiology, and Molecular Biology, The Pennsylvania State University)
Abstract not available online - please check the printed booklet.
Friday 01:15-01:30pm: Synthesis of mini-lariat RNA and use as a microRNA mimic for RNA interference
Stephanie Mack (Chemistry, Carnegie Mellon University), Jey Sabith Ebron (Biological Sciences, Cleveland State University), Girish C. Shukla (Biological Sciences, Cleveland State University), Subha R. Das (Chemistry, Carnegie Mellon University)
Abstract:
After the excision of intronic sequences as lariats in splicing, their fate is varied. Some introns are completely degraded into constituent nuclear bases and nucleotides recycled, while others enter into biogenesis and other pathways related to regulatory RNAs. MicroRNAs (miRNAs) derived from lariat introns, or mirtrons, represent a substantial amount of the total microRNA in a cell. These lariat introns are processed by lariat debranching enzyme, then enter the miRNA biogenesis pathway independent of drosha to generate miRNA duplexes that contain a guide and passenger strand. Of these, the guide strand is incorporated into the silencing complex for RNAi. Based on these lariat introns, here we synthesize single guide strands as mini-lariats for use as miRNA mimics. These mini-lariats are the size of a mature microRNA guide strand (19-21 mers) and are synthesized on the solid phase through the inclusion of a photo-cleavable protecting group at the 2' position of a desired residue. Following, photo-deprotection, the 2'-hydroxyl cyclizes with an activated 5'-phosphoramidite. Solid phase synthesis also allows for the incorporation of modified residues, which increase the stability of the RNA. These mini-lariats, with loops much smaller than natural lariat RNA, are opened up by debranching enzyme in vitro and effective in RNAi based knockdowns in mammalian cells.
Keywords: RNA interference, micro RNA
Friday 01:30-01:45pm: RiboCAT, a new RNA probing data analysis tool: Development and application to probing secondary structure of HTLV-1 5’UTR
Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University), William Cantara (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University), Weixin Wu (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University)
Abstract:
Human T-cell leukemia virus type-1 (HTLV-1) is the only retrovirus other than HIV known to cause human disease. Despite the well-known importance of genomic RNA (gRNA) elements in other retroviruses, comparatively little is known about the structure and function of HTLV-1 gRNA. We used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of the functionally important HTLV-1 5’ untranslated region (5’UTR). To facilitate processing of the large amount of data generated by capillary electrophoresis sample analysis, RiboCAT (Ribonucleic acid Capillary-electrophoresis Analysis Tool) and RiboDOG (RiboCAT Data Output Generator) were developed as user-friendly, Microsoft Excel-based tools. Relative to the most popular current software, QuShape, these new tools significantly reduce the time required for manual intervention during data analysis. This is accomplished with improvements to the algorithms used in RNA probing data processing and through the open architecture of Excel, which allows for simple user intervention. To validate these new tools, the secondary structure of the HIV-1 5′UTR was first determined by SHAPE, matching the results of previous work (Wilkinson et al, PLoS Biol, 2008). Finally, SHAPE/RiboCAT was used to determine the secondary structure of the HTLV-1 5’UTR, revealing RNA functional elements that include a new putative dimerization initiation site, the architecture of the primer binding site, and sites of HTLV-1 matrix protein binding.
References:
Wilkinson KA, Gorelick RJ, Vasa SM, Guex N, Rein A, Mathews DH, Giddings MC, Weeks KM. 2008. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. PLoS Biol 6: e96.
Keywords: RNA Probing, SHAPE, HTLV-1
Friday 01:45-02:00pm: Structural control of cap-independent translation in Enterovirus 71
Michele Tolbert (Department of Chemistry, Case Western Reserve University), Christopher E. Morgan (Department of Chemistry, Case Western Reserve University), Mei-Ling Li (Department of Biochemistry and Molecular Biology, Rutgers Robert Wood Johnson Medical School ), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)
Abstract not available online - please check the printed booklet.
Friday 02:00-02:15pm: Title not available online - please see the printed booklet.
Eric J. Strobel (Chemical and Biological Engineering, Northwestern University, Evanston, IL), Kyle E. Watters (Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY), Angela M. Yu (Tri-Institutional Program in Computational Biology and Medicine, Cornell University, Ithaca, NY, Weill Cornell Medical College, New York, NY, Memorial Sloan-Kettering Cancer Center, New York, NY), John T. Lis (Molecular Biology and Genetics, Cornell University, Ithaca, NY), Julius B. Lucks (Chemical and Biological Engineering, Northwestern University, Evanston, IL)
Abstract not available online - please check the printed booklet.
Friday 02:15-02:30pm: Construction and Characterization of G0-G4 RNA Dendrimers with Phi29 3WJ Modules
Mario Vieweger (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA.), Ashwani Sharma (Nanobiotechnology Center; Markey Cancer Center; 3Department of Pharmaceutical Sciences; University of Kentucky, Lexington, KY 40536, USA.), Farzin Haque (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA.), Peixuan Guo (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA.)
Abstract:
We report the design and self-assembly of five generations (G0-G4) of monodisperse, globular, and nanoscale-sized RNA dendrimers using the ultrastable three-way junction (3WJ) motif of bacteriophage phi29 as building block. This series of dendrimer generations is assembled around a central, square-shaped RNA core with a 3WJ motif at each corner (G0). Incorporation of overhanging sticky-ends allows radial growth in a controlled and step-wise manner by addition of 3WJ layers up to G4. All five generations can be efficiently self-assembled using one-pot synthesis into monodisperse samples, which is a significant advantage over conventional dendrimers that typically require step-wise synthesis or produce heterogeneous particle sizes. Products can further be purified using Sucrose Gradient Ultracentrifugation to yield highly purified and homogeneous samples, as demonstrated by gel electrophoresis and AFM imaging. Information from structure prediction, AFM and DLS suggests a compact globular shape in solution with diameters ranging from 2.8 nm (G0) to 26 nm (G4). Thermal gradient fluorescence and gel electrophoresis (TGGE) reveal a bi-phasic assembly/disassembly mechanism in which the outer most layers form around a preformed thermodynamically stable core. This feature allows thermodynamic control over release and degradation of the external 3WJ strands that can be modified to contain targeting and therapeutic moieties. Upon incorporation of folate on the peripheral branches, RNA dendrimers showed high binding and internalization into cancer cells. RNA dendrimers are envisioned to have a major impact in targeting, disease therapy, molecular diagnostics and bioelectronics in the near future.
References:
1. Guo P. The emerging field of RNA nanotechnology. Nature Nanotechnology. 2010. 5:833-42.
2. Shu D, Shu Y, Haque F, Guo P. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology. 2011. 6:658-67.
3. Sharma A, Haque F, Pi F, Shlyakhtenko LS, Evers BM, Guo P. Controllable Self-assembly of RNA Dendrimers. Nanomedicine: Nanotechnology, Biology, and Medicine. 12:835–844 (2016).
Keywords: RNA Nanotechnology, dendrimers, Phi29 3WJ
Friday 02:30-02:45pm: Single molecule Förster resonance energy transfer study of labeled small molecule and G-quadruplex
Parastoo Maleki (Department of Physics, Kent State University), Y. MA (Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology), K. Iida (Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology), K. Nagasawa (Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology), Hamza Balci (department of physics, Kent State University)
Abstract not available online - please check the printed booklet.
Friday 03:15-03:30pm: RBFOX2 Controls a Developmental Splicing Switch of the Hippo Pathway Regulator Neurofibromin 2
Cole Lewis (Department of Biochemistry and Medical, University of Illinois, Urbana-Champaign), Waqar Arif (Department of Biochemistry and Medical, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry and Medical, University of Illinois, Urbana-Champaign)
Abstract:
The Hippo signaling pathway is a major regulator of cellular proliferation and organ size. Here we identify an evolutionarily conserved alternative splicing switch in Neurofibromin 2 (NF2), an upstream regulator of the Hippo pathway, during postnatal liver development. Inclusion of NF2 exon 15 (E15) increases in murine hepatocytes after birth and introduces an early stop codon, resulting in an NF2 isoform (NF2-2) with a truncated C-terminal domain (CTD). Three-dimensional structural modeling demonstrated that in contrast to the fetal isoform (NF2-1), NF2-2 is not predicted to form intramolecular interactions between the CTD and the N-terminal FERM domain. As these interactions inhibit NF2’s tumor suppressor activity, NF2-2 is expected to constitutively antagonize cellular proliferation in quiescent adult hepatocytes. To identify the cis-regulatory elements and trans-acting factors controlling this event in hepatocytes, we engineered a dual-color fluorescent reporter to monitor E15 splicing in real-time and at single-cell resolution. Computational analysis of ENCODE eCLIP data from human liver cell line revealed that RBFOX2 binds to a highly conserved region in the intron immediately downstream of E15. Systematic mutational analysis of the cluster of three RBFOX2 binding sites identified a hexamer UGCAUG sequence that drives RBFOX2-mediated NF2 E15 inclusion. Furthermore, using siRNA-knockdown and adenoviral overexpression in mouse and human hepatocytes, we demonstrate that RBFOX2 is both necessary and sufficient for producing the adult NF2-2 splice isoform. Conditional knockout of Rbfox2 gene in mouse livers resulted in near complete skipping of E15, confirming the in vivo requirement for RBFOX2 in maintaining the adult NF2 splicing pattern. Altogether, our data show that by controlling a developmental splicing switch of an upstream regulator of the Hippo signaling pathway, RBFOX2 may serve to fine-tune the proliferation potential of adult hepatocytes.
Keywords: splicing, Hippo signaling pathway
Friday 03:30-03:45pm: Stressing SMN splicing for a cure
Catey Dominguez (Center for Childhood Cancer, Ohio State University), Dawn Chandler (Center for Childhood Cancer, Ohio State University)
Abstract not available online - please check the printed booklet.
Friday 03:45-04:00pm: Title not available online - please see the printed booklet.
Jordan Powers (Biochemistry Program, St. Bonaventure University), Nikhil Gowda (Department of Biology, St. Bonaventure University), Xiao Ning Zhang (Biochemistry Program, St. Bonaventure University)
Abstract not available online - please check the printed booklet.
Friday 04:00-04:15pm: Ptbp2 regulates an alternative splicing network necessary for germ cell development
Molly M. Hannigan (Center for RNA Molecular Biology, Case Western Reserve University), Leah L. Zagore (Center for RNA Molecular Biology, Case Western Reserve University), Donny D. Licatalosi (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract not available online - please check the printed booklet.
Friday 04:15-04:30pm: A-to-I editing within the C. elegans nervous system
Sarah N. Deffit (Medical Sciences Program, Indiana University, Bloomington, IN 47405), Pranathi Vadlamani (Medical Sciences Program, Indiana University, Bloomington, IN 47405), Brian A. Lee, Emily C. Wheeler, Gene W. Yeo (Bioinformatics and Systems Biology Program, University of California, San Diego, La Jolla, CA 92093), Heather A. Hundley (Medical Sciences Program, Indiana University, Bloomington, IN 47405)
Abstract:
The conversion of adenosine (A) to inosine (I) by ADAR enzymes is one of the most prevalent forms of RNA editing, occurring in all metazoa. Inosine is a biological mimic of guanosine, and thus, A-to-I editing alters coding potential, splicing patterns and small RNA mediated gene silencing. In mammals, A-to-I editing is most abundant in the central nervous system, where a number of ion channels and receptors are edited in coding regions, thus changing amino acid sequence and protein function. These edits are important for proper neuronal function as aberrant editing of ion channels and receptors occur in a number of neuropathological diseases, including brain tumors, ALS and Alzheimers disease. Loss of ADARs in model organisms also affects neuronal function, however the substrates that are responsible for these phenotypes are unknown. To attempt to identify these substrates, we performed the first unbiased assessment of editing in the C. elegans nervous system. FACS was used to purify neural cells from which the neural transcriptome was isolated and the neural editome identified. Consistent with previous editing site identification in whole worms, most neural editing events were found in noncoding regions such as 5’ and 3’ UTRs; however, a few coding editing events were identified. Furthermore, we identified editing dependent and independent effects on gene expression. Editing of clec-41, a gene involved in the immune response and chemotaxis, altered its expression level, with neural cells deficient in editing having 75% reduced expression of clec-41. Interestingly, this was specific for neural cells as whole worm lysates deficient in editing express similar levels of clec-41 as their wildtype counterparts. Loss of ADARs is associated with reduced chemotaxis in C. elegans and our studies identified several edited targets that regulate this biological function including clec-41. Future studies assessing the role of editing in regulating these proteins may shed light onto the mechanisms resulting in chemotaxis defects in worms deficient in editing. In this study we developed a novel methodology for cell/tissue-type specific identification of editing targets, opening the door for future studies assessing tissue-specific factors that regulate A-to-I RNA editing.
Keywords: A-to-I RNA editing, ADAR, Neuron
Friday 04:30-04:45pm: A global view of RNA-protein interactions reveals novel root hair cell fate regulators
Shawn W. Foley (Department of Biology, Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA USA), Sager J. Gosai, Nur Selamoglu, Amelia C. Solitti, Fevzi Daldal (Department of Biology, University of Pennsylvania, Philadelphia, PA USA), Dorothee Staiger (Department of Molecular Cell Physiology, Bielefeld University, Bielefeld, Germany), Dongxue Wang, Roger B. Deal (Department of Biology, Emory University, Atlanta, GA USA ), Brian D. Gregory (Department of Biology, Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA USA)
Abstract:
The Arabidopsis root epidermis is comprised of two cell types: hair cells and nonhair cells, which differentiate from the same precursor cell. This cell fate decision is partially dependent on environmental cues, as root hair cells uptake both water and nutrients from the surrounding soil. Previous studies have focused on understanding the transcriptional regulators that differ between these cell types, identifying transcription factors that are necessary for hair or nonhair cell fate. Despite these advances, little is known about cell type specific post-transcriptional regulation. RNA-binding proteins (RBPs) tightly regulate all post-transcriptional regulatory processes, such as splicing, polyadenylation, stability, and microRNA biogenesis and targeting. RBPs bind to interaction sites on target RNAs via sequence and secondary structure specific interactions, thereby functioning to promote or inhibit splicing events in a cell-type specific manner. Therefore, identifying both the RBPs and RBP interaction sites that distinguish these two cell types will deepen our understanding of the post-transcriptional programs that determine root epidermal cell fate.
To address this question, we utilized our protein interaction profile sequencing (PIP-seq) technique to globally probe the RNA-protein interaction sites in root hair and nonhair cell nuclei. Using these nuclear data, we identify enriched protein-bound RNA sequences. These sequences allowed us to identified two RBPs, one that promotes and one that inhibits root hair cell fate. Follow up experiments reveal that overexpressing the root hair cell promoting RBP in plants results in an abundance of phosphate stress response genes, indicating that this protein activates the phosphate stress response pathway thereby leading to an increase in root hair cell number. In total, these data identify novel functions of two known RBPs, and illuminate an understudied aspect of plant root development.
Keywords: RNA-protein interactions, post-transcriptional regulation, Plant development
Friday 04:45-05:00pm: Alternate Exon Junction Complexes potentiate branched nonsense-mediated mRNA decay pathways
Justin Mabin (Department of Molecular Genetics, The Ohio State University), Lauren Woodward (Department of Molecular Genetics, The Ohio State University), Mengxuan Jia and Vicki Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Robert Patton (Department of Physics, The Ohio State University), Ralf Bundschuh (Department of Physics, Division of Hematology, College of Medicine, Center for RNA Biology, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University)
Abstract:
Pre-mRNA splicing deposits the exon junction complex (EJC) ~24 nucleotides upstream of most exon-exon junctions in a sequence-independent manner. The EJC core consists of eIF4AIII, Y14 and Magoh. This core stably associates with RNA and serves as an interaction platform for more dynamic peripheral EJC proteins that direct mRNA export, localization, translation and nonsense-mediated mRNA decay (NMD). To date, the specificity of the peripheral EJC proteins in target RNA selection and the resulting impact on mRNA fate remains largely unknown. We recently reported that MLN51, a protein presumed to be an EJC core factor, and several peripheral EJC proteins are sub-stoichiometric in EJCs purified from human cells. We have now discovered that MLN51 and RNPS1, two EJC proteins previously implicated in NMD, exist in two mutually exclusive stable EJCs in human and mouse cells. Biochemical and proteomic analysis of the purified MLN51 and RNPS1 complexes from HEK293 cells shows that the two alternate EJCs share the three EJC core proteins but have many unique components. Importantly, alternate EJCs associate differentially with NMD proteins Upf2 and Upf3b suggesting that these alternate complexes could lie at the root of previously documented Upf2- and/or Upf3-independent NMD branches. Surprisingly, we find that the alternate EJCs have very similar binding patterns on endogenous mRNAs including those targeted for NMD, and yet many NMD targeted mRNAs are more sensitive to RNPS1 depletion than MLN51 depletion. Taken together, our data suggests that EJCs undergo sequential remodeling from RNPS1- to MLN51-containing complexes. While both complexes are likely to be active in NMD, RNPS1-EJC may represent the early-acting NMD branch and MLN51-EJC may define the late-acting branch. These branches could also differ in Upf factor requirements. We are currently testing these and other models to understand how the unexpected EJC composition dynamics that we discovered impacts mRNA fate.
Keywords: Exon-Junction Complex, Nonsense-mediated mRNA decay, Post-transcriptional gene regulation
Saturday 08:30-08:45am: Title not available online - please see the printed booklet.
Michelle R. Gibbs (Department of Microbiology, The Ohio State University), Kyung-Mee Moon (Department of Biochemistry and Molecular Biology, University of British Columbia), Menglin Chen (Department of Microbiology, The Ohio State University), Rohan Balakrishnan (Department of Microbiology, The Ohio State University), Leonard J. Foster (Department of Biochemistry and Molecular Biology, University of British Columbia), Kurt Fredrick (Department of Microbiology, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 08:45-09:00am: The trans-acting protein Erb1 facilitates multiple remodeling events during 60S ribosomal subunit assembly
Salini Konikkat (Dept. of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA), John L. Woolford (Dept. of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA)
Abstract not available online - please check the printed booklet.
Saturday 09:00-09:15am: Mechanistic insights into RAN translation of C9orf72-associated GGGGCC repeat expansions
Katelyn M. Green (Department of Neurology, Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI), Michael G. Kearse (Department of Neurology, University of Michigan, Ann Arbor, MI; Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA), Amy Krans (Department of Neurology, University of Michigan, Ann Arbor, MI), Peter K. Todd (Department of Neurology, Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI; Veteran Administration Medical Center, Ann Arbor, MI. )
Abstract not available online - please check the printed booklet.
Saturday 09:15-09:30am: Translational recoding in Human Disease
Vivek Advani (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Mary McMahon (Department of Urology, University of California, San Francisco), Jordan Aoyama (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Joesph Briggs (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Davide Ruggero (Department of Urology, University of California, San Francisco), Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park)
Abstract:
Translational recoding has recently emerged as an important mechanism of post-transcriptional gene regulation. Prior studies have explained how Programmed -1 Ribosomal Frameshifting (-1 PRF) fits within this paradigm. Analysis of predicted -1 PRF signals across 20 genomes suggests that it is a universal mechanism(1). The overwhelming majority of “genomic” -1 PRF events are predicted to direct translating ribosomes to premature termination. These -1 PRF signals function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway(2)(3). We have explored the significance of the connection between -1 PRF and NMD on telomere maintenance in yeast(4). In published work we have shown that human mRNA encoding Ccr5p harbors a -1 PRF signal which functions as an mRNA destabilizing element through NMD(5). In the current work we are exploring how abnormalities in ribosome function, implicated in congenital and acquired syndromes, broadly classified as ‘ribosomopathies’, contribute to disease pathogenesis. Preliminary findings using X-linked Dyskeratosis Congenita (X-DC) and Spinocerebellar ataxia 26 family (SCA26) as models suggest that these genetically inherited defects result in translational fidelity defects (i.e. changes in rates of -1 PRF, +1 PRF, stop codon recognition), with attendant effects on mRNA abundance and gene expression. The observations that global dysregulation of -1 PRF has deleterious effects on gene expression leads us to hypothesize that ‘-1 PRF plays an important role in regulating cellular gene expression’. In silico analysis predicts ~10% of the genes in humans have at least one functional -1 PRF signal which translates to 1,943 high probability candidates in the human genome. Through Found in Translation (FIT) Undergraduate Research Program, we have validated -1 PRF signals encoded by over 70 human genes involved in immune signaling, cancer, anemias and other diseases.
References:
1. Belew AT et.al. PRFdb: a database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals. BMC Genomics 2008 Jan.
2. Belew AT, et.al. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Res.2010 Nov;39 2799–808.
3. Plant EP et.al. A programmed -1 ribosomal frameshift signal can function as a cis-acting mRNA destabilizing element. Nucleic Acids Res. 2004 Jan 3 32(2):784–90.
4. Advani VM et.al. Yeast telomere maintenance is globally controlled by programmed ribosomal frameshifting and the nonsense-mediated mRNA decay pathway. Transl. 2013 Apr 1
5. Belew AT et.al. Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway. Nature. 2014;512:265–9
Keywords: Translational Recoding, -1 PRF, Ribosomopathies
Saturday 09:30-09:45am: Title not available online - please see the printed booklet.
Jackson B. Trotman (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University), Andrew J. Giltmier (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University), Chandrama Mukherjee (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University), Daniel R. Schoenberg (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 09:45-10:00am: The DEAD-box protein Dhh1p couples translation elongation to mRNA decay
Ying-Hsin Chen (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 ), Sophie Martin (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 ), Aditya Radhakrishnan (Program in Molecular Biophysics, Johns Hopkins University School of Medicine), Najwa AlHusaini (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 ), Rachel Green (Program in Molecular Biophysics, Johns Hopkins University School of Medicine), Jeff Coller (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 )
Abstract:
mRNA degradation regulates the steady-state levels of mRNAs. In eukaryotic cells, the major mRNA decay pathway is initiated by removal of 3’ polyA tail, followed by 5’ decapping and exonucleolytic digestion in a 5’ to 3’ direction. Our labs recently measured the global decay rate of mRNAs and found that mRNA stability correlates well with codon usage. The transcripts with a higher ratio of optimal codons are more stable compared to those composed with non-optimal codons. We also demonstrate that codon optimality impacts ribosome movement on mRNAs. How is translation elongation coupled to mRNA decay is the subject of our current work.
The DEAD-box protein Dhh1p is a known regulator of mRNA decapping and has suggested roles in translational control. In current study we demonstrate that Dhh1p is a sensor of codon optimality- consolidating an mRNA into decay if elongation rate is slow. First, we find that mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. We find that these effects on the mRNA decay are sensitive to the concentration of Dhh1p in the cell and the number of slowly moving ribosomes on an mRNA. Second, biochemical pull-downs show that Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. Finally, over-expression of Dhh1p leads to the accumulation of ribosomes specifically on mRNAs with low codon optimality in ribosome profiling experiments.
These findings are consistent with a model that Dhh1p can bind on transcripts with slower translational elongation rate and act to promote mRNA decay.
References:
1. Vladimir Presnyak, N. A.-H., Ying-Hsin Chen, Sophie Martin, Nathan, Morris, N. K., Sara Olson, David Weinberg, Kristian E. Baker, Brenton R. & Graveley, a. J. C. Codon optimality is a major determinant of mRNA stability. Cell (2015).
2. Sweet, T., Kovalak, C. & Coller, J. The DEAD-box protein Dhh1 promotes decapping by slowing ribosome movement. PLoS Biol (2012).
Keywords: Dhh1p, mRNA decay, codon optimality
Saturday 10:00-10:15am: The CCR4-1 deadenylase plays a major role regulating malarial transmission from host to vector
Kevin J. Hart (Center for Malaria Research, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park), Elyse E. Munoz (Center for Malaria Research, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park), Michael P. Walker (Center for Malaria Research, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park), Dean Taylor (Center for Malaria Research, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park), Mark Kennedy (Center for Malaria Research, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park), Scott E. Lindner (Center for Malaria Research, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park)
Abstract:
The transmission of the malaria parasite between mosquitoes and mammals requires translational repression to ensure that only the proper proteins are expressed at the right time, while still allowing the parasite to prepare the mRNAs it will need for the next developmental stage. With relatively few known specific transcription factors (ApiAP2 family) that may specifically initiate gene transcription, Plasmodium also regulates the stability and turnover of transcripts to provide more comprehensive gene regulation. We and others have demonstrated that the parasite uses both translational repression and transcript degradation mechanisms to achieve this control. Transcript degradation in eukaryotes typically begins with the removal of the Poly-A tail by deadenylases, especially CCR4 and Caf1 that are found in the Caf1-CCR4-Not complex. We have bioinformatically identified four CCR4-domain containing proteins in the Plasmodium yoelii genome that are conserved across Plasmodium species that may provide specialized functions. Genetic deletion of PyCCR4-1, which we found associates with the Caf1-CCR4-Not complex in cytosolic granules, results in significantly fewer exflagellating male gametes and decreased transmission to the mosquito. Comparative RNA sequencing in gametocytes showed a threefold decrease in CDPK4, a known regulator of exflagellation in microgametocytes, along with a decrease in transcripts (e.g. NEK4, CelTOS, PSOP Family) that play important roles in early mosquito stage development and that match the observed phenotypes. It is clear from these data and the work of others that Plasmodium encodes specific deadenylases for the targeted degradation of specific mRNAs to promote and control facets of its development and transmission.
Keywords: CCR4, Translational Repression, Malaria
Saturday 10:45-11:00am: Translation quality control signals coordination of multiple stress responses in Saccharomyces cerevisiae
Kyle Mohler (Microbiolgy, The Ohio State University), Rebecca Mann (OSBP, The Ohio State University), Tammy Bullwinkle, Kyle Hopkins, Noah Reynolds (Microbiolgy, The Ohio State University), Michael Polymenis (Biochemistry and Biophysics, Texas A&M University), Lin Hwang, Kym Faull (Psychiatry and Biobehavioral Sciences, UCLA), Michael Ibba (Microbiolgy, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 11:00-11:15am: Title not available online - please see the printed booklet.
Katherine M. McKenney (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA), Ian M.C. Fleming (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA), Kirk W. Gaston (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, OH 45221, USA ), Pat A. Limbach (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, OH 45221, USA ), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA), Juan D. Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA)
Abstract not available online - please check the printed booklet.
Saturday 11:15-11:30am: Trypanosoma brucei RAP1 suppresses VSG switching by suppressing levels of telomeric transcript levels and RNA:DNA hybrids
Vishal Nanavaty (Biological, Geological and Environmental Sceinces, Cleveland State University), Ranjodh Sandhu (Department of Microbiology and Genetics, University of California, Davis), Sanaa Jehi (Department of Molecular and Experimental Medicine, The Scripps Research Institute), Unnati Pandya (Department of Medicine, Division of Translational Medicine, New York University School of Medicine), Bibo Li (Biological, Geological and Environmental Sceinces, Cleveland State University)
Abstract:
Telomeres are the nucleoprotein complexes present at linear chromosome ends. They protect natural chromosome ends from illegitimate repair and recombination, maintain a proper length for continued cell proliferation, and silence subtelomeric genes. Despite of having a heterochromatic structure, telomeres are transcribed into long non-coding RNAs called TERRA in many organisms. In yeast and human cells high levels of TERRA expression has been shown to increase the amount of RNA:DNA (R-loop) hybrids at the telomere, which in turn increases homologous recombination (HR) locally. It was previously shown that in Trypanosoma brucei, a parasite causing the fatal sleeping sickness in humans, telomeres are transcribed. However, T. brucei TERRA functions are unexplored. Antigenic variation is an obligatory mechanism for a long-term survival of T. brucei inside its mammalian host. T. brucei expresses VSG as its major surface antigen and regularly switches its VSG coat to evade the host immune response. Although T. brucei genome has more than 2,500 VSG genes and pseudogenes, it only expresses one VSG from one of 15 subtelomeric VSG expression sites (ESs) at any time. VSG switching can be transcriptional (in situ switch) or DNA HR-mediated (gene conversion or reciprocal DNA crossover). We previously found that T. brucei RAP1, a telomere protein, is essential for silencing subtelomeric VSGs. Here we found that a transient depletion of TbRAP1 increases the VSG switching frequency, and the majority of the switchers arose through HR-mediated VSG gene conversion. We also observed increases in TERRA levels and telomeric RNA:DNA hybrid amounts in TbiRAP1 depleted cells. R-loops are sensitive to RNAseH, which cleaves the RNA strand of the DNA:RNA hybrid. Ectopic overexpression of RNaseH1 in TbRAP1 RNAi cells resulted in reduction of the telomeric R-loops back to the WT level and suppressed the elevated VSG switching frequency phenotype. Therefore, we propose that increased TERRA and R-loop levels in TbRAP1-depleted cells mediate DSB-induced VSG gene conversion and VSG switching.
Keywords: Telomere Transcript - TERRA, RNADNA hybrids, VSG switching
Saturday 11:30-11:45am: Characterization of the human DEAD-box helicase DDX5
Zheng Cindy Xing (Biochemistry Department, Purdue University; Purdue Center for Cancer Research), Elizabeth Tran (Biochemistry Department, Purdue University; Purdue Center for Cancer Research)
Abstract:
DEAD-box proteins are a class of RNA helicases that function as RNA chaperones and RNA-protein complex (RNP) remodeling enzymes. Previous studies in our lab have revealed that the DEAD-box helicase Dbp2 in Saccharomyces cerevisiae functions in gene regulation by promoting co-transcriptional mRNP assembly and by antagonizing long non-coding RNA (lncRNA) activity (1-4). Moreover, Dbp2 is an active RNA helicase and ATPase in vitro (1, 3), suggesting that Dbp2 can modulate RNA secondary structures and remodel RNPs in vivo.
DDX5 is the mammalian ortholog of Dbp2 that plays roles in tissue differentiation and is frequently overexpressed in cancers (5). This enzyme is involved in multiple steps of RNA metabolism and transcription regulation (5), however, the mechanism of action and precise molecular roles in specific cellular processes of DDX5 remain unknown. To address these questions, we asked if DDX5 and Dbp2 are biochemically and functionally conserved. We now show that DDX5 unwinds RNA duplexes faster than Dbp2 and the mammalian specific C-terminal extension (CTE) of DDX5 is required for its higher activity. DDX5 displays slightly higher RNA binding affinity and a significantly higher on-rate than Dbp2. Moreover, DDX5 has minimal RNA annealing activities at Apo and ADP-bound state, whereas Dbp2 shows appreciable annealing activities under both conditions. Finally, DDX5 complements the overall growth and gene expression defects of dbp2∆. The mammalian/avian specific CTE of DDX5 is not required for complementation. Thus, DDX5 is an enzymatically enhanced RNA helicase that is functionally equivalent to Dbp2, suggesting a common role for this enzyme in mRNP assembly and lncRNA functions.
References:
1. Ma WK, Cloutier SC, Tran EJ. J Mol Biol 2013; 425:3824–38.
2. Ma WK, Paudel BP, Xing Z, Sabath IG, Rueda D, Tran EJ. J Mol Biol 2016; 428:1091–106.
3. Cloutier SC, Wang S, Ma WK, Petell CJ, Tran EJ. PLoS Biol 2013; 11:32–4.
4. Cloutier SC, Wang S, Ma WK, Al Husini N, Dhoondia Z, Ansari A, Pascuzzi PE, Tran EJ. Mol Cell 2016;
61:393–404.
5. Fuller-Pace FV. Biochimica et biophysica acta 2013; 1829(8):756-763.
Keywords: DEAD-box helicase, Biochemical conservation, Functional conservation
Saturday 11:45-12:00pm: Role of Argonaute as an interrogator in target RNA cleavage
Daniel Dayeh (The Ohio State University, Center for RNA Biology), Kotaro Nakanishi (The Ohio State University, Center for RNA Biology)
Abstract not available online - please check the printed booklet.
Saturday 12:00-12:20pm: Title not available online - please see the printed booklet.
Jessica A. Brown (Chemistry & Biochemistry, University of Notre Dame), Charles G. Kinzig (Molecular Biophysics & Biochemistry, Yale University/HHMI), Suzanne J. DeGregorio (Molecular Biophysics & Biochemistry, Yale University/HHMI), Joan A. Steitz (Molecular Biophysics & Biochemistry, Yale University/HHMI)
Abstract not available online - please check the printed booklet.
Saturday 12:20-12:40pm: miR-574-5p Plays a Cardioprotective Role in Heart Failure
Jiangbin Wu (Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA), Ricky Chan (Computational Biology Core, Institute for Computational Biology, Case Western Reserve University, Cleveland, OH, USA), Peng Yao (Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA)
Abstract:
Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. One major etiology of CVD is dysregulation of cardiac gene expression by transcription factors (TFs), microRNAs (miRNAs) and RNA-binding proteins (RBPs). Cardiac hypertrophy and heart failure (HF) are accompanied by changes in the expression of specific miRNAs in pathological cardiac remodelling1. miRNAs are small non-coding RNAs involved in gene expression regulation via miRNA-induced silencing complex (miRISC)2,3. TFs are usually poor small-molecule drug targets. However, miRNAs can act as unique therapeutic agents because they can target multiple TFs in the same pathological pathway. Here, we investigated the function and molecular mechanisms of miR-574-5p in cardiac hypertrophy and HF. We showed that miR-574-5p was enriched in murine HF tissues versus normal hearts. In vitro, transfection of miR-574-5p mimics in mouse cardiomyocytes (CM) protected the cells from isoproterenol (ISO, a β-adrenergic agonist)-induced hypertrophy and hypoxia-serum starvation-induced cell death. In vivo, miR-574 knockout (KO) mice exhibited an advanced cardiac hypertrophy phenotype associated with increased fibrosis and enlarged CM, compared to wild-type (WT) C57BL/6 mice after subcutaneous ISO injection and transverse aortic constriction (TAC) surgery. miRACE analysis indicated that miR-574-5p repressed mRNA translation of three pro-hypertrophic Mef2 TFs: Mef2a, Mef2c and Mef2d. Protein expression of Mef2 TFs and mRNA transcription of their downstream hypertrophic and fibrotic marker genes was significantly induced in miR-574 KO mice compared to WT mice under hypertrophic stress. In parallel, RNA-Seq analysis revealed that a pro-apoptotic RBP Mex3d was a major target down-regulated at the level of mRNA stability. Together, these findings uncover miR-574-5p as a novel cardioprotective miRNA that regulates cardiac gene expression and modulates pathological cardiac hypertrophy and remodelling in CM.
References:
1. van Rooij, E., et al. 2006. A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proceedings of the National Academy of Sciences of the United States of America 103:18255-18260.
2. Bartel, D.P. 2009. MicroRNAs: target recognition and regulatory functions. Cell 136:215-233.
3. Yao, P., Eswarappa, S.M. & Fox, P.L. 2015. Translational control mechanisms in angiogenesis and vascular biology. Current atherosclerosis reports 17:506.
Keywords: miRNA, RNA-binding protein, cardiac disease