Poster abstracts
1. Investigating the RNA-binding properties of the Larp1 La-Module
Hiba A. Al-Ashtal (Biological Sciences, University of Pittsburgh), Courtney Rubottom (Biological Sciences, University of Pittsburgh), Andrea J. Berman, PhD. (Biological Sciences, University of Pittsburgh)
Abstract not available online - please check the printed booklet.
2. Determining the Role of ADR-1 as a Key Cofactor in RNA Editing.
Haider Al-Awadi (Medical Sciences Program, Indiana University School of Medicine), Suba Rajendren (Genome, Cell and Developmental Biology Program, Indiana University), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine)
Abstract:
RNA editing is a normal cellular process in which cells make modifications to specific nucleotide sequences within an RNA molecule. The most prevalent form of RNA editing in animals is an Adenosine (A)-to-Inosine (I) base deamination. ADARs (adenosine deaminases acting on RNA) are a family of enzymes that catalyze the hydrolytic deamination of Adenosine-to-Inosine within double stranded RNA (dsRNA). A-to-I RNA editing is essential in mammals, whereas Caenorhabditis elegans lacking ADARs are viable. When RNA editing is faulty, tumors, schizophrenia, ALS, and other neurological disorders may result. My project focuses on characterizing the A-to-I editing machinery from C. elegans. Previous results from our laboratory indicated that ADR-2 is the editing enzyme in worms, but ADR-1 promotes editing by ADR-2 in vivo. ADR-1 is a double-stranded RNA binding protein that is related to the ADAR family, but lacks deamination ability. Using purified recombinant proteins, I am testing how ADR-1 affects RNA editing in vitro. Using a Thin Layer Chromatography (TLC) assay and radiolabeled ATP, editing levels can be observed and assessed in the presence and absence of recombinant ADR-1 and ADR-2 (produced in insect cells). The dsRNA substrate used is the lam-2 3’ UTR, a known C. elegans edited mRNA. TLC is a highly sensitive method for detecting Adenosine monophosphates (AMP) and Inosine monophosphates (IMP). I will determine the rate at which the deamination reaction occurs. To verify the interaction of the proteins is necessary for editing, various mutants will be used and editing levels are observed. The binding domains of ADR-1 and ADR-2 are mutated to determine the interaction of the proteins to the RNA and its effect on RNA editing. More insight behind the mechanism for RNA editing will allow us to regulate the deamination mechanism, which can lead to treatment.
Keywords: ADARs, Deamination, Celegans
3. Processing of Ribo-T rRNA ends.
Nikolay A. Aleksashin (Center for Biomolecular Sciences, University of Illinois at Chicago), Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago)
Abstract:
Correct processing of rRNA ends is one of the most important event of ribosome assembly. Ribo-T (ribosomes with tethered subunits) is built on a hybrid 16S/23S scaffold and its assembly is expected to deviate significantly from the normal ribosome biogenesis pathways. Therefore, the reason for the potential limitations of translational activity of Ribo-T could be the incorrect processing of rRNA ends. Moreover, because of the unique Ribo-T properties, this type of chimeric rRNA is an interesting model for studying the ribosome assembly. The main goal of this work is to examine the processing of the ends of Ribo-T rRNA.
By using 3’ RACE we observed that the processing of Ribo-T rRNA ends is less efficient in comparison with ribosomes with untethered subunits. A significant fraction of cellular Ribo-T carries 1-3 nt extensions at the 5’ end, whereas 3’ end of Ribo-T with the natural anti-Shine-Dalgarno (anti-SD) sequence is processed to the completion. Interestingly, altering the wild type anti-SD to a different sequence (the change required to generate an orthogonal translation system) resulted in the poorly processed 3’ ends. In this case a fraction of orthogonal Ribo-T carried 33 nt 3’ extensions.
Our results show that rRNA with significant perturbations of the structure or with unnatural sequences in a close proximity to the ends could still be processed, but less successfully.
Keywords: Ribo-T, rRNA, Processing
4. Codon optimality impacts translation rate
Najwa Alhusaini (Center for RNA Molecular Biology, Case Western Reserve University), Gavin Hanson (Center for RNA Molecular Biology, Case Western Reserve University), Jeff Coller (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract not available online - please check the printed booklet.
5. An investigation into the regulation of metK (SAM synthetase): an atypical S-box family member
Geoge M. Allen (Department of Microbiology, Center for RNA Biology, The Ohio State University), Vineeta A. Pradhan (Department of Microbiology, Center for RNA Biology, The Ohio State University), Frank J. Grundy (Department of Microbiology, Center for RNA Biology, The Ohio State University), Tina M. Henkin (Department of Microbiology, Center for RNA Biology, The Ohio State University)
Abstract not available online - please check the printed booklet.
6. Improved Non-Redundant datasets for RNA 3D Structure Annotation and Data Integration Pipeline to support RNA Science
Sri D. Appasamy (Department of Biological Sciences, Bowling Green State University), Blake Sweeney (Department of Biological Sciences, Bowling Green State University), James Roll, Craig L. Zirbel (Department of Mathematics and Statistics, Bowling Green State University), Jamie J. Cannone (Center for Computational Biology and Bioinformatics, University of Texas at Austin), Poorna Roy, Maryam Hosseini, Neocles B. Leontis (Department of Chemistry, Bowling Green State University)
Abstract:
The number and size of RNA 3D structures being deposited in the Protein Data Bank (PDB) due to advances in RNA structure determination methods and interest generated by functional diversity of RNA molecules has resulted in a pressing need for automated methods to annotate, compare and analyze these structures. We have developed and maintain an RNA 3D structure annotation pipeline in collaboration with Nucleic Acid Database (NDB), and recently overhauled it to take advantage of the new mmCIF structure format for large structures. All output is available on our website rna.bgsu.edu. One can now view annotations of basepairs and other pairwise interactions in all RNA-containing 3D structures, updated each week. The pipeline now groups together RNA structures by molecule, sequence, and geometry at the level of RNA chains to produce “equivalence classes.” We have improved the methodology for selecting a representative from each equivalence class to produce non-redundant (NR) lists of 3D structures. The NR lists are the building blocks of the RNA 3D Motif Atlas, an online database that contains automatically-classified internal and hairpin loop RNA 3D motifs. Work is in progress to integrate new visualization components and RSR values from PDB to facilitate the analysis and evaluate the reliability of the annotations in this database. Apart from these, we have developed several web-based tools for identifying and studying the conservation RNA 3D motifs in sequences. A new server called JAR3D allows users to match the sequences of RNA internal loop and hairpin loop to motif groups in the Motif Atlas, providing a mechanism for the identification of RNA 3D motifs in novel RNA sequences, even in the absence of exact sequence matches to known RNA motifs. Another tool that is closely related to RNA sequences is R3D-2-MSA, which allows the user to look up known sequence variants of nucleotides at given positions in RNA 3D structures. Collectively, these tools serve as valuable resources for investigating diverse questions regarding the relationship between RNA 3D structure and sequence.
Keywords: RNA 3D motifs, RNA 3D structure annotation pipeline, non-redundant (NR) list of 3D structures
7. HEPATOCYTE-SPECIFIC DELETION OF A SPLICING REGULATORY PROTEIN CAUSES SPONTANEOUS AND SEVERE NONALCOHOLIC STEATOHEPATITIS IN MICE
Waqar Arif (University of Illinois, Urbana-Champaign), Suthat Liangpunsakul (University of Illinois, Urbana-Champaign), Sarath Janga (University of Illinois, Urbana-Champaign), Auinash Kalsotra (University of Illinois, Urbana-Champaign)
Abstract not available online - please check the printed booklet.
8. Functional and structural basis for selective substrate binding by the exonuclease Rrp6p
Armend Axhemi (Center for RNA Molecular Biology & Department of Biochemistry, Case Western Reserve University, Cleveland OH), Deepak Sharma, Sukanya Srinivasan, Ulf-Peter Guenther (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland OH), Elizabeth V. Wasmuth (Structural Biology Program & Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Sloan Kettering Institute, New York, NY ), Christopher D. Lima (Structural Biology Program & Howard Hughes Medical Institute, Sloan Kettering Institute, New York, NY ), Eckhard Jankowsky (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland OH)
Abstract not available online - please check the printed booklet.
9. G-quadruplex driven functional modulation in the human piwiinteracting RNAs
Sumirtha Balaratnam (Department of Chemistry and Biochemistry, Kent State University), MadaraHettiArachchilage (Department of Biological Science,Kent State University,)
Abstract:
The piwi interacting RNAs (piRNAs) are small non-coding RNAs mostly 24-32 nucleotides in length. Among all types of non-coding RNAs, piRNAs are by far the most numerous, existing only in animals. The piRNAs are defined by their specific binding to the PIWI proteins, a requirement for their function. The piRNAs do not have conserved secondary structure, because piRNA sequences are not known to contain any conserved motifs. Using bioinformatics analysis, we discovered the presence of putative G-quadruplex (GQ)-forming sequences in human piRNAs that is higher in number compared to the piRNA pools of other organisms that were analyzed (exclude Chicken). We investigated the propensity to form the G-quadruplex structure in one (piRNA 48164) of the potential GQ forming sequence in human. Circular dichroism and RNase T1 footprinting data confirmed the formation of stable G-quadruplex structure by piRNA 48164. GQ formation led to inhibiting the piRNA-target complementary base pairing which consequently affects target gene silencing. Further studies are underway to analyze the effect of PIWI protein binding on GQ forming piRNAs. These studies begin to unravel the role of GQ in piRNA function.
References:
PengZhang, et al, piRBase: a web resource assisting piRNA functional study, Database, 2014, 1–7
Oleg Kikin,et al, QGRS Mapper: a web-based server for predicting G-quadruplexesin nucleotide sequences, Nucleic Acids Res, 2006,676-682.
MikikoC. Siomi, et al, PIWI-interacting small RNAs: the vanguard of genome defence, Nat Rev Mol Cell Bio,2011,246-258
Keywords: piRNAs, G-quadruplex, function modulation
10. Several IFN-induced long noncoding RNAs regulate the antiviral response
Marina Barriocanal (Department of Gene Therapy and Regulation of Gene Expression, CIMA, University of Navarra), Victor Segura (Department of Bioinformatics, CIMA), Saba Valadkhan (Department of Molecular Biology and Microbiology, CWRU), Puri Fortes (Department of Gene Therapy and Regulation of Gene Expression, CIMA, University of Navarra)
Abstract:
Long noncoding RNAs (lncRNAs) have been described to have important roles in several processes such as cell differentiation and proliferation among others. However, the antiviral role of lncRNAs has remained largely unknown. We have identified several Interferon (IFN) Stimulated long noncoding RNAs (lncRNAs), that we call ISRs, by transcriptome analysis of cells treated or not with IFN.
Interestingly, some of these ISRs are located in the genome close to IFN stimulated genes (ISGs). lncBST2/BISPR, ISR12 and ISR8 are located close to BST2/tetherin, IL6 and IRF1 respectively. Downregulation of BISPR causes a decrease in the levels of BST2, indicating that BISPR is a positive regulator of BST2 expression. ISR12 seems to be a negative regulator of the expression of the distant ISG GBP1. Depletion of ISR12 does not affect its neighboring gene IL6. Instead, it leads to increased levels of the IFN induced gene GBP1 and, therefore, to decreased replication of Hepatitis C virus (HCV) in hepatocellular cell lines. Finally, clones with altered ISR8 expression obtained using the CRISPR-Cas system do not establish a successful IFN response. They fail to induce expression of several ISGs regulated by IRF1. This suggests that ISR8 works as a positive regulator of the IFN pathway.
In summary, our results show that lncRNAs induced after IFN treatment may function as positive or negative regulators of the IFN response. Molecular mechanism of the ISR-mediatd regulation of IFN response is currently under investigation.
Keywords: lncRNAs, IFN
11. Using high-throughput microscopy coupled with machine learning to decipher regulatory pathways for the RNA helicase Dbp2
Zachary T. Beck (Department of Biochemistry, Purdue University), Ben Grys (Department of Molecular Genetics, University of Toronto), Brenda Andrews (Department of Molecular Genetics, University of Toronto), Elizabeth J. Tran (Department of Biochemistry, Purdue University)
Abstract:
The ability of an organism to sense the current status of available nutrients is key to survival and fecundity. Responses to changes in the environment require highly specific and coordinated alterations in gene expression. Dbp2 is a bona fide ATP-dependent RNA helicase in Saccharomyces cerevisiae that associates directly with actively transcribed chromatin to promote co-transcriptional packaging and processing of mRNAs and ribosomal RNAs.1,2 Under normal growth conditions when carbon sources are readily available, Dbp2 is concentrated in the nucleus. However, when carbon sources are limited, such as during glucose deprivation, Dbp2 is rapidly relocalized to cytoplasm.3 Utilizing high-throughput confocal microscopy (HTC) of a synthetic gene array (SGA), we were able to track the localization of Dbp2 in response to +/- glucose conditions in over 3000 mutant yeast strains. Images were analyzed with CellProfiler to quantitatively determine the localization of Dbp2 in relation to reference markers in the cytoplasm and nucleus. Known genetic and physical interaction data of genes identified as hits from the screen were obtained from the Saccharomyces Genome Database. This list was then compiled into interaction pathways using Cytoscape to construct a visual representation of the glucose responsive network for Dbp2. Interestingly, our analyses show three general cellular processes associated with glucose-dependent Dbp2 localization: kinase signaling, sugar metabolism, and ribosome assembly. We are in the process of independently validating the results and constructing a model of nutrition-based environmental sensing. The glucose-dependent redistribution of Dbp2 represents the first time a DEAD-box helicase has been tied directly to sensing extracellular carbon source cues. Importantly, the human counterpart of Dbp2, termed hDDX5, has recently been linked to normal glucose metabolism, a process that is drastically altered in cancer cells. Thus, our studies, have direct implications to our understanding of DDX5 and mechanisms which promote tumor cell metabolism.
References:
1. Cloutier SC, Ma WK, Nguyen LT, Tran EJ. The DEAD-box RNA helicase Dbp2 connects RNA quality control with repression of aberrant transcription. J Biol Chem 2012; 287:26155–66.
2. Ma WK, Cloutier SC, Tran EJ. The DEAD-box protein Dbp2 functions with the RNA-binding protein Yra1 to promote mRNP assembly. J Mol Biol 2013; 425:3824–38.
3. Beck ZT, Cloutier SC, Schipma MJ, Petell CJ, Ma WK, Tran EJ. Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae. Genetics 2014; 198:1001–14.
Keywords: Dbp2, helicase, metabolism
12. Identification and characterization of S-box riboswitches that regulate at the level of translation initiation
Divyaa Bhagdikar (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus, OH 43210 ), Frank J. Grundy (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus, OH 43210 ), Tina M. Henkin (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus, OH 43210 )
Abstract not available online - please check the printed booklet.
13. Activation of early postnatal splicing program by ESRP2 is essential for liver regeneration.
Amruta Bhate (Biochemistry, University of Illinois Urbana-Champaign), Joe Seimetz, Waqar Arif, Sushant Bangru, Kevin Yum, Edrees Rashan (Biochemistry, University of Illinois Urbana-Champaign), Sayeepriyadarshini Anakk (Molecular and Integrative Physiology, University of Illinois Urbana-Champaign), Russ. P. Carstens (Medicine and Genetics, University of Pennsylvania), Auinash Kalsotra (Biochemistry and Medical Biochemistry, University of Illinois Urbana-Champaign)
Abstract:
The mammalian liver has the unique capacity to undergo robust regeneration in response to damage. This regenerative capacity was previously attributed to facultative stem cell populations, but several recent lineage-tracing studies have shown that virtually all new hepatocytes are derived from pre-existing ones. However, the underlying principles that drive reprogramming of a quiescent hepatocyte into proliferative state remain elusive. We performed high-resolution RNA-sequencing of quiescent and toxin-injured mouse livers, identifying extensive remodeling of the hepatic transcriptome: including genes related to cell cycle regulation, proliferation, RNA modification, and metabolic processes. Surprisingly, the remodeling process is accompanied by numerous splice isoform transitions, a majority of which switch to an early postnatal pattern, suggesting hepatocytes reprogram their transcriptome towards an immature state to become proliferative. Furthermore, we demonstrate that dynamic regulation of Epithelial splicing regulatory protein 2 (ESRP2) upon toxin-injury is essential for activating the early postnatal splicing program within hepatocytes. ESRP2 protein levels decline during the injury phase but are fully restored upon toxin removal. The livers of ESRP2 knockout mice, compared to controls, exhibit excessive hepatocyte proliferation upon injury, but are unable to return to a physiologically mature state following recovery. Remarkably, forced expression of ESRP2 during toxin exposure blocks the hepatocyte proliferation and results in premature death. We conclude that after sustaining damage, an early postnatal splicing regulatory network is redeployed through dynamic regulation of ESRP2 within adult hepatocytes to facilitate liver regeneration.
Keywords: ESRP2, liver regeneration, alternative splicing
14. Three major remodeling events within assembling ribosomes are required for irreversible removal of a pre-rRNA spacer sequence
Stephanie Biedka (Department of Biological Sciences, Carnegie Mellon University), Jelena Jakovljevic (Department of Biological Sciences, Carnegie Mellon University), Shan Wu (School of Life Sciences, Tsinghua University), Ning Gao (School of Life Sciences, Tsinghua University), John Woolford (Department of Biological Sciences, Carnegie Mellon University)
Abstract:
Three interdependent processes drive ribosome biosynthesis in vivo: pre-rRNA folding and processing and binding of ribosomal proteins to the pre-rRNA. In yeast, ribosome assembly is facilitated by the actions of ~200 protein assembly factors. Assembly is hierarchical and progresses through establishment and restructuring of RNP interaction networks. A major goal at this time is to establish what these networks are, how they are remodeled, and how their reconstruction drives assembly forward in an efficient and accurate manner. We have focused on the events required for one particular remodeling event- irreversible removal of the ITS2 spacer RNA from the pre-60S subunit. Our approach has been to isolate mutants that cannot carry out this pre-rRNA processing event and assay effects of these mutations on pre-ribosome protein composition. Critical to our analysis was our recent determination of near atomic resolution cryo-EM structures of three consecutive late nuclear pre-60S subunit assembly intermediates. From these structures we were able to identify RNP networks affected in these mutants. Based on their mutant pre-rRNA processing phenotype, ~30 ribosomal proteins and assembly factors are known to be required for cleavage at the C2 site to initiate removal of ITS2. Analysis of these mutants revealed three major remodeling events that are necessary for C2 cleavage. (1) A group of assembly factors bound proximal to ITS2 must be released. (2) Several assembly factors that bind to the nascent peptidyl transferase center must associate with the pre-60S subunit. (3) Seven ribosomal proteins surrounding the exit of the polypeptide exit tunnel must become stably associated with the pre-60S subunit. We believe that these three events reveal the existence of a complicated checkpoint that prevents pre-60S subunits with improperly structured functional centers from undergoing the irreversible step of ITS2 removal.
Keywords: ribosome assembly, pre-ribosome remodeling, pre-rRNA processing
15. Title not available online - please see the printed booklet.
Abigail Bieker (Biology, Lewis University), Elizabeth Przekwas (Biology, Lewis University), Mallory Havens (Biology, Lewis University)
Abstract not available online - please check the printed booklet.
16. Characterization of U5NU Binding by Vaccinia Capping Enzyme
Rachel Boldt (SUNY Buffalo), Dr. Paul Gollnick (SUNY Buffalo)
Abstract not available online - please check the printed booklet.
17. Azido Modified NTP as chemical handles for multiplex analysis of tRNA digestion products
Kayla Borland (Chemistry UC), Pat Limbach (Chemistry UC)
Abstract:
Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the gold standard in transfer ribonucleic acid (tRNA) modification identification and mapping. LC-MS/MS can provide both qualitative and quantitative data depending on sample preparation conditions. A common practice in LC-MS/MS in the field of proteomics is the use of multiplexing, which allows multiple biological samples to be analyzed simultaneously. Previously, our group demonstrated multiplexed LC-MS/MS analysis for tRNA modification mapping using 16O/18O-labeled water to differentiate biological samples. The strategy is limited to binary sample mixtures and the 2 Da label difference can often lead to confusion in sample identification for multiply charged digestion products.
To overcome these limitations, we are exploring the use of poly adenosine polymerase (PAP), which – under optimized conditions – can add one 2’ azido modified nucleotide to the 3’-terminus of tRNA digest products. The addition of this azido-modified nucleotide can allow for the use of click chemistry to uniquely tag each sample. The mass shift of each sample is much larger than that produced from 18O labeling depending on the tag added to each biological sample. Here we report preliminary studies focused on optimized click reaction conditions for tag addition to azido modified incorporated substrate digestion products.
Keywords: azido modified dNTP, poly adenosine polymerase
18. Using SELEX to Identify Aptamers That Bind To E. coli for Biosensor Development
Hailey Bowen (Department of Chemistry SIUE), Nicolette Geron (Department of Chemistry SIUE), Natalie Schleper (Department of Chemistry SIUE), Eric Bomba (Department of Chemistry SIUE), Mina Sumita (Department of Chemistry SIUE)
Abstract not available online - please check the printed booklet.
19. Title not available online - please see the printed booklet.
Kara M. Braunreiter (Molecular Genetics, The Ohio State University), Madeline Parker (Molecular Genetics, The Ohio State University), Dr. Susan E. Cole (Molecular Genetics, The Ohio State University)
Abstract not available online - please check the printed booklet.
20. Versatile RNA Tetra-U Helix Linking Motif as a Toolkit for Nucleic Acid Nanotechnology
My N. Bui (Department of Chemistry, Ball State University ), Emil F. Khisamutdinov (Department of Chemistry, Ball State University )
Abstract:
Remarkable molecular recognition, structural, and functional properties of RNA molecules make them one of the most promising materials to pattern matter with nanoscale accuracy. RNA nanotechnology implies this molecule to engineer addressable nanostructures in one, two, and three dimensions. Resulting RNA nano-scaffolds have been used to precisely position functional moieties including siRNAs, RNA aptamer, ribozymes, proteins, fluorophores into deliberately designed patterns. Although the RNA nanotechnology field is emerging, the notion of production of RNA nanoparticles is no longer novel and despite all advantages, the RNA biomaterial is fragile and prone to enzymatic degradation and hydrolysis. This makes believe that RNA is not particularly convenient molecule to handle in laboratory settings. In the current report, we demonstrate computer assisted de-novo design and application of versatile RNA tetra-uracil (tetra-U) motif as a toolkit to overcome some major obstacles in RNA nanotechnology. We demonstrate that the tetra-U RNA module can be implemented to construct various nanoparticles (NPs) ranging from equilateral triangle, rectangle, pentagon, and hexagon shapes. In particular, we have extensively studied triangular nano-scaffold and demonstrated that the complex can be readily prepared from RNA, DNA or a mixture of RNA and DNA strands representing the power of simplicity of the RNA tetra-U (DNA tetra-T) motif. The fabrication of hybrid nanostructures offers multiple benefits including fine-tunable stability in blood serum, thermodynamic stability, immunostimulatory activity and notably, reduced total cost of production compared to RNA and modified RNA counterparts. The hybrid NPs remained functional as conjugating the triangles with siRNA strands targeting GFP shows significant knock-down of protein expression in human breast cancer cells. The reported work paves the way for the exploration of economically favorable RNA tetra-U toolkit for construction of fine-tunable hybrid RNA/DNA nanostructures to fulfill the needs of the rapidly developing field of RNA nanotechnology.
Keywords: RNA 3D motif, Bionanotechnology, RNA nanoparticles
21. Synthesis and Characterization of Nucleic Acid Aptamers Targeted at Aspergillus Surface Carbohydrates
Jessica Bush (Department of Chemistry, Albion College), Megan Sheridan (Department of Chemistry, Albion College), David Engelke (Department of Chemistry, University of Michigan ), Christopher Rohlman (Department of Chemistry, Albion College)
Abstract:
Aspergillus is a common fungus found naturally throughout the world. Normally these spores are inhaled and processed within the body without any negative consequences, however prolonged exposure to high quantities of Aspergillus can cause allergic or toxic symptoms and infection in immuno-suppressed individuals (Hummel et al., 2006). Traditional detection methods for Aspergillus are difficult and invasive including biopsies of cerebral lesions and extraction of cerebrospinal fluid, and frequently yield negative results (Hummel et al., 2006). The high specificity and affinity binding capabilities of aptamers make them a potent diagnostic and therapeutic tool. Aptamers can be selected to target and identify a wide variety of infectious diseases (McKeague and DeRose, 2012). By selecting for specific nucleic acid aptamers and fluorescently labeling these molecules, we aim to develop a new detection method for Aspergillosis that is more sensitive and less invasive. As a means for developing and evaluating the selection process several carbohydrate targets were studied. DNA aptamers were synthesized by binding a randomized N15 DNA sequence to cassettes to target carbohydrates. Once bound washing processes eliminated nonbinding DNA segments followed by an elution process and asymmetric PCR to produce a single stranded pool of aptamer candidates for the next round of selection. Mobility shift assays have also been used to further refine the aptamer pool. In parallel, F-modified RNA aptamers targeted at the Aspergillus cell surface carbohydrate beta-D-glucan were selected from an N40 randomized template pool. RNA aptamers were generated using in vitro transcription of the template pool utilizing F-NTPs. Successful aptamers were selected through nine successive rounds of binding to beta-D-glucan, reverse transcription, and cDNA template pool amplification. Binding characteristics as well as characterization of the aptamer pool sequence diversity are currently in progress.
References:
Challenges and Opportunities for Small Molecule Aptamer Development
Maureen McKeague and Maria C. DeRosa (2012) Journal of Nucleic Acids volume 2012 doi:10.1155/2012/748913
Detection of Aspergillus DNA in Cerebrospinal Fluid from Patients with Cerebral Aspergillosis by a Nested PCR Assay M. Hummel, B. Spiess, K. Kentouche, S. Niggemann, C. Bohm, S. Reuter, M. Kiehl, H. Morz, R. Hehlmann, and D. Buchheidt (2006) Journal of Clinical Microbiol 44(11): 3989–3993.
Keywords: Aptamer, Carbohydrate
22. Role of Unique C-terminal Domain in an Aminoacyl-tRNA Trans-editing Protein from Arabidopsis thaliana
Jun-Kyu Byun (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University), Marina Bakhtina (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University)
Abstract:
Aminoacyl-tRNA synthetases (ARSs) play a central role in maintaining high fidelity of protein synthesis, which is essential for cell viability. Due to the structural similarity of amino acids, some ARSs are error prone and mispair non-cognate amino acids with specific tRNA. Thus, to prevent mistranslation, quality control mechanisms are required. Prolyl-tRNA synthetase (ProRS) can misactivate and mischarge noncognate alanine to form Ala-tRNA-Pro. Some bacterial ProRSs possess an editing domain that can deacylate Ala-tRNA-Pro, but some bacteria and all eukaryotes lack this editing domain in the context of ProRS. Instead, a free-standing editing domain homolog, ProXp-ala, is encoded in many organisms and has been shown to hydrolyze Ala-tRNA-Pro in trans. Here, we report that ProXp-ala from plants has a unique C-terminal extension domain that is missing from bacterial and higher eukaryotic ProXp-ala domains. To determine the function of the C-terminal extension in plant ProXp-ala, we designed and purified a truncated Arabidopsis thaliana ProXp-ala variant (At Delta;C-ProXp-ala). Circular dichroism spectroscopy showed that the secondary structure of ProXp-ala does not appear to be perturbed upon truncation. In vitro deacylation of Ala-tRNA-Proby both wild-type (WT) and At Delta;C-ProXp-ala showed that the truncation resulted in an 15-fold decrease in the deacylation rate. To establish whether this was primarily due to a binding defect, deacylation assays were performed with increasing concentrations of ProXp-ala. Based on the recovery of near WT levels of ProXp-ala deacylation with increasing amounts of the truncated construct, we conclude that the C-terminal domain in plant ProXp-ala has a critical role in binding the tRNA substrate. Future studies will focus on identifying the tRNA elements that are recognized by the unique C-terminal extension of plant ProXp-ala.
References:
1. Das, Mom, et al. "Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation." Nucleic acids research 42.6 (2014): 3943-3953.
2. Vargas-Rodriguez, Oscar, and Karin Musier-Forsyth. "Exclusive use of trans-editing domains prevents proline mistranslation." Journal of Biological Chemistry 288.20 (2013): 14391-14399.
Keywords: trans-editing , aminoacyl-tRNA synthetase, ProXp-ala
23. Title not available online - please see the printed booklet.
Susan Campbell, Yueting Xu, Conner Bardine, Austin Cosgrove (Biochemistry Department at Allegheny College), Ivelitza Garcia (Chemistry Department at Allegheny College)
Abstract not available online - please check the printed booklet.
24. A genome-wide alternative polyadenylation and gene expression analysis provides molecular insights into the abiotic stress response in sorghum
Manohar Chakrabarti (Dept. of Plant and Soil Sciences, University of Kentucky ), Laura de Lorenzo (Dept. of Plant and Soil Sciences, University of Kentucky ), Salah E. Abdel-Ghany (Department of Biology, Colorado State University), Anireddy S.N. Reddy (Department of Biology, Colorado State University), Arthur G. Hunt (Dept. of Plant and Soil Sciences, University of Kentucky )
Abstract:
Sorghum [Sorghum bicolor (L.) Moench] is a major cereal and bioenergy crop and is grown widely in some of the harshest agro-climatic regions in the world, such as in South Asia and Sub-Saharan Africa. In spite of its importance, not much is known about the transcriptional and post-transcriptional regulation of abiotic stress responses in sorghum. Recent studies have unearthed the role of alternative polyadenylation (APA) in diverse developmental and physiological processes by virtue of its effects on gene expression, mRNA stability, translatability and subcellular localization. Under this backdrop, we have undertaken a genome-wide APA and gene expression analysis in sorghum seedlings that have been subjected to several abiotic stresses. The genome-wide APA analysis revealed that under different stress treatments, the relative levels of mRNA isoforms derived from APA at locations within protein-coding regions and 5΄UTRs increased significantly, and isoforms derived from polyadenylation within introns increased slightly. Conversely, the levels of mRNA isoforms with poly (A) sites in annotated 3΄-UTRs decreased significantly in stressed plants compared with controls. Our analysis also identified poly (A) clusters and genes depicting APA under abiotic stresses. A comparison of gene expression analysis using PATs, mapped to the ‘gene’ with those mapped only to the ‘3΄UTR’, suggested that there can be false positives among the differentially expressed genes from the ‘gene’ dataset, which led us to perform gene expression analysis with the PATs, mapped only to the ‘3΄UTR’ to generate a catalog of functional gene expression. The gene expression analysis revealed that sorghum responds faster to the salinity and heat stress as compared to the drought stress, however, at prolonged exposure all three stresses impacted gene expression significantly. Interestingly, prolonged drought and salt treatments led to a significant down regulation of several core histone genes. Altogether, these analyses suggested a novel possible mode of epigenetic control of abiotic stress responses, and they also revealed a widespread role for APA in regulating abiotic stress responses in this important cereal crop.
Keywords: Sorghum, abiotic stress, alternative polyadenylation
25. Structural studies on the Salmonella enterica htrA RNA thermometer using SHAPE analysis
Edric K. Choi (Department of Chemistry and Biochemistry, Denison University), Rachel M. Mitton-Fry (Department of Chemistry and Biochemistry, Denison University)
Abstract not available online - please check the printed booklet.
26. Investigating How Repressive Chromatin Modification Is Initiated at Transposons
Sarah G. Choudury (Department of Molecular Genetics, The Ohio State University), Andrea D. McCue (Department of Molecular Genetics, The Ohio State University), Alissa Cullen (Department of Molecular Genetics, The Ohio State University), R. Keith Slotkin (Department of Molecular Genetics, The Ohio State University)
Abstract not available online - please check the printed booklet.
27. Multi-color high-resolution fleezers development and measurement of RNA polymerase transcription
Cho-Ying Chuang (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48864, USA), Matthew Zammit (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48864, USA), Abhishek Mazumder (Waksman Institute and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA), Richard H. Ebright (Waksman Institute and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA), Matthew J. Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48864, USA)
Abstract:
Here we present developments in multi-color high-resolution fleezers instrumentation and methods. We have developed a high-resolution fleezers instrument with three-color confocal fluorescence microscopy. To reduce photobleaching due to traps, timeshared dual optical traps were interlaced and synchronized with three fiber coupled fluorescence excitation lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting fluorescence detectors. We show a number of new fluorescence detection schemes useful both for measurement and instrument alignment including: We have developed a fleezers compatible method to image inorganic fluorescent quantum dots. Dramatically reduced photobleaching compared to organic fluorophores allows fleezers imaging and precise fluorophore localization. To remove the fluorescence background from beads, we combined a multi-channel flow chamber and internal hairpin binding construct to perform 'cryptic binding' experiments. We have measured fluorophore photobleaching lifetimes with a set of fluorophores for a variety of excitation powers in a range of buffer conditions. Finally, we present preliminary RNA polymerase transcription and fluorescence measurements. We show that the high-resolution fleezers instrument accurately measures transcription for greater than 1 kb with no difficulties. We discuss fluorescence challenges.
References:
Comstock MJ, et al. (2015) Direct observation of structure-function relationship in a nucleic acid-processing enzyme. Science 348(6232):352–354
Keywords: RNA polymerase, single molecule fluorescence, optical trapping
28. High resolution mapping and dynamic visualization of R-loops
Sara Cloutier (Department of Biochemistry, Purdue University), Elizabeth Tran (Department of Biochemistry, Purdue University; Purdue University Center for Cancer Research)
Abstract not available online - please check the printed booklet.
29. A novel mouse model of rhabdomyosarcoma underscores the dichotomy of splice variant MDM2-ALT1 function in vivo
Daniel Comiskey, Aishwarya Jacob (Molecular, Cellular, & Developmental Biology, The Ohio State University), Brianne Sanford, Andrew Goodwin (Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital), Aixa S. Tapia-Santos, Tom Bebee, Matas Montes (Molecular, Cellular, & Developmental Biology, The Ohio State University), Haley Steiner, Jessica Grieves, Prosper Boyaka, Krista La Perle (Department of Veterinary Biosciences, The Ohio State University), Dawn Chandler (Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital)
Abstract not available online - please check the printed booklet.
30. Genome-wide analysis of alternative polyadenylation landscapes modulated during plant-pathogen interactions in Arabidopsis thaliana
Laura de Lorenzo (Department of Plant and Soil Sciences. University of Kentucky), Gah-Hyun Lim (Department of Plant Pathology. University of Kentucky), Pradeep Kachroo (Department of Plant Pathology. University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences. University of Kentucky)
Abstract:
In the past few years, several studies have highlighted significant roles of alternative polyadenylation (APA) in the control of physiological events such as cellular proliferation, differentiation, transformation, and organ development. This is true for APA events that lead to non-canonical mRNA isoforms (APA sites located in intron, 5’-UTR and coding regions), and affect the transcripts and their translation products. We hypothesized that alternative polyadenylation might also play an equally important role in regulation of host gene expression post pathogen infection in Arabidopsis. Using poly(A) site analysis coupled to high throughput sequencing, we examined polyadenylation events after bacterial, fungal and viral infections. This included both virulent and avirulent combination of bacterial [Pseudomonas syringae (Pst)] and viral [Turnip Crinkle Virus (TCV)] pathogens and a virulent isolate of necrotrophic fungal pathogen Botrytis cinerea. We found that virulent Pst and TCV infections led to an increase in the usage of non-canonical mRNA isoforms. Interestingly, avirulent response to Pst and TCV showed a general decrease in the production of non-canonical mRNA isoforms. Moreover, although APA profile changes after virulent infections were mainly associated with genes implicated in biotic and abiotic stress responses and electron transport, avirulent infections led to changes in genes encoding for receptor proteins and those implicated in signal transduction. Surprisingly, poly(A) site usage after Botrytis infection overlapped with that of the avirulent responses. A plasticity in transcription factors in APA genes expressed in any pathogen condition was also found. Together, these findings suggest that APA is induced in response to pathogen infection and might play an important role in fine-tuning of transcripts contributing to pathogen response.
References:
Supported by NSF Award 1243849
Keywords: Alternative polyadenylation, High throughput sequencing, Biotic stress
31. Impact of Fhit on lung cancer linked mRNAs
Daniel L. Kiss (Center for RNA Biology, Department of Biological Chemistry and Pharmacology, The Ohio State University), Daniel del Valle-Morales (Center for RNA Biology, Department of Biological Chemistry and Pharmacology, The Ohio State University), Stefano Volinia (Department of Morphology, Surgery, and Experimental Medicine, University Ferrara), Jenna Karas, Kay Huebner (Department of Cancer Biology and Genetics, The Ohio State University), William Baez, Ralf Bundschuh (Center for RNA Biology, Department of Physics, Department of Chemistry and Biochemistry, 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.
32. Characterization of a putative R2 retrotransposon in the vertebrates Lampetra aepyptera and Lethenteron appendix.
Rex Meade Strange (Biology Department, University of Southern Indiana), L. Peyton Russelburg (Molecular Biology, University of Utah), Kimberly J. Delaney (Biology Department, University of Southern Indiana)
Abstract:
R2 retrotransposons are a unique class of transposon that move via an RNA intermediate through eukaryotic genomes. These elements have an exquisitely specific genomic location: they are only capable of inserting themselves in the middle of the 28S rDNA gene, thus providing thousands of potential genomic insertion points. The R2 gene is transcribed as part of the larger rDNA cassette and cleaves itself from the pre-rRNA transcript via a ribozyme encoded in its 5’UTR. R2 retroelements have been extensively characterized in arthropods, but there has been almost no description of R2 elements in vertebrate genomes. We have putatively identified an R2 retrotransposon in the genomes of the lamprey species Lampetra aepyptera and Lethenteron appendix. We are in the process of characterizing these putative transposons via sequencing, locus quantification via qPCR, and a measure of transposition activity via 5’ end profiling. Characterization of this gene will lead to biochemical analysis of the structure and function of the R2 protein in vertebrates as well as a look at the phylogenetic conservation of the HSV-like ribozyme that regulates it’s cleavage from the rRNA transcript and thus plays an important role in transposition.
Keywords: retrotransposon, ribozyme, R2
33. The Emerging Landscape of Small, Noncoding RNA Processing
Samantha Dodbele (Ohio State Biochemistry Program, The Ohio State University; Center for RNA Biology, The Ohio State University, Department of Chemistry and Biochemistry, The Ohio State University), Blythe Moreland (Center for RNA Biology, The Ohio State University; Department of Physics, The Ohio State University), Yicheng Long (Ohio State Biochemistry Program, The Ohio State University; Center for RNA Biology, The Ohio State University, Department of Chemistry and Biochemistry, The Ohio State University), Spencer Gardner (Department of Chemistry and Biochemistry, The Ohio State University), Ralf Bundschuh (Center for RNA Biology, The Ohio State University; Department of Chemistry and Biochemistry, The Ohio State University; Department of Physics, The Ohio State University), Jane Jackman (Ohio State Biochemistry Program, The Ohio State University; Center for RNA Biology, The Ohio State University, Department of Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the printed booklet.
34. Mechanism of Human Lysyl-tRNA Synthetase/tRNALys Primer Recruitment and Packaging into HIV-1
Alice Duchon (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State Un), Nathan Titkemeier (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State Un), Corine St. Gelais (Department of Veterinary Biosciences, Center for RNA Biology, Center for Retroviral Research, The Ohio State University), Li Wu (Department of Veterinary Biosciences, Center for RNA Biology, Center for Retroviral Research, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State Un)
Abstract:
The primer for reverse transcription in HIV-1, human tRNALys3, is selectively packaged into virions along with tRNALys1,2. We have previously reported that human lysyl-tRNA synthetase (hLysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also packaged into HIV-1 and the presence of both host cell factors is required for optimal viral infectivity. How the hLysRS/tRNALys complex is diverted from its normal cellular location into HIV-1 particles is unknown. hLysRS is normally part of a dynamic mammalian multisynthetase complex (MSC). In recent years, hLysRS has been shown to be mobilized from the MSC and to function in a wide variety of non-translational pathways that involve nuclear localization, membrane binding, and secretion. Here, we show that the expression of hLysRS is unaltered upon HIV-1 infection of HEK293T and CD4+ HuT/CCR5+ T cells, suggesting that the hLysRS species packaged is recruited from an existing pool of LysRS. Immunofluorescence and deconvolution microscopy imaging reveal that hLysRS is released from the MSC and relocalized to the nucleus upon HIV-1 infection. Pretreatment of cells with a MEK inhibitor known to block specific phosphorylation of LysRS and release from the MSC, results in reduced nuclear localization and importantly, reduced HIV-1 infectivity. Knockdown of endogenous LysRS resulted in a reduction of progeny virion infectivity while rescue of infectivity was achieved with a phosphomemetic mutant. Taken together, these studies shed light on the mechanism of tRNALys3 primer recruitment into HIV-1 and suggest a new avenue for targeting a host cell factor that is essential for HIV-1 infection.
Keywords: HIV, LysRS, tRNA
35. Mass spectrometryanalysis of the Paf1 Complex in rtr1 yeast
K. Nichole Dyer (Biology Department, DePauw University ), Lynn M Bedard (Biology Department, DePauw University), Amber L. Mosley (Biochemistry Department, Indiana University School of Medicine)
Abstract not available online - please check the printed booklet.
36. Title not available online - please see the printed booklet.
Jey Sabith Ebron, Jagjit Singh (Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA.), Eswar Shankar (Department of Urology, Case Western Reserve University & University Hospitals Case Medical Center, Cleveland, Ohio, USA), Kavleen Sikand (Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA.), Daniel Lindner (Department of Cellular and Molecular Medicine and Cancer Biology, Lerner Research Institute, 9100 Euclid Avenue, Cleveland Clinic Foundation, Cleveland, Ohio, USA), Sanjay Gupta (Department of Urology, Case Western Reserve University & University Hospitals Case Medical Center, Cleveland, Ohio, USA), Girish C. Shukla (Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA.)
Abstract not available online - please check the printed booklet.
37. Orthogonal Strand Displacement and Translation Control Using Chimeric γPNA
Cole Emanuelson (Department of Chemistry Mellon College of Science Carnegie Mellon University), Taylor Canady (Department of Chemistry Mellon College of Science Carnegie Mellon University), April Berlyoung (Department of Chemistry Mellon College of Science Carnegie Mellon University), Marcel Bruchez (Department of Chemistry Mellon College of Science Carnegie Mellon University), Bruce Armitage (Department of Chemistry Mellon College of Science Carnegie Mellon University)
Abstract not available online - please check the printed booklet.
38. Title not available online - please see the printed booklet.
Caleb Embree (Department of Biology, Ball State University), Douglas Bernstein (Department of Biology, Ball State University)
Abstract not available online - please check the printed booklet.
39. Proline-rich antimicrobial peptide Apidaecin exhibits a unique mode of inhibition of translation
Tanja Florin (Center for Biomolecular Sciences, University of Illinois at Chicago), Cristina Maracci (Department of Physical Biochemistry, Max Planck Institute, Goettingen, Germany), Dorota Klepacki (Center for Biomolecular Sciences, University of Illinois at Chicago), Marina Rodnina (Department of Physical Biochemistry, Max Planck Institute, Goettingen, Germany), Nora Vazquez-Laslop (Center for Biomolecular Sciences, University of Illinois at Chicago), Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago)
Abstract not available online - please check the printed booklet.
40. Title not available online - please see the printed booklet.
Jane K. Frandsen (Ohio State Biochemistry Program, Center for RNA Biology), Anna V. Sherwood (Molecular Cellular and Developmental Biology, Center for RNA Biology), Alexandar L. Hansen (CCIC NMR Facility), Frank J. Grundy (Department of Microbiology, Center for RNA Biology), Mark P. Foster (Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, Center for RNA Biology), Tina M. Henkin (Department of Microbiology, Ohio State Biochemistry Program, Molecular Cellular and Developmental Biology, Center for RNA Biology)
Abstract not available online - please check the printed booklet.
41. MetaComb: Meta-Transcriptomic Alignment with a Combined Genome
David E Frankhouser (Biomedical Sciences Graduate Program, The Ohio State University), Max Westphal, Alex Pelletier, Angela Urdaneta. Paige Stump, Peter Shields (Comprehensive Cancer Center, The Ohio State University), Peally Yan (Division of Hematology, The Ohio State University), Huiling He , Albert de la Chapelle (Department of Molecular Virology, Immunology & Medical Genetics, The Ohio State University), Carmine Sonzone (Molecular, Cellular, and Developmental Biology, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University)
Abstract:
The ability to detect bacterial and viral RNA embedded within the host transcriptome is a growing area of research and clinically important to understand their roles in human cancers. Currently, there are a few tools, one being SURPI, that can quantify metatranscriptomic (MT) reads in human RNA sequencing (RNAseq) data. The computational demands, lack of flexibility in study design, and limited accuracy in bacterial detection of these tools limit their applicability, especially for studies that include clinical trials. MetaComb, a computational method for detecting and quantifying MT reads in RNAseq data is both flexible and devoid of these common limitations.
MetaComb aligns reads from an RNAseq experiment to a combined host+microbial database. This allows reads from homologous sequences to be kept when their best alignment is to the microbial sequence. Reads are aligned using the fast, industry standard RNAseq aligner STAR. MetaComb is the only MT tool that leverages paired-end (PE) sequencing technology. We have demonstrated that PE reads increases the unique non-host MT alignment by two-fold. We were able to affirm the sensitivity and specificity of MetaComb-derived MT reads with BLAST. MetaComb cannot only quantify bacterial RNA content at the species level, but also at the genus or higher taxonomic levels. Quantification at higher taxonomic levels increase the number of included reads by reducing instances of multi-mapping that result from sequence homology between related species and strains. As proof-of-principle we validated MetaComb-derived quantification using real-time quantitative PCR. In summary, MetaComb provides: 1) increased accuracy of microbial RNA detection, 2) a flexible workflow well suited to clinical studies, and 3) the ability to quantify at any taxonomic level. MetaComb therefore allows researchers to identify microbial RNA content with minimal computational and manpower investment.
Keywords: metatranscriptomic, RNA-seq, metatranscriptomic-specific pipeline
42. Investigating ribosome processivity
Erin An (Washington and Lee University), Kyle Friend (Washington and Lee University)
Abstract not available online - please check the printed booklet.
43. Delineating Rbfox-regulated splicing networks critical for vertebrate muscle development
Thomas L. Gallagher (Molecular Genetics, The Ohio State University), Zachary T. Morrow (Molecular Genetics, The Ohio State University), Swanny A. Lamboy Rodriguez (Department of Biology, University of Puerto Rico in Humacao), Marcus H. Stoiber, James B. Brown, Susan E. Celniker, John G. Conboy (Life Sciences Division, Lawrence Berkeley National Lab), Sharon L. Amacher (Molecular Genetics, The Ohio State University)
Abstract:
Rbfox RNA binding proteins are regulators of phylogenetically-conserved alternative splicing events important for neuromuscular function. To investigate rbfox gene function, we knocked down two zebrafish rbfox genes expressed in muscle, rbfox1l and rbfox2, and showed that double morphant embryos have skeletal and cardiac muscle defects and changes in splicing of bioinformatically-predicted Rbfox target exons (Gallagher et al., 2011). Using CRISPR/Cas9 technology, we now have generated rbfox1l and rbfox2 single and compound null mutants. Whereas rbfox1l and rbfox2 single mutant embryos have normal myofibers and contractility, rbfox1l-/-; rbfox2-/- double mutant embryos have disorganized myofibrils coupled with complete paralysis. Despite complete loss of contractility in double mutants, neuromuscular junctions and fiber type specification appear normal. Splicing analysis reveals that predicted Rbfox target exons are down-regulated in rbfox1l-/-; rbfox2-/- double mutants. Interestingly, compound mutant combinations have different effects: rbfox1l-/-; rbfox2+/- mutants have wavy myofibers and undergo seizures upon touch-evoked stimulation, whereas rbfox1l+/-; rbfox2-/- mutants are indistinguishable from single mutants. Together, these data suggest that levels of Rbfox factors are critical for the coordinated regulation of an alternative splicing program essential for muscle function. In order to globally define the Rbfox-regulated splicing program, we used RNA-Seq to identify Rbfox target exons that are misregulated in Rbfox-deficient muscle and have identified compelling downstream candidates. Our goal is to understand how the network of Rbfox-regulated splicing events impacts muscle development and function that in the long term will allow us to predict and evaluate therapeutic approaches for splicing defects that lead to human disease.
References:
Gallagher TL, Arribere JA, Geurts PA, Exner CR, McDonald KL, Dill KK, Marr HL, Adkar SS, Garnett AT, Amacher SL, Conboy JG. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions. Dev Biol 2011, Nov 15;359(2):251-61.
Keywords: rbfox, splicing, muscle
44. EJC core protein Rbm8a is critical for muscular and neural development in zebrafish
Pooja Gangras (Department of Molecular Genetics, OSU), Thomas L. Gallagher (Department of Molecular Genetics, OSU), Kiel T. Tietz (Department of Molecular Genetics, OSU), Natalie C. Deans (Department of Molecular Genetics, OSU), Sharon L. Amacher (Department of Molecular Genetics, OSU), Guramrit Singh (Department of Molecular Genetics, OSU)
Abstract not available online - please check the printed booklet.
45. lincNXT1 modulates basal-like breast cancer invasiveness through regulating the expression of THBD
Omid Gholamalamdari (Department of Cell and Developmental Biology, UIUC), Deepak Singh (Department of Cell and Developmental Biology, UIUC), Tina Moazezi (Department of Cell and Developmental Biology, UIUC), Supriya G. Prasanth (Department of Cell and Developmental Biology, UIUC), Jian Ma (Computational Biology Department, CMU), Kannanganattu V. Prasanth (Department of Cell and Developmental Biology, UIUC)
Abstract:
Genome Wide Association Studies have identified genomic regions associated with different traits, including cancer. Interestingly 88% of these regions falls in noncoding portion of genome. In addition to regulatory elements, long non-coding RNAs(lncRNAs) are present in these regions. lncRNAs have been shown to play regulatory roles in different cellular processes including cell cycle, genome imprinting, nuclear organization, cell fate, development, and signaling. High-throughput RNAseq data reveal that the expression of lncRNAs is spatiotemporally regulated, and is dysregulated in cancers, including basal-like breast cancer. However, roles of lncRNAs in basal-like breast cancer are largely unknown. Here we have identified lncRNAs with potential cis regulatory function in basal-like breast cancer. We’ve characterized lincNXT1 as a cis regulator of Thrombomodulin (THBD) in basal-like breast cancer. Our results indicate that lincNXT1 levels are positively correlated with THBD mRNA in basal-like patients. lincNXT1 downregulation decreases cellular THBD levels, and therefore adhesion properties of cells and increases migrative and invasive properties of basal-like mammary epithelial cells. We anticipate our results pave the road for better diagnostic tools and targeted therapies in basal-like breast cancer.
References:
1. Esteller, M. (2011) Non-coding RNAs in human disease. Nat. Rev. Genet.
2. Quinodoz, S. & Guttman, M. (2014) Long noncoding RNAs: an emerging link between gene regulation and nuclear organization. Trends Cell Biol.
3. Tripathi, V., Ellis, J., Shen, Z., Song, D. & Pan, Q. (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol. Cell.
4. Xing, Z., Lin, A., Li, C., Liang, K., Wang, S., Liu, Y., Park, P. K., et al. (2014) lncRNA Directs Cooperative Epigenetic Regulation Downstream of Chemokine Signals. Cell
5. Hindorff, L., MacArthur, J., Morales, J. & Junkins, H. (2014) A Catalog of Published Genome-Wide Association Studies.
Keywords: Long non-coding RNA, Basal-like breast cancer, THBD
46. Title not available online - please see the printed booklet.
Andrew J. Giltmier (Biological Chemistry and Pharmacology), Jackson B. Trotman (Biological Chemistry and Pharmacology), Dr. Daniel R. Schoenberg (Biological Chemistry and Pharmacology)
Abstract not available online - please check the printed booklet.
47. Investigating regulation of eukaryotic gene expression by programmed -1 ribosomal frameshifting
Mina Griffioen (Cell biology and molecular genetics University of Maryland College Park), Liya Ket (Cell biology and molecular genetics University of Maryland College Park), Tania Mamdouhi (Cell biology and molecular genetics University of Maryland College Park), Arvin Massoudi (Cell biology and molecular genetics University of Maryland College Park), Vivek Advani (Cell biology and molecular genetics University of Maryland College Park), Jonathan D. Dinman (Cell biology and molecular genetics University of Maryland College Park)
Abstract:
Translational recoding refers to exception to the rules of translation. Programmed -1 ribosomal frameshifting (-1 PRF) is one such translational recoding mechanism first identified in some RNA viruses and later described in higher eukaryotes(1). A canonical ‘genomic’ -1 PRF signal directs an elongating ribosome to a -1 frame premature termination codon (PTC), thereby resulting in rapid degradation of the message through nonsense mediated mRNA decay (NMD) pathway. There exist an inverse relationship between -1 PRF efficiency and mRNA abundance(2). In silico analysis predicts that approximately 10% of genes in humans have at least one predicted -1 PRF signal(3). We have identified functional frameshift signals in mRNAs encoding human ATG7, EIF5B, BCL2L11, RASA2, and MAP4K3; as confirmed by bicistronic reporter assays. These are involved in translational initiation, apoptosis, cell stress, and cell signaling pathways. Additionally, preliminary data suggests that these -1PRF sequences function as mRNA destabilizing elements. Bioinformatic analysis suggests that microRNAs might be involved in regulating -1PRF efficiencies encoded by these messages.
References:
Jacobs JL, et. al. Identification of functional, endogenous programmed -1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae. NAR. 2007 Jan 12; 35(1):165–74.
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. 2011 Apr 1; 39(7):2799–808.
3. Belew AT, et. al. PRFdb: a database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals. BMC Genomics. 2008 Jan ;9(1):339.
Keywords: -1PRF, NMD, miRNA
48. Medicago truncatula LINC complexes and their potential role in root symbioses
Anna Hare Newman Griffis (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University, Columbus, OH, USA), Katherine Beigel (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA), Myriam Charpentier (Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK), Giles E.D. Oldroyd (Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK), Iris Meier (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University, Columbus, OH, USA)
Abstract:
Several crop plants’ productivity depends on root symbionts that provide the plants with vital nutrients. One of the most important events in symbiosis establishment is the initiation of interactions between plant roots and soil-dwelling microbes, including rhizobia and arbuscular mycorrhizal fungi (AMF). The role of nuclear migration in these processes has long been implied, but never definitively established. Recently, LINC complexes, consisting of SUN and KASH proteins, have been shown to be components of metazoan nuclear movement machinery. While SUN proteins are widely conserved, no animal KASH protein homologs exist in plants. Working in the model plant Arabidopsis, our lab recently discovered the first plant KASH proteins, which are involved in processes ranging from root hair and pollen tube nuclear movement to oomycete defense. However, Arabidopsis is a host to neither rhizobia nor to AMF. Therefore, to study the role of LINC complexes in nuclear movement during symbiosis establishment, we have adopted a reverse genetics approach in the model legume Medicago truncatula. We have identified 12 genes encoding putative Medicago LINC complex components via bioinformatic tools. Here, we will show subcellular localization data for these LINC complex candidates from transient expression assays in Nicotiana benthamiana. Further candidate validation by co-immunoprecipitation with appropriate putative binding partners (i.e. SUN-KASH interactions) will also be presented. Tnt1 retrotransposon insertion mutant lines have been obtained for 8 of the 12 putative LINC complex component genes. Results from phenotypic analyses performed on mutant lines, focusing on potential symbiosis and/or nuclear movement defects, will also be shown.
Keywords: symbiosis, Medicago, nuclear envelope
49. Alternative Polyadenylation as a Potential Mechanism Responding to Drought Stress in Arabidopsis thaliana
Guijie Hao (Deparment of Plant and Soil Sciences), Arthur G. Hunt (Deparment of Plant and Soil Sciences)
Abstract:
mRNA polyadenylation is an important step in eukaryotic gene expression. Polyadenylation impacts gene expression through determining the coding and regulation potential of the mRNA, especially the mRNAs of genes that may be polyandenylated at more than one position. Alternative polyadenylation (APA) has potential effect to gene regulation and function. In Arabidopsis, APA may be linked with stress responses through a polyadenylation complex subunit (CPSF30) that is inhibited by calmodulin and by disulfide remodeling. Calcium signaling (through calmodulin) and disulfide remodeling (through the generation of reactive oxygen species) are typically associated with various stress responses in plants, suggestive of a possible connection.
In order to explore this, a study was done to examine the effects that an artificial drought stress (triggered by growth of plants in mannitol) has on genome-wide poly (A) site choice. We treated wild type Arabidopsis with different concentrations of mannitol, for varying lengths of time. RNA was extracted after harvesting the whole seedlings and poly (A) tag libraries were prepared and sequenced on the Illumina platform. The sequence data was analyzed to assess gene expression and alternative polyadenylation. The gene expression analysis demonstrated genes from certain GO categories are up- or down- regulated by the simulated drought stress; these categories include responses to hormone stimulus, secondary metabolism, transmembrane transport, regulation of transcription, and protein phosphorylation. Moreover, the results showed that drought stress caused significant changes in the usage of poly (A) sites that lie within 5’-UTRs; specifically, the poly (A) site profiles for a number of genes were shifted to increased usage of sites within 5’ UTRs, site that would truncate the mRNA.
These results indicate that drought stress alters poly (A) site choice for many genes, and may play negative regulatory roles for genes that possess poly (A) sites that fall within 5’-UTRs. They also suggest that APA plays an important role in the responses of Arabidopsisto drought stress. Future studies will include 3’-RACE confirmation of selected genes, and comparisons of the effects of drought stress with other abiotic and biotic stress.
References:
1.Arthur G Hunt. The Arabidopsis polyadenylation factor subunit CPSF30 as conceptual link between mRNA polyadenylation and cellular signaling. Current Opinion in Plant Biology 2014, 21:128–132
2.Lutz CS, Moreira. Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression. Wiley Interdiscip Rev RNA. 2011 Jan-Feb; 2(1):23-31
3.Zhang J, Addepalli B, Yun KY, Hunt AG, Xu R, Rao S, Li QQ, Falcone DL. A polyadenylation factor subunit implicated in regulating oxidative signaling in Arabidopsis thaliana. PLoS One 2008, 3:e2410
Keywords: Alternative polyadenylation, drought stress, Arabidopsis thaliana
50. Title not available online - please see the printed booklet.
Xinying Zong, Qinyu Hao, Omid Gholmalamdari (University of Illinois at Urbana-Champaign), Ritu Chaudhary, Xiao Ling Li, Ashish Lal (NCI, NIH), Sasan Hashemi , Sarath Janga (Indiana & Purdue University), Sven Diederichs (German Cancer Research Center), Anindya Bagchi1, Jian Ma2 (1University of Minnesota; 2Carnegie Mellon University), Supriya G. Prasanth, Kannanganattu V. Prasanth (University of Illinois at Urbana-Champaign)
Abstract not available online - please check the printed booklet.
51. Hypoxic induction of c-Myc and microRNAs to inhibit nephron progenitor differentiation
Shelby L. Hemker (Pediatrics, University of Pittsburgh), Andrew Clugston (Pediatrics, University of Pittsburgh; Developmental Biology, University of Pittsburgh), Dennis Kostka (Developmental Biology, University of Pittsburgh), Sunder Sims-Lucas (Pediatrics, University of Pittsburgh), Jacqueline Ho (Pediatrics, University of Pittsburgh)
Abstract:
Placental insufficiency causes fetal hypoxia, reduced blood flow to the kidneys, and is the main cause of intrauterine growth restriction (IUGR). IUGR and fetal hypoxia adversely affect kidney development, resulting in congenital anomalies of the kidney and urinary tract (CAKUT), the leading cause of renal failure in children. We propose that oxygen tension in the developing kidney is precisely controlled to facilitate the normal development of nephrons, the functional unit of the kidney. To investigate how different oxygen levels alter gene expression of the developing kidney, we transcriptionally profiled embryonic day 12.5 mouse kidneys cultured in either 1%, 5%, or 21% O2. As expected, the Hypoxia Inducible Factor (HIF) transcriptional network—which regulates the expression of many genes in response to cellular hypoxia—was up-regulated in the kidneys cultured in 1% O2. Furthermore, there is increased expression of early kidney development markers and decreased expression of genes expressed by maturing kidney epithelial cells in the 1% O2 kidney cultures, suggesting an impairment in differentiation. This was associated with evidence for increased activity of the c-Myc pathway, which is known to be an important regulator of proliferation in nephron progenitor cells. Finally, several hypoxia-responsive microRNAs were demonstrated to be differentially expressed, and Ingenuity Pathways Analysis was used to predict differentially expressed miRNA-mRNA target pairs in hypoxia. Together, this data suggests that c-Myc and microRNAs are important HIF effectors that inhibit nephron progenitor differentiation in kidney development, in response to varying oxygen tension.
Keywords: microRNA, RNA sequencing, kidney development
52. Inhibition of telomerase by new purine analogs
Wilnelly Hernandez-Sanchez (Department of Pharmacology, Case Western Reserve University), Wei Huang (Department of Pharamacology, Case Western Reserve University), Anthony J. Berdis (Department of Chemistry, Case Western Reserve University), Derek J. Taylor (Department of Pharamacology,Department of Biochemistry, Case Western Reserve University)
Abstract not available online - please check the printed booklet.
53. Identifying the regulatory role of the DNA entry-exit site of the nucleosome in transcription termination
A. Elizabeth Hildreth (Department of Biological Sciences, University of Pittsburgh), Karen M. Arndt, PhD (Department of Biological Sciences, University of Pittsburgh)
Abstract:
Eukaryotic chromatin is a restrictive barrier to RNA polymerase II, which transcribes protein coding and some noncoding RNAs. Chromatin consists of repeating nucleosomes, approximately 147 basepairs of DNA surrounding an octamer of histone proteins. Transcription is controlled by factors that modify nucleosomes, allowing Pol II to contact otherwise occluded DNA. The mechanisms by which chromatin is modified are well understood in regard to transcription initiation and elongation. Despite a few studies showing a requirement for select histone modifications and chromatin remodelers, little else is known about the role of chromatin at the final termination step. The goal of our work is to elucidate this role using Saccharomyces cerevisiae. We used a plasmid library encoding mutant histones and a termination reporter to identify 12 residues in histones H3, H4, and H2A required for proper termination. Interestingly, many of these residues reside in or near the nucleosome DNA entry-exit site. This surface coordinates the first 30 basepairs of DNA, thus regulating stability and accessibility of the nucleosome. Analysis thus far reveals improper nucleosome occupancy at candidate loci and defective placement of a transcription-coupled histone modification. Recent results suggest that increased elongation rate may also contribute to the observed termination defect in some mutants. These data implicate the DNA entry-exit site as an important player in the regulation of transcription.
Keywords: transcription, termination, chromatin
54. Further characterization of the roles of DDX41, a DEAD-box RNA helicase, in blood cancer and the spliceosome
James M. Hiznay (Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University), Chantana Polprasert (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Hideki Makishima (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Eckhard Jankowsky (Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University), Jaroslaw P. Maciejewski (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Richard A. Padgett (Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University)
Abstract:
Members of the DEAD-box RNA helicase family have prominent roles throughout RNA metabolism. In addition to unwinding RNA duplexes, these proteins can remodel RNA-protein complexes and serve as scaffolds on which ribonucleoproteins assemble. Members of this protein family play a prominent role in the splicing of pre-mRNAs, where they serve as key components of the spliceosome. Sequencing of tumors from myelodysplastic syndrome (MDS) patients has revealed mutations—both germline and tumor-specific—in DDX41. MDS is a type of blood neoplasm that occurs in the elderly; it is typified by incomplete differentiation of erythrocytes, platelets, and certain leukocytes. A bevy of DDX41 mutations in MDS patients has been uncovered in the past three years. The most common DDX41 germline mutation causes a frame shift in the protein and results in a nonfunctional polypeptide. The most common tumor-specific mutation, R525H, affects the ATP-binding site and abrogates the ATPase activity of the protein. People with DDX41 mutations develop MDS younger and have a poorer prognosis than those without mutations. While DDX41 appears essential for cell growth, little is known of its role in the spliceosome or myeloid neoplasms. RNA-seq analysis of mutant patient tumors has shown a number of genes with altered spliced isoform ratios. Knockdown of DDX41 in cells results in the inhibition of splicing of a subset of introns. RNA CLIP-seq analysis of tagged DDX41 discovered preferential crosslinking to both U2 and U12 snRNAs as well as to 5’ and 3’ splice site regions of pre-mRNAs. Proteomic analysis of proteins co-precipitated using tagged DDX41 revealed numerous splicing factors, particularly from spliceosomal C complexes. Similar analyses using the R525H mutant protein showed reduced association with several key factors, including PRPF8 and SF3B1, which are also mutated in myeloid neoplasms. RNA co-immunoprecipitation experiments performed in an in vitro splicing assay with recombinant, tagged DDX41 evinced an enrichment of the lariat intron product. These data support our hypothesis that DDX41 is a core spliceosome component whose altered expression can affect the splicing of key tumor suppressor genes, which in turn may promote leukemogenesis.
Keywords: DDX41, RNA splicing, MDS
55. RNA evolution in an oxidizing Environment: The mammalian mitochondrial ribosome
Poorna Roy (Department of Chemistry, Bowling Green State University), Maryam Hosseini (Department of Chemistry, Bowling Green State University), Eric Westhof (Architecture et Ractivit de lARN, Universit de Strasbourg, Institut de biologie molculaire et cellulaire du CNRS), Marie Sissler (Architecture et Ractivit de lARN, Universit de Strasbourg, Institut de biologie molculaire et cellulaire du CNRS), Neocles Leontis (Department of Chemistry, Bowling Green State University)
Abstract:
Mitochondria evolved from bacterial symbionts of early eukaryotes and have their own DNA and ribosomes. Mammalian mitochondrial (mmt) ribosomes translate just 13 polypeptides, all of which belong to the electron transport chain (ETC), located in the inner mitochondrial membrane (IMM), which is the source of reactive oxygen species (ROS). Thus mmt-ribosomes are exposed to a highly oxidizing environment and turn over every 4-5 hours, at a rate ~24 times faster than for cytosolic ribosomes (Gelfand and Attardi, 1981). Hence, we hypothesize that mitochondria are subject to strong selection for ribosomes of reduced sensitivity to oxidative damage. Comparison of mmt- and bacterial rRNA shows large-scale loss of peripheral rRNA in both mmt-ribosomal subunits and a parallel increase in the sizes and numbers of r-proteins. Analysis of recent cryo-EM structures of the mmt-SSU shows that all rRNA elements that directly contact tRNA and mRNA are nonetheless retained, but large numbers of exposed G’s in hairpin, internal and multi-helix junction “loops,” that are highly conserved in bacterial SSU (>99%), are replaced in mmt-rRNA by bases less sensitive to oxidation, A, C, or U, as revealed by structure-correlated sequence analysis of high quality rRNA sequence alignments (http://rna.bgsu.edu/r3d-2-msa, Cannone et al. 2015). We present a detailed analysis of differences between bacterial and mmt ribosomes in sequence conservation, RNA-RNA and RNA-protein interactions correlated with solvent accessible surface area, to assess this hypothesis. Our analyses show that mitochondria-specific r-proteins and expansions of bacterial r-protein homologues (1) substitute for truncated rRNA helices while preserving the mutual orientations of the remaining helices in 3D space (2) compensate missing RNA-RNA interactions, (3) reduce the solvent accessibility of exposed bases in rRNA loops, and (4) stabilize 3D motifs lacking Gs that are conserved in bacteria.
References:
Gelfand, R. and Attardi, G. (1981) “Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable.” Mol Cell Biol. 1981; 1(6): 497–511.
Cannone, J.J., Sweeney, B.A., Petrov, A.I., Gutell, R.R., Zirbel, C.L., and Leontis, N. (2015) “R3D-2-MSA: the RNA 3D structure-to-multiple sequence alignment server.” Nucleic Acids Res. 43(W1):W15-23. doi: 10.1093/nar/gkv543
Keywords: Evolution, ROS, Mitochondria
56. Assessment of candidate factors required for RNA editing in Physarum polycephalum mitochondria
Jillian Houtz (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University), Jonatha Gott (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract not available online - please check the printed booklet.
57. Cellular adaptation using the m1G9 tRNA modification and its catalyst, Trm10
Nathan Howell (Ohio State University), Abi Hubacher (Ohio State University), Aiswarya Krishnamohan (Ohio State University), Emily Kuiper (Emory University), Jane Jackman (Ohio State University)
Abstract:
In addition to its accepted role as a critical adaptor molecule between nucleic acids and proteins, tRNA is central to a novel regulatory system in the cell, where changes in cellular environment are sensed by tRNA-modifying enzymes, resulting in perturbations to the tRNA pool and consequent cellular adaptation, potentially through altered translational output. To better understand the role that tRNA modification enzymes play in this system, we study the function and mechanism of Trm10, homologs of which constitute a broadly conserved family of tRNA modification enzymes responsible for m1G9 modification in tRNA. We seek to determine the functional significance of the m1G9 modification, as well as how Trm10 distinguishes substrate tRNAs from a larger pool of potential substrates. To do this, we have explored Trm10 substrate specificity by converting a Type II, non-substrate tRNA into a Type I-like substrate using domain-swapping mutations. Additionally, the impact of m1G9 deficiency is assessed using genetic techniques as well as HPLC analysis of bulk RNA and individual tRNA species.
Keywords: tRNA modifications, enzymology, dynamic modifications
58. Exploring the role of tRNA modifications in cytotoxicity of the anti-cancer drug 5-fluorouracil
Abigail R. Hubacher (Dept of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Dept of Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the printed booklet.
59. miR-146a directly regulates expression of 5-lipoxygenase activating protein (FLAP) in lung cancer cells
Joseph R. Iacona (Microbiology, Biochemistry and Molecular Genetics, Rutgers Biomedical and Health Sciences-Newark, NJ), Nicholas J. Monteleone (Microbiology, Biochemistry and Molecular Genetics, Rutgers Biomedical and Health Sciences-Newark, NJ), Carol S. Lutz (Microbiology, Biochemistry and Molecular Genetics, Rutgers Biomedical and Health Sciences-Newark, NJ )
Abstract:
Arachidonic acid can be converted into prostaglandins (PGs) or leukotrienes (LTs) by cyclooxygenase (COX-1 and COX-2) or 5-lipoxygenase (5-LO) enzyme activity, respectively. PGs and LTs are lipid signaling molecules that have been implicated in various diseases, including multiple cancers. 5-LO and its activating protein (FLAP) work together in the first conversion step of LT production. Previous work has suggested a role for 5-LO and FLAP in cancer development and progression. MicroRNAs (miRNAs) are small RNA molecules that negatively regulate gene expression at the post-transcriptional level. Several studies have shown that both 5-LO and FLAP expression are controlled in this manner. Here, we show that FLAP protein is overexpressed in lung cancer cells compared to normal lung cells. One miRNA in particular, miR-146a, is predicted to target FLAP. Transfection of synthetic miR-146a in lung cancer cells can repress endogenous FLAP mRNA and protein expression, and reporter assays indicate this regulation occurs through a direct interaction between miR-146a and the FLAP 3ʼ UTR. Intriguingly, miR-146a can also repress endogenous 5-LO mRNA and protein expression, though in an indirect manner. This concerted downregulation of 5-LO and FLAP also results in decreased cancer cell LTB4 production. This study and previous work from our lab suggest miR- 146a can regulate both PG and LT production in lung cancer cells by directly targeting the 3ʼ UTRs of COX-2 and FLAP.
Keywords: miRNA, gene expression, regulation
60. Title not available online - please see the printed booklet.
Zachary Iezzi (Allegheny College), Conner Bardine, Haley Englert, Ivelitza Garcia (Allegheny College)
Abstract not available online - please check the printed booklet.
61. Investigation of IRES-mediated Translation of PUMA mRNA, Initiation Factor Requirements and Search for IRES-Trans Acting Factors (ITAFs)
AMRA ISMAIL (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University), Shuai Zhao (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University), Crystal M. Weyman (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University), Anton A. Komar (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University)
Abstract:
Canonical translation initiation in majority of cellular mRNAs occurs by a cap-dependent/scanning mechanism, whereby the 43S preinitiation complex binds to the mRNA 5’ terminal cap structure and scans the 5’UTR of mRNA in search of the initiation codon. Internal Ribosome Entry Sites (IRESs) are cis-acting elements, located in the 5’UTRs of some viral and cellular mRNAs that facilitate direct recruitment of the 40S ribosomal subunits near the AUG codon. IRES-mediated translation initiation may proceed without the help of many canonical initiation factors but may require additional IRES-trans-acting factors (ITAFs).
The proapoptotic Bcl-2 family member PUMA has been previously shown to contain an IRES element that is active under conditions of eIF2-α phosphorylation and hypophosphorylation of eIF4E-BP that inhibits cap-dependent translation.
We further investigated the mechanism for PUMA mRNA recruitment to the ribosome and found that unlike class III or IV viral IRESs, PUMA IRES is not able to bind 40S ribosomal subunits directly. We also analyzed PUMA IRES initiation factor requirements and found that PUMA IRES requires intact eIF4G and eIF4A for its activity, which is reminiscent of the hepatitis E virus (HEV) IRES requirements. RNA affinity pull down assays and mass spectrometric analysis identified Hsp70 as one of the proteins that binds PUMA IRES with high affinity. Gel shift assays confirmed that the binding is specific. Further in vitro studies reveal that IRES-mediated translation of PUMA mRNA is enhanced in the presence of Hsp70 protein.
Keywords: Internal Ribosome Entry Sites, ITAF (IRES-trans acting factors)
62. Title not available online - please see the printed booklet.
Jingyi Fei (1University of Illinois at Urbana-Champaign, Department of Physics, Center for the Physics of Living Cells), Mahdieh Jadaliha (University of Illinois at Urbana-Champaign, Department of Cell and Developmental Biology), Tyler Harmon (Washington University in St. Louis, Department of Physics), Isaac T.S. Li (University of Illinois at Urbana-Champaign, Department of Physics), Kannanganattu V. Prasanth (University of Illinois at Urbana-Champaign, Department of Cell and Developmental Biology), Taekjip Ha (University of Illinois at Urbana-Champaign, Institute for Genomic Biology)
Abstract not available online - please check the printed booklet.
63. Rules of RNA Specificity of hnRNP A1 Revealed by Global and Quantitative Analysis of its Affinity Distribution
Niyati Jain (Department of Chemistry, Case Western Reserve University, Cleveland, OH), Hsuan-Chun Lin (Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH), Christopher E. Morgan (Department of Chemistry, Case Western Reserve University, Cleveland, OH), Michael E. Harris (Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University, Cleveland, OH)
Abstract:
The selectivity of many RNA binding proteins (RBPs) is more complex than
specific versus non-specific. Heterogeneous nuclear ribonucleoprotein A1
(hnRNP A1) is a multipurpose RBP involved in normal and pathologic RNA
metabolism. Transcriptome-wide mapping and in vitro evolution identify
consensus motifs; however, such data do not reveal how surrounding RNA
sequence and structural context modulate affinity. We determined the affinity of
hnRNP A1 for all possible sequence variants (n=16,384) of the HIV ESS3 7-nt
apical loop. Analysis of the affinity distribution identifies the optimal motif 5’-
YAG-3’ and shows how its copy number, position in the loop and loop structure
modulate affinity. We show that specificity is determined by association rate
constants, and that variants lacking the minimal sequence motif bind competitively with consensus RNA.
Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding
how it discriminates between alternative competing RNA ligands in vivo.
Keywords: RNA binding proteins, hnRNP A1
64. Microscopically visible P-bodies contribute little to miRNA-mediated gene silencing
Sethu Pitchiaya (Department of Chemistry, University of Michigan, Ann Arbor, MI, USA), Ameya Jalihal (Department of Chemistry, University of Michigan, Ann Arbor, MI, USA), Marcio D.A. Mourao (Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA), Santiago Schnell (Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA), Nils G. Walter (Department of Chemistry, University of Michigan, Ann Arbor, MI, USA)
Abstract:
microRNA (miRNA) bound messenger RNAs (mRNAs) localize to processing bodies (PBs), which are sub-cellular, membrane-less foci enriched in RNA processing enzymes, as a cause or consequence of post-transcriptional gene silencing. To gain a deeper understanding of the hitherto unobserved dynamics of miRNA:PB interactions and the contribution of PBs to miRNA-mediated gene silencing, we used a modified version of our recently developed iSHiRLoC (intracellular Single molecule High-Resolution Localization and Counting) technique and combined the resulting data with molecular diffusion modeling. miRNAs and PBs were labeled with spectrally distinct fluorescent probes so that single functional miRNAs could be localized and tracked with respect to single PBs inside human cells with ~30 nm spatial accuracy at 100 ms time resolution. iSHiRLoC revealed that only a small fraction of miRNAs, independent of the abundance of their mRNA targets, localize to PBs, with the majority of PBs containing only one or two fluorophore labeled miRNA molecules. Surprisingly, higher mRNA target abundance led to a smaller fraction of miRNAs stably co-localizing with PBs, suggesting that PBs may play a significant role in sequestration of target-free miRNAs. Moreover, molecular diffusion modeling predicted that PB aggregation into fewer, but larger (microscopically visible) particles should diminish their contribution towards gene silencing. Supporting this observation, aggregation and immobilization of PBs in cultured cells via hypertonic treatment suppressed miRNA mediated gene silencing. Taken together, our data suggest that microscopically visible PBs minimally contribute to gene silencing and perhaps play a role in target-independent miRNA sequestration. Our single-molecule systems biology approach promises to map the dynamic interaction network of the RNA silencing pathway to reveal crucial mechanistic details and assess their impact on cellular gene expression.
Keywords: miRNA, Processing Bodies, Super Resolution Microscopy
65. Interaction between Human Glutamyl-Prolyl Aminoacyl-tRNA Synthetase Linker Domain and HIV-1 Matrix: Implications for HIV-1 Infectivity
Danni Jin, Nathan Titkemeier, Alice Duchon (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Corine St. Gelais (Center for RNA Biology, Center for Retrovirus Research, and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Yiping Zhu (Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY, 10032), Li Wu (Center for RNA Biology, Center for Retrovirus Research, and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Stephen P. Goff (Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY, 10032), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)
Abstract not available online - please check the printed booklet.
66. The role of eukaryotic ribosomal protein uS9 (S16) in translation
Supriya Jindal (Center for Gene Regulation in Health and Disease, Department of Biological, Geological & Environmental Sciences, Cleveland State University), Arnab Ghosh, Nishant Singh, Anton A. Komar (Center for Gene Regulation in Health and Disease, Department of Biological, Geological & Environmental Sciences, Cleveland State University)
Abstract not available online - please check the printed booklet.
67. Title not available online - please see the printed booklet.
Paul Kelly (Molecular, Cellular, and Developmental Biology Program, The Ohio State University), Tammy Bullwinkle (Department of Microbiology, The Ohio State University), David Ardell (Program in Quantitative Systems Biology, University of California, Merced), Roger Linington (Department of Chemistry, Simon Fraser University), Abhay Satoskar (Department of Microbiology and the Department of Pathology, The Ohio State University), Michael Ibba (Department of Microbiology, The Ohio State University)
Abstract not available online - please check the printed booklet.
68. Title not available online - please see the printed booklet.
Alan C. Kessler (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Sneha Kulkami (Biology Centre, Institute of Parasitology, University of South Bohemia, Czech Republic), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Zdenk Paris (Biology Centre, Institute of Parasitology, University of South Bohemia, Czech Republic), Juan D. Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University)
Abstract not available online - please check the printed booklet.
69. A programmed ribosomal frameshifting defect potentiates the transforming activity of the JAK2-V617F mutation
Yousuf A. Khan (Department of Cell Biology and Molecular Genetics, University of Maryland. College Park MD 20742), Sergey O. Sulima (KU Leuven Department of Oncology, Leuven, Belgium; VIB Center for the Biology of Disease, Leuven, Belgium), Zachary Flickinger, John E. Jones, Vivek M. Advani (Department of Cell Biology and Molecular Genetics, University of Maryland. College Park MD 20742), Kim de Keersmaecker (KU Leuven Department of Oncology, Leuven, Belgium; VIB Center for the Biology of Disease, Leuven, Belgium), Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland. College Park MD 20742)
Abstract not available online - please check the printed booklet.
70. Fine-tunable Nucleic Acid nano-Scaffolds Designed from Flexible Tetra-U Helix Linking Module
My N. Bui (Department of Chemistry, Ball State University ), Emil F. Khisamutdinov (Department of Chemistry, Ball State University )
Abstract not available online - please check the printed booklet.
71. A tool for fusion detector post-analysis coverage visualization of chimeric RNA-seq data
Jackson A Killian (Department of Physics, The Ohio State University), Taha Topiwala (Department of Physics, The Ohio State University), Alex Pelletier (Department of Physics, The Ohio State University), David Frankhouser (Biomedical Sciences Graduate Program, The Ohio State University), Pearlly Yan (The Ohio State University Comprehensive Cancer Center and Division of Hematology, Department of Internal Medicine), Ralf Bundschuh (Department of Physics, Department of Chemistry and Biochemistry, Division of Hematology, Center for RNA Biology, The Ohio State University)
Abstract:
Gene fusions often occur in cancer cells and in some cases can even be the cause of the onset of the disease. Correct identification of oncogenic gene fusions thus has implications for targeted treatment in future therapy courses. Recognition of this potential along with the advent of RNA-seq has led to the development of a myriad of sequencing-based fusion detection tools designed to detect novel fusion transcripts in RNA data. However, given the same input, many of these detectors will find different fusion points or claim different sets of supporting data. Furthermore, the rate at which these tools falsely detect fusion events in data varies greatly. This discrepancy between tools underscores the fact that computational methods still cannot perfectly evaluate evidence; especially when provided with small amounts of supporting data as is typical in fusion detection. If data is provided in an easily digestible form, humans are more proficient at drawing conclusions. We thus have developed a visualization tool, FuSpot, that puts the power to decide false positives in the researcher’s hands, saving time and resources by cutting down on costly and labor intensive PCR validation. FuSpot is given the coordinates of a candidate fusion junction and a set of reads aligning in the vicinity of the candidate fusion junction. FuSpot then realigns the reads simultaneously to genomic sequences as well as the sequences of the nearest exons for both candidate fusion gene partners and visualizes them relative to the reference sequences on either side of the fusion point so that researchers can quickly visually confirm if their data does indeed support the presence of the fusion. We apply our visualization tool to a publicly available dataset and provide examples of true as well as false positives reported by commonly used fusion detection tools.
Keywords: Fusion, Visualization, RNA-Seq
72. Studies of RNA nucleoside platination and destabilization of glycosidic bond
Bett Kimutai (Chemistry Department, Wayne State University), Chenchen He (Chemistry Department, Wayne State University), Xun Bao (Chemistry Department, Wayne State University), Christine S. Chow (Chemistry Department, Wayne State University)
Abstract not available online - please check the printed booklet.
73. Trypanosoma brucei utilizes dual-coding genes through RNA editing
Laura Kirby (Microbiology and Molecular Genetics, Michigan State University), Yanni Sun (Computer Science and Engineering, Michigan State University), Donna Koslowsky (Microbiology and Molecular Genetics, Michigan State University)
Abstract:
Trypanosoma brucei is a disease causing parasite that lives in mammalian hosts and is transmitted by the bite of a tsetse fly. This salivarian trypanosome is exclusively extracellular in the vertebrate host and can continuously replicate within the bloodstream for months, escaping the host’s immune response by switching surface glycoproteins. During growth in the glucose-rich bloodstream, the mitochondrion is down-regulated and lacks a functional electron transport chain. This unique life cycle leads to two main problems: 1) the lack of selection for genes involved in mitochondrial energy production and 2) repeated population bottlenecks, each time they undergo an antigenic switch. These conditions should lead to the rapid accumulation of mutations in the mitochondrial genes required for survival in the insect vector. In our analyses of the gRNA transcriptome, we have identified alternative terminal gRNAs (the last gRNA in the editing cascade) for the CR3 transcript that generate transcripts using different open reading frames. Surprisingly, the alternative reading frame is 9 amino acids longer than the previously identified ORF. Subsequent analyses of the pan-edited mitochondrial genes indicate that several contain extended dual ORFs, and thus could be dual-coding. We hypothesize that RNA editing is a unique mechanism that can be used to gain access to multiple ORFs, and that the overlapping of genetic information helps combat deleterious genetic drift that would occur during growth under non-selective conditions. Using Illumina deep sequencing, we have identified alternative forms of CR3 and ND3 which align their start codons with two different ORFs, and we have identified the alternative gRNAs that direct these edits.
Keywords: Dual-coding genes, RNA-editing
74. The relationship of mRNA 5’ ends generated by cap homeostasis to translation
Daniel L. Kiss (Center for RNA Biology, Department of Biological Chemistry and Pharmacology,The Ohio State University, Columbus, Ohio 43210), William D Baez (Center for RNA Biology, Department of Physics, and Division of Hematology,The Ohio State University, Columbus, Ohio 43210), Bernice Agana (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210), Vicki H Wysocki (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210), Ralf Bundschuh (Center for RNA Biology, Department of Physics, and Division of Hematology,The Ohio State University, Columbus, Ohio 43210), Daniel R Schoenberg (Center for RNA Biology, Department of Biological Chemistry and Pharmacology,The Ohio State University, Columbus, Ohio 43210)
Abstract:
Until recently, removal of the 5’ cap structure was thought to be an irreversible step leading to the degradation of the decapped mRNA. Our lab has discovered and characterized a cytoplasmic capping enzyme (CE) complex that includes capping enzyme and a 5’ monophosphate RNA kinase assembled on the two most C-terminal SH3 domains of the scaffold protein Nck1. This complex promotes the stability and translation of specific mRNAs by restoring the 5’ cap onto their decapped forms. Previous work used a line of cells expressing an inducible dominant negative form of CE (K294A) to identify cytoplasmic capping targets. Uncapped forms of these mRNAs accumulate in non-translating mRNPs and retain translationally competent poly(A) tails. Data from capped analysis of gene expression (CAGE) was used to refine experiments using 5’ RACE to identify the 5’ ends of these transcripts. In agreement with the published CAGE data, these results show a subset of mRNAs can be recapped at multiple locations to generate 5’ truncated mRNAs. This was examined further by ribosome profiling to ascertain the prevalence and functional effects of cap homeostasis across the translating transcriptome. Results will be presented that show decapping and recapping (cap homeostasis) is a mechanism by which cells regulate the translation of a subset of mRNAs.
Keywords: cytoplasmic capping, cap homeostasis, sequencing
75. Regulation of HIV-1 genomic RNA 5’-UTR conformation
Jonathan Kitzrow (Chemistry and Biochemistry, The Ohio State University ), Erik Olson (Chemistry and Biochemistry, The Ohio State University ), Karin Musier-Forsyth (Chemistry and Biochemistry, The Ohio State University )
Abstract:
HIV-1 packages its genetic material into assembling virions as a dimer of full-length genomic RNA (gRNA). HIV-1 gRNA packaging is facilitated through interactions between the nucleocapsid (NC) domain of the HIV-1 Gag protein and specific structural elements in the 5’-untranslated region (5’UTR) of gRNA. Full-length HIV-1 gRNA also serves as the template for Gag/Gag-Pol polyprotein translation. A conformational change in the 5’UTR has previously been proposed to govern a switch from translation competent to packaging competent gRNA. NMR studies have shown that the U5 region of the 5’UTR can interact with both the dimer initiation signal (DIS) and Gag start codon (AUG). These interactions selectively expose either the DIS (packaging competent form) or AUG (translation competent form) elements in a mutually exclusive manner. In this work, a Förster resonance energy transfer (FRET)-based assay has been designed to probe the HIV-1 5’UTR conformation in both the monomeric and dimeric state. We hypothesize that Gag, NC, and/or tRNALys3 primer binding may shift the 5’UTR conformational equilibrium and this will also be tested.
Keywords: HIV-1, gRNA, FRET
76. Mechanistic features of the tRNA m1G9 SPOUT methyltransferase, Trm10
Aiswarya Krishnamohan (Ohio State Biochemistry Program, Dept. of Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Dept. of Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the printed booklet.
77. Function of a putative trans-editing factor, ProXp-x, in Rhodopseudomonas palustris
Lexie Kuzmishin (The Ohio State Biochemistry Program, Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Ziwei Liu (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Jo Marie Bacusmo (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, and Center for RNA Biology, The Ohio State University)
Abstract:
Aminoacyl-tRNA synthetases (ARSs) attach specific amino acids to cognate tRNAs; this process is a critical checkpoint in protein synthesis. Due to the similarity in size and structure of many amino acids, ARSs are known to misactivate both proteinogenic and non-protein amino acids. Thus, editing mechanisms are needed to prevent widespread mistranslation. In addition to specific tRNAPro editing mediated by the insertion (INS) domain present in most bacterial prolyl-tRNA synthetases (ProRS), single-domain trans-editing factors structurally homologous to INS have evolved in some organisms. To date, INS-like trans-editing proteins have been shown to act on specific tRNAs mischarged with proteogenic amino acids. Here, we show that Rhodopseudomonas palustris ProXp-x, a previously uncharacterized INS homolog, displays robust editing of tRNAs mischarged with the non-protein amino acids α-aminobutyrate (2-Abu) and D-Thr in vitro. In vivo experiments have demonstrated that 2-Abu is toxic to an E. coli strain encoding an editing-defective valyl-tRNA synthetase, and expression of R. palustris ProXp-x rescues cell growth. Similarly, preliminary in vivo experiments indicate D-Thr is toxic to E. coli when overexpressing threonyl-tRNA synthetase, and co-expression of R. palustris ProXp-x can rescue the growth defect . An R. palustris ProXp-x deletion strain has been constructed and experiments are underway to characterize growth phenotypes under various conditions . Metabolomics studies are also in progress to further understand which environmental conditions cause intracellular accumulation of 2-Abu and D-Thr . Based on our results, we hypothesize that editing by ProXp-x may mitigate misincorporation of non-protein amino acids under environmental conditions or stresses in which non-protein amino acids pose a greater threat to the cell.
Keywords: trans-editing, aminoacyl-tRNA synthetases, Rhodopseudomonas palustris
78. Fluorescence Enhancing Aptamers in Biosensing Applications
Volition La (University of Waterloo, Canada), Thorsten Dieckmann (University of Waterloo, Canada)
Abstract:
Presently, an aptamer that binds to a derivative of thiazole orange is being studied. Once bound, the fluorescence of the ligand is enhanced up to 1200 fold with a detection limit of in the pmol range. The use of this aptamer for use in a lateral flow assay is being explored due to the high signal to noise ratio conferred by this system. The kinetics of the binding will be studied using the Open SPR system.
SPR is a powerful tool that can be used to study the binding kinetics of biomolecules in a real-time fashion. The malachite green aptamer is used as a model system to explore aptamer binding to its ligand that is immobilized on a gold surface. It has been shown that the kinetics of the binding interaction is greatly affected by the identity and concentration of the positive ions in solution through isothermal titration calorimetry.
References:
1. Dolgosheina E V, Jeng SCY, Panchapakesan SSS, Cojocaru R, Chen PSK, Wilson PD, Hawkins N, Wiggins PA, Unrau PJ: RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking. ACS Chem. Biol. 2014, 9:2412–2420.
Da Costa JB, Andreiev AI, Dieckmann T: Thermodynamics and Kinetics of Adaptive Binding in the Malachite Green RNA Aptamer. Biochemistry 2013, 52:6575–6583.
Bernard Da Costa J, Dieckmann T: Entropy and Mg(2+) control ligand affinity and specificity in the malachite green binding RNA aptamer. Mol Biosyst 2011, 7:2156–2163.
Keywords: Aptamer, Lateral flow, SPR
79. LARP1 on TOP: molecular recognition of the 5’TOP motif by LARP1
Roni M. Lahr (University of Pittsburgh, Department of Biological Sciences), Hiba A. Al-Ashtal (University of Pittsburgh, Department of Biological Sciences), Andrea J. Berman (University of Pittsburgh, Department of Biological Sciences)
Abstract not available online - please check the printed booklet.
80. Characterizing the role of the DEAD-box protein Dbp2 in RNA 3’ end processing
Yu-Hsuan Lai (Department of Biochemistry, Purdue University), Siwen Wang (Department of Biochemistry, Purdue University), Elizabeth J. Tran (Department of Biochemistry, Purdue University; Purdue University Center for Cancer Research)
Abstract:
RNA helicases functions in all aspects of RNA biology through remodeling of RNA structures and ribonucleoprotein (RNP) complexes. Among them, DEAD-box (DDX) proteins constitute the largest RNA helicase family in eukaryotes, playing critical roles at different levels of gene expression, including transcription, splicing, and RNA transport. Deregulation of many human DDX genes has been observed in various types of cancers and neurological diseases. However, the in vivo functions and precise RNA binding targets of individual DEAD-box helicases still remain largely unknown. Previously, studies from our laboratory have demonstrated that a DEAD-box protein in Saccharomyces cerevisiae, Dbp2, is an ATP-dependent RNA helicase in vitro. In vivo, Dbp2 functions in transcription termination, as loss of DBP2 results in a transcriptional read-through defect in a model gene locus(1). Our group also showed that Dbp2 facilitates assembly of single-stranded RNA-binding proteins onto polyadenylated mRNA, suggesting that Dbp2 may utilize its RNA helicase activity to promote RNP assembly and termination of nascent transcripts(2).
To determine the relationship between Dbp2 binding and transcription termination, we undertook a genome wide approach. First, we utilized individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) combined with Next-Gen sequencing (iCLIP-seq). After removal or adaptor sequences and PCR duplicates, we have more than 20 million mappable reads in all three replicates, with a mapping rate of more than 95%. This resulted in identification of 1950 mRNAs as Dbp2 binding targets. Second, we conducted RNA-seq to identify RNA 3’ end extensions in dbp2Δ cells compared to the wild type. This revealed that around 1000 genes express extended transcripts in dbp2Δ cells. Among these aberrantly expressed mRNAs, ~40% are also bound by Dbp2. The Fisher’s exact test indicates that the binding of Dbp2 and the read-through defect in dbp2Δ are significantly correlated, suggesting that Dbp2 modulate mRNA 3’ end processing through direct binding on the transcripts. This work will enable us to determine the precise function of Dbp2 in co-transcriptional processes.
References:
1. Cloutier SC et al. J Biol Chem. 2012
2. Ma WK et al. J Mol Biol. 2013
Keywords: DEAD-box helicases, transcription termination, iCLIP
81. Construction and inspection of the polypeptide exit tunnel in the yeast 60S ribosomal subunit
Amber J. LaPeruta (Carnegie Mellon University), Dan M. Wilson (Carnegie Mellon University), John L. Woolford (Carnegie Mellon University)
Abstract:
Assembly of 60S ribosomal subunits in eukaryotes proceeds in a hierarchical fashion involving the entry and exit of approximately 80 assembly factors. Generation of mature 60S particles requires the timely construction of multiple functional centers, which may require passing quality control checkpoints. Among many of these functional centers is the polypeptide exit tunnel (PET). Recent cryo-EM structures of pre-ribosomal particles purified using Nog2-TAP, Arx1-TAP and Nmd3-TAP have given us new insights into the mechanisms involved in the assembly of this complex and interactive conduit which, until now, have remained elusive. In all three of these structures a theme arises: the C-terminal extensions of three assembly factors (first Nog1 followed by Rei1 then Reh1) probe the entire length of the exit tunnel, reaching to the peptidyl transferase center (PTC). These tails appear to interact with the rRNA lining of the PET and the tentacle-like extensions of ribosomal proteins L17 (μL22) and L4 (μL4), which form a constriction site in the PET. The mutually exclusive occupation of the PET by various assembly factors from Nog2’s lifetime onward raises several hypotheses. The C-terminal extensions of Nog1, Rei1 and Reh1 may function as scaffolds for the folding of rRNA in and around the exit tunnel. Alternatively, they may act as tunnel inspectors or prevent premature function of incomplete ribosomes. To gain insight into the function of these extensions and the assembly of the PET as a whole, we are manipulating the C-terminal extensions of Nog1, Rei1 and Reh1 as well as the β-hairpin of L17 and the extended loop of L4 in S. cerevisiae using deletions, substitutions, and the addition of bulky moieties. These mutant strains are being assessed for growth, polysome profile, and rRNA processing defects as well as by purification and analysis of pre-ribosomal composition.
Keywords: Ribosome, Polypeptide Exit Tunnel, Assembly
82. Physiochemical insights into an intrinsically disordered domain of hnRNP A1
Jeffrey D. Levengood (Case Western Reserve University, Department of Chemistry), Christopher E. Morgan (Case Western Reserve University, Department of Chemistry), Blanton S. Tolbert (Case Western Reserve University, Department of Chemistry)
Abstract:
The hnRNP A1 protein plays a key role in eukaryotic RNA processing events. It 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. The G-CTD, which is a low complexity sequence domain, has not been very well characterized. The G-CTD is essential to the regulation of hnRNP A1 activity as it is responsible for protein-protein interactions with other regulatory proteins.
A preliminary view of the structure of full length hnRNP A1 has been obtained through SAXS-scored structural modeling. This model revealed the C-terminal domain to be a long, unstructured, disordered polypeptide chain extending away from the UP1 domain. This was confirmed by NMR HSQC data. When compared to full length hnRNP A1, the residues for UP1 showed minimal chemical shifts. Similar results were obtained for a comparison between full length hnRNP A1 and the G-CTD itself.
Recent research has shown the G-CTD to be a low complexity sequence 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 mRNPs stalled in translation. The mRNA binding properties of hnRNP A1 provide it with the ability to sequester away stalled transcripts in this manner. In order to elucidate the role of hnRNP A1 in this process, we are studying the conditions under which it undergoes LLPS and the role RNA binding plays in mediating it. We have found LLPS is induced by low salt and low temperatures. The effects on LLPS in the presence of HIV-1 ESS3, a RNA structural element previously shown to regulate splicing, are also being examined.
Keywords: RRM, Stress Granules, Splicing Regulation
83. Controllable self-assembly of molecularly defined RNA tetrahedrons for cancer targeting and therapeutics delivery
Hui Li (Division of Pharmaceutics and Pharmaceutical Chemistry; Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute; The Ohio State University), Kaiming Zhang (Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine), Fengmei Pi, Sijin Guo, Dan Shu (Division of Pharmaceutics and Pharmaceutical Chemistry; Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute; The Ohio State University), Luda Shlyakhtenko (Department of Pharmaceutical Sciences, University of Nebraska Medical Center), Wah Chiu (Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine), Peixuan Guo (Division of Pharmaceutics and Pharmaceutical Chemistry; Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute; The Ohio State University)
Abstract:
Since RNA shows an integer of 11 nucleotides per helix turn of 360°, in comparison to DNA that shows a non-integer of 10.5 nucleotides per helix turn, we propose that different sizes of RNA 3D structures can be constructed with precise control and the size of the particle will increase linearly by increasing the nucleotide number as multiples of eleven. Herein, we used the ultra-stable pRNA-3WJ motif with controllable angles and arm lengths to construct tetrahedral architectures composed purely of RNA via one-pot bottom-up assembly with high efficiency and thermal stability. By introducing arm sizes of 22 bp and 55 bp, respectively, we constructed two RNA tetrahedrons with similar global contour structure but with different sizes of 8 nm and 17 nm, respectively. AFM and cryo-EM imaging clearly demonstrated the 3D shapes of RNA tetrahedrons that were consistent with the designs. The RNA tetrahedrons were also highly amenable to functionalization. Fluorogenic RNA aptamers, ribozyme, siRNA, and protein-binding RNA aptamers were integrated into the tetrahedrons by simply fusing the respective sequences with the tetrahedral core modules. Upon systemic injection, 2'-F modified RNase resistant RNA tetrahedrons harboring EGFR aptamers specifically targeted orthotopic breast tumors without getting trapped in healthy organs. The favorable biodistribution highlight their potentials as safe and specific drug delivery vectors. The reported design principles can be extended to construct higher order polyhedral RNA architectures for various applications in nanomedicine and nanotechnology.
References:
1. Guo P. The emerging field of RNA nanotechnology. Nature Nanotechnology. 2010; 5:833.
2. Shu D, Shu Y, Haque F, Abdelmawla S and Guo P. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology. 2011; 6:658.
3. Li H, Lee T, Dziubla T, Pi F; Guo S, Xu J, Li C, Haque F, Liang, X-J, Guo P. RNA as a Stable Polymer to Build Controllable and Defined Nanostructures for Material and Biomedical Applications. Nano Today. 2015; 10:631.
4. Li H, Zhang K, Pi F, Guo S, Shlyakhtenko L, Chiu W, Shu D, Guo P. Controllable Self-Assembly of RNA Tetrahedrons with Precise Shape and Size for Cancer Targeting. Advanced Materials. 2016;28:7501.
Keywords: RNA Nanotechnology, Bacteriophage Phi29 pRNA, RNA Nanoparticles
84. Alternative RNA splicing accompanies loss of ELL2 in mouse B cells
Zhitao Liang (Immunology, University of Pittsburgh), Sage Smith (Immunology, University of Pittsburgh), Nolan Carew (Immunology, University of Pittsburgh), Ashley Dunham (Immunology, University of Pittsburgh), Christine Milcarek (Immunology, University of Pittsburgh)
Abstract:
The ELL2 transcription elongation factor is induced by Lipopolysaccharide treatment of B cells and is crucial for the subsequent processing of secretory-specific immunoglobulin heavy chain mRNA (1). A conditional knockout of ELL2 in mouse B cells shows reduced amounts of: IgH secretory mRNA, serum Ig, the secretory protein apparatus, as well as antibody secreting and long-lived plasma cells (2). ELL2 thus drives Ig mRNA production and the unfolded protein response leading to Ig secretion. Human mutants in ELL2 give rise to myeloma (a tumor of long lived plasma cells), reduced serum Ig, and increased susceptibility to infectious diseases.
To investigate a potential role for ELL2 in myeloma formation, we have investigated alternative splicing in B cell development. Using mRNA seq and ChIP-seq we have identified several alternatively spliced ELL2 target genes in the ELL2 conditional knockout B cells. One interesting alternatively spliced gene is XafI, the full length version of which normally interacts with XIAP to modulate cell growth. In many types of tumor cells exon 4 of XafI is frequently deleted by splicing, as it is in ELL2 conditional knockout cells. We are determining if the splicing variations in XafI contribute to changes in the growth properties of the ELL2 knockout cells and possibly to myeloma. Modifications of the RNAP-II elongation by ELL2 thus may have far-reaching consequences in the immune system.
References:
1. Martincic, K., S. A. Alkan, A. Cheatle, L. Borghesi, and C. Milcarek. 2009. Transcription elongation factor ELL2 directs immunoglobulin secretion in plasma cells by stimulating altered RNA processing. Nat Immunol 10:1102-1109.
2. Park, K. S., I. Bayles, A. Szlachta-McGinn, J. Paul, J. Boiko, P. Santos, J. Liu, Z. Wang, L. Borghesi, and C. Milcarek. 2014. Transcription elongation factor ELL2 drives immunoglobulin secretory specific mRNA production and the unfolded protein response. J. Immunol. 193:4663-4674.
Keywords: Splicing, Transcription, B cell development
85. Structural analyses of human and mouse NEAT1 lncRNAs suggest long-range RNA interactions contribute to paraspeckle architecture
Yizhu Lin (Carnegie Mellon Universty, Department of Biological Sciences), Gemma May (Carnegie Mellon Universty, Department of Biological Sciences), Joel McManus (Carnegie Mellon Universty, Department of Biological Sciences)
Abstract:
In the past decade, lncRNAs have been increasingly recognized as important regulators of gene expression. Because they don’t encode proteins, lncRNA structures and protein interactions are believed to be important for their functions. One relatively abundant lncRNA, NEAT1, functions in the formation of paraspeckles - nuclear bodies that have been implicated in multiple stress responses and diseases. NEAT1 has two isoforms produced by alternative 3’ end processing. The short isoform (NEAT1_v1) is 3.7 kb and contains a polyA tail, while the long isoform (NEAT1_v2) is 23 kb in length and has a 3’ end produced by RNase P cleavage. Though the NEAT1 primary sequence is not well conserved, the two isoforms and their function in paraspeckle formation were observed in both human and mouse cells. NEAT1 has a highly ordered spatial organization within the paraspeckle, in which NEAT1_v1 and both the 5’ and 3’ ends of NEAT1_v2 are localized to the periphery. The central sequences of NEAT1_v2 are found within the core 1 . The structural features that maintain this spatial organization remain unknown, however it was suggested that interactions among proteins bound to NEAT1 may help maintain paraspeckle structure 1 .
We combined experimental RNA secondary structure probing (Mod-seq 2 ) and computational structural analyses to investigate the structural features of NEAT1. The full length human and mouse NEAT1_v1 structures were probed using SHAPE and Mod-seq. By comparing the structures of human and mouse NEAT1, we identified conserved structural features. Our results indicate NEAT1_v1 is highly folded, and several regions have specific local secondary structural signatures conserved between human and mouse. Further computational analyses suggest that the 5’ end of NEAT1_v2 (or NEAT1_v1) and the 3’end of NEAT1_v2 form long-range base pairs with each other, which may contribute to the co-localization of NEAT1 5’ and 3’ ends. This interaction was verified in vitro using an RNA intermolecular gel-shift assay. Our results suggest that the secondary structure of NEAT1 and the long-range interactions among NEAT1 transcripts may have important architectural functions in paraspeckle formation.
References:
1. Souquere, S., Beauclair, G., Harper, F., Fox, A. & Pierron, G. Highly ordered spatial organization of the structural long noncoding NEAT1 RNAs within paraspeckle nuclear bodies. Mol Biol Cell 21, 4020–4027 (2010).
2. Talkish, J., May, G., Lin, Y., Woolford, J. L. & McManus, C. J. Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA 20, 713–20 (2014).
Keywords: NEAT1, RNA structure, lncRNA
86. Capsid and SP1 domains of HIV-1 Gag contribute to genomic RNA binding selectivity
Shuohui Liu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Erik D. Olson (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Ioulia Rouzina (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH)
Abstract:
HIV-1 Gag is a polyprotein consisting of matrix (MA), capsid (CA), nulceocapsid (NC), and p6 domains, in addition to two short spacer peptides, SP1 and SP2. Both NC and MA domains are highly basic nucleic acid binding domains. How Gag selectively packages its cognate genomic RNA (gRNA) over the vast excess of cellular RNAs is not understood. Here, we study the effect of Gag mutations on its in vitro ability to distinguish between various specific and non-specific fragments of gRNA. We have previously shown that WT HIV-1 Gag binds the Psi RNA (packaging signal) and a non-Psi RNA with similar affinity at physiological salt (~150 mM NaCl), but that the salt dependence of these two binding events differs dramatically (Webb, JA, et al, RNA 2013). Gag binds Psi RNA with a strong non-electrostatic binding component and a small effective charge (Zeff~5), which we hypothesize is due to an NC-only binding mode. In contrast, HIV-1 Gag binds non-Psi RNA with a very weak non-electrostatic component and a larger effective charge (Zeff~9) that is consistent with a contribution from both NC and MA domains. In new work, a WM to AA mutation in the CA C-terminal domain, which eliminates Gag dimerization, reduces Psi RNA binding selectivity while retaining the NC-only binding mode, similar to a Gag variant lacking the MA domain. Interestingly, a Gag deletion mutant in which the CA domain is replaced by a 16-residue flexible linker, retains partial selectivity for Psi. Another linker variant, which additionally lacks SP1, almost completely loses selectivity. SP1 has been shown to be capable of undergoing a concentration-dependent random coil to helix transition, and is helical in the immature virus particle. Taken together, our observations suggest that the selectivity of Gag for gRNA may be regulated by a global Gag conformational change triggered by specific Psi RNA binding, and additionally requires CA domain protein-protein contacts, as well as a local SP1 conformational switch.
References:
Webb, Joseph A., et al. "Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging." RNA 19.8 (2013): 1078-1088.
Keywords: HIV-1, Gag, genomic RNA
87. Regulation of the translation by eukaryotic translation initiation factor 4B
Xiaozhuo Liu (Department of Biological Sciences, The State University of New York at Buffalo), Sarah E. Walker (Department of Biological Sciences, The State University of New York at Buffalo)
Abstract:
Eukaryotic translation initiation factors (eIFs) interact with the ribosome, mRNA elements and protein cofactors to regulate both global levels of translation, as well as the levels and isoforms of proteins translated from specific mRNAs. The RNA Recognition Motif (RRM)-containing translation initiation factor eIF4B was historically known as an RNA-binding protein, and thought to enhance eIF4F unwinding activities by binding to single-stranded regions of unwound capped mRNAs. Our previous work demonstrated that the RRM and RNA-binding activities of eIF4B are actually dispensable for growth and translation, and further showed that eIF4B binds directly to the small (40S) ribosomal subunit to promote changes in the mRNA entry channel. Here we have analyzed the effects of eIF4B on cellular fitness under ~1400 different conditions and found that eIF4B promotes growth in response to various stresses. The results of these studies highlight the importance of eIF4B in adapting to external conditions, and allow us to tie mechanical functions of a translation factor to specific phenotypes.
Keywords: Eukaryotic translation initiation factors
88. STRUCTURAL basis for selectivity by aminoacyl-tRNA trans-editing factors
Xiao Ma (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Eric M. Danhart, Marina Bakhtina, Will Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Lexie Kuzmishin, Brianne Sanford (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Ziwei Liu, Bradley C. Howard (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Marija Kouti, Ronald Micura (Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University), Karin Musier-Forsyth, Mark P. Foster (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for charging cognate amino acids on their corresponding tRNAs, which are then shuttled to the ribosome by EF-Tu so that the amino acid can be incorporated into the elongating peptide chain during protein translation. The structural features of the aaRSs catalytic domains provide a high degree of selectivity for the correct amino acid; however, given the similar chemical properties of many of the amino acids, errors in tRNA charging can occur, thus leading to the mistranslation of the genetic code. In addition to the selectivity provided by the synthetases, in many organisms, there are editing enzymes that function in trans to hydrolyze mischarged tRNAs to maintain the high fidelity of translation. ProXp-ala, a homolog of the editing domain from ProRS, can deacylate the mischarged tRNA, Ala-tRNAPro. To determine how ProXp-ala is selective for Ala-tRNAPro and doesn’t deacylate Pro-tRNAPro or Ala-tRNAAla, NMR and site-directed mutagenesis mapping are performed with uncharged microhelixPro and a non-hydrolyzable, amide-linked Ala-microhelixPro substrate analog. Relaxation experiments show that an α-helix, exhibiting dynamics on the ps-ns timescale, is less mobile with Ala-microhelixPro, but remains dynamics when bound to the uncharged microhelixPro. We propose that conformational selection by ProXp-ala allows substrate sampling and Ala-tRNAPro-specific induced-fit binding via a flexible helix. Another ProRS editing enzyme, Yeak, has been shown in vitro to deacylate multiple mischarged tRNAs (Ser-tRNAAla/Lys/Thr and Thr-tRNALys/Ile/Val). To identify the interaction between Yeak and its multiple substrates, we are using X-ray crystallography and NMR to identify the structural basis for broad selectivity of this generalist enzyme.
References:
1.Das M., Vargas-Rodriguez O., Goto Y., Novoa, E., Pouplana L., Suga H., Musier-Forsyth K., (2014). Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation. Nucl. Acids Res. 42 (6):3943-3953.
2.Ling J, Reynolds N, Ibba M (2009) Aminoacyl-tRNA synthesis and translational quality
control. Annu Rev Microbiol 63:61–78.
3.Liu Z., Vargas-Rodriguez O., Goto Y., Novoa, E., Pouplana L., Suga H., Musier-Forsyth K., (2015). Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation. PNAS May 12, 2015 vol. 112 no. 196027-6032
Keywords: trans-editing, NMR, RNA-protein interaction
89. A study of aminoglycosides binding to 16S rRNA
Prabuddha Madubashitha (Department of Chemistry, Wayne State University, Detroit, MI 48202), Evan Jones (Department of Chemistry, Wayne State University, Detroit, MI 48202), Laimar Garmo (Department of Chemistry, Wayne State University, Detroit, MI 48202), Christine S. Chow (Department of Chemistry, Wayne State University, Detroit, MI 48202)
Abstract:
Adverse health effects including ototoxicity is one of the major challenges of using aminoglycosides (AG) as antibiotics. In developing AG-based antibiotics with low ototoxic potential, knowledge of the binding of a particular AG to its target at the molecular level is important. The objective of our study is to gain a better understanding of the binding affinity and drug-induced structural changes of classical and modified AGs with helix 44 (h44) at the aminoacyl-tRNA site (A site) of 16S rRNA. The interactions between AGs and h44 constructs from E. coli, wild-type human mitochondria, and human mitochondrial mutant were studied by using a fluorescence-based assay and nuclear magnetic resonance spectroscopy. The data reveal a difference between binding of AG analogues to wild-type human mitochondrial h44 compared to E. coli and human mitochondrial mutant h44 constructs, which can be used to explain the observed AG-related ototoxicity. These results will lead to a better understanding of drug-rRNA interactions at the molecular level.
Keywords: aminoglycosides, ototoxicity, mitochondria
90. Identifying mRNA Targets of Nanos in Drosophila melanogaster
Mohammad Marhabaie (Department Molecular Genetics and Department of Cancer Biology and Genetics, The Ohio State University), Sung Y. Kim (Department Molecular Genetics and Department of Cancer Biology and Genetics, The Ohio State University), Tammy Wharton (Department Molecular Genetics and Department of Cancer Biology and Genetics, The Ohio State University), Sushmita Ghosh (Department Molecular Genetics and Department of Cancer Biology and Genetics, The Ohio State University), Robin P. Wharton (Department Molecular Genetics and Department of Cancer Biology and Genetics, The Ohio State University)
Abstract:
Nanos (Nos) is a translational regulator that specifies abdominal segmentation of the embryo by repressing a single mRNA, hunchback (hb) in the posterior. Ectopic Nos represses a second mRNA, bicoid (bcd), in the anterior. In addition to controlling body patterning, Nos regulates other, as yet unknown mRNAs to govern diverse processes including maintenance of germline stem cells, various aspects of primordial germ cell biology, and dendritic morphology.
To identify mRNAs regulated by Nos, we have expressed a Upf1-Nos fusion protein in flies, on the assumption that the chimera would degrade rather than repress targeted mRNAs. Several lines of evidence suggested this is indeed the case; for example, embryos from females expressing Upf1-Nos have neither bcd nor hb mRNA in the anterior and develop a bicaudal body plan. Quantitation of staged embryos reveals that hb mRNA is depleted ~140-fold from Upf1-Nos embryos.
We next used RNA-seq to identify mRNAs depleted in Upf1-Nos embryos, compared to control embryos expressing Upf1 or Nos alone. These experiments reveal that 2834 mRNAs, 42% of the maternal transcriptome, are significantly depleted by Upf1-Nos. We are currently focusing on the pathways that are over-represented among abundant mRNAs that are highly depleted by Upf1-Nos, testing the biological role of Nos-dependent regulation in vivo.
Keywords: Nanos, mRNA translation
91. Context Specific Action of Linezolid and Chloramphenicol
James Marks (Center for Biomolecular Sciences, University of Illinois at Chicago), Amira Kefi (Center for Biomolecular Sciences, University of Illinois at Chicago), Dorota Klepacki (Center for Biomolecular Sciences, University of Illinois at Chicago), Krishna Kannan (Center for Biomolecular Sciences, University of Illinois at Chicago), Nora Vazquez-Laslop (Center for Biomolecular Sciences, University of Illinois at Chicago), Alexander Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago)
Abstract:
The generally accepted mechanism of action of the classic antibiotic chloramphenicol (CHL) and the newer drug linezolid (LNZ) is inhibition of peptide bond formation. Because both antibiotics bind in the A-site of the peptidyl transferase center (PTC), they obstruct accommodation of aminoacyl-tRNAs and, thus, it is assumed that they prevent the formation of any peptide bond. Thereby, it is anticipated that exposure of bacterial cells to CHL or LZD should non-specifically and equally efficiently arrest translating ribosomes at any codon along mRNA.
We employed the genome-wide technique of ribosome profiling to examine drug-induced changes in the distribution of ribosomes along cellular mRNAs. Neither of the drugs produced the expected uniform arrest of ribosomes on mRNA. Strikingly, the data revealed that the antibiotic-arrested ribosomes preferentially carried a nascent peptide whose penultimate amino acids were Ala, and less frequently Ser or Thr. Subsequent biochemical experiments in a cell-free translation system confirmed that neither one of these compounds has a global inhibitory effect but instead, block formation of only specific peptide bonds, based on the sequence context of the protein being made.
Remarkably, the CHL mediated programmed translation arrest which regulates inducible drug resistance in bacteria, exploits the same principle: the arresting sequence of the regulatory nascent peptides contains an Ala or Thr residues in their penultimate positions.
Our findings suggest that the mechanisms of action of this important class of antibiotics have been largely misunderstood and reveal that the nascent peptide, in a sequence specific manner, affects interactions of the substrates with the ribosomal A site and define the preferential sites of action of the PTC inhibitor by either promoting the antibiotic binding or slowing the rate of accommodation of aminoacyl-tRNAs.
References:
Oh E, et al. (2011) Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147(6):1295-1308.
Lovett PS (1996) Translation attenuation regulation of chloramphenicol resistance in bacteria - A review. Gene 179:157-162.
O'Shea JP, et al. (2013) pLogo: a probabilistic approach to visualizing sequence motifs. Nat Methods 10(12):1211-1212.
Leach KL, et al. (2007) The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria. Mol Cell 26(3):393-402.
Dunkle JA, Xiong L, Mankin AS, Cate JH (2010) Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc Natl Acad Sci USA 107(40):17152-17157.
Keywords: Antibiotic, Ribosome, Translation
92. Title not available online - please see the printed booklet.
Sophie Martin (Center for RNA molecular biology, Case Western Reserve University, Cleveland, OH 44106), Jeff Coller (Center for RNA molecular biology, Case Western Reserve University, Cleveland, OH 44106)
Abstract not available online - please check the printed booklet.
93. Understanding substrate specificity of tRNAHis guanylyltransferase (Thg1)
Ashanti Matlock (Chemistry and Biochemistry), Jane Jackman (Chemistry and Biochemistry)
Abstract not available online - please check the printed booklet.
94. Oncogenic effects of unprocessed microRNAs
Phillip J. McCown (Department of Biological Sciences, Purdue University), Hana Kubo (Department of Biological Sciences, Purdue University), Andrea L. Kasinski (Department of Biological Sciences, Purdue University)
Abstract not available online - please check the printed booklet.
95. Programmed frameshifting generates a copper transporter and a copper chaperone from the same gene
F. Sezen Meydan (Center for Biomolecular Sciences, University of Illinois at Chicago, IL, USA), Dorota Klepacki, Subbulakshmi Karthikayen, Tonu Margus (Center for Biomolecular Sciences, University of Illinois at Chicago, IL, USA), Paul Thomas (Proteomics Center of Excellence, Northwestern University, Chicago, IL, USA), John Edward Jones, Yousuf Khan, Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA), Nora Vazquez-Laslop, Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago, IL, USA)
Abstract:
Ribosome profiling of E. coli cells showed an abrupt interruption of translation at the 70th codon of the copA gene that encodes a copper transporter. Our in vivo and in vitro experiments showed that the interruption of copA translation is due to a novel case of -1 programmed ribosomal frameshifting (PRF). The PRF within copA results in production of the full-size 834 amino acid-long transporter CopA and a 70 amino acid-long truncated product that we named CopA(Z) from a single gene. The PRF within copA gene occurs with a high efficiency nearing 50%. We have identified three main elements stimulating the -1 PRF of copA: (1) the presence of the slippery sequence CCC-AAA-G, (2) a downstream mRNA pseudoknot, and (3) the sequence of the CopA nascent chain in the ribosomal exit tunnel. Our phylogenetic analysis revealed that the stimulatory signals of -1 PRF in copA are evolutionarily conserved among a wide variety of bacteria, suggesting that the PRF is a common mechanism of regulating expression of this gene, which confers fitness advantage. Indeed, co-growth experiments demonstrated that cells carrying mutations that disrupt the slippery sequence, and hence lack the ability to produce CopA(Z) lose in competition with wt cells when challenged by toxic concentrations of copper. Since E. coli lacks an independent gene encoding for a copper chaperone, we suggest that this role is achieved by the PRF product CopA(Z) in E. coli and other bacteria. Strikingly, the same slippery sequence CCC-AAA-G is also observed in ATP7B gene encoding the CopA homolog in human. The dual luciferase reporter assays in HEK293T cells demonstrated that this sequence together with a predicted downstream pseudoknot is indeed able to induce -1 frameshifting with about 12% efficiency. Moreover, a similar arrangement of SS and a downstream pseudoknot is found in ATP7B homologs of higher primates. It is possible that in these organisms, -1 PRF could be involved in generating a copper transporter and a copper chaperone, with related but distinct functions in copper management. Our findings hence illuminate a unique translation regulation system crucial for the copper homeostasis in bacteria and likely in human and other eukaryotes.
Keywords: programmed frameshifting, ribosome profiling, pseudoknot
96. Revisiting the catalytic mechanism of the hammerhead ribozyme
Aamir Mir (Department of Biochemistry, Purdue University), Ji Chen (Department of Biochemistry, Purdue University), Kyle Robinson (Department of Biochemistry, Purdue University), Emma Lendy, Jaclyn Goodman (Purdue University), David Neau (Department of Chemistry and Chemical Biology, Cornell University, Northeastern Collaborative Access Team, Argonne National Laboratory), Barbara L. Golden (Department of Biochemistry, Purdue University)
Abstract:
The hammerhead ribozyme catalyzes the site specific cleavage of a single phosphodiester bond within its RNA substrate. Originally discovered in plant viroids, thousands of hammerhead-like sequences have been found in all domains of life. The cleavage mechanism of the hammerhead ribozyme has been studied extensively but many questions have persisted. The crystal structure of the ribozyme suggests that G12 is the general base as it is well positioned to activate the 2’-hydroxyl of C17 for an in-line nucleophilic attack on the scissile phosphate. Yet, the pH dependence of the G12A mutant is similar to that of the wild-type ribozyme and it is unclear how G12 is activated for proton transfer. In addition, discrepancies exist between the crystal structure and the biochemical data concerning the role of divalent metals in the catalytic mechanism. The crystal structures do not show a divalent metal within the active site of the hammerhead ribozyme. Biochemical data suggest that divalent metals greatly accelerate the hammerhead reaction rate and appear to interact with the pro-Rp oxygen of the scissile phosphate. We studied different hammerhead mutants using both kinetics and X-ray crystallography to address these discrepancies. Our results suggest that conformational changes and two divalent metals are playing important roles in the catalysis of the hammerhead ribozyme.(1-2) Furthermore, based on the pH-rate profile and the divalent metal specificity switch observed in the G12A mutant, we propose that the imino tautomeric species of A12 serves as the general base in G12A hammerhead ribozyme.
References:
1. Mir, A., Chen, J., Robinson, K., Lendy, E., Goodman, J., Neau, D., and Golden, B. L. (2015) Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction, Biochemistry 54, 6369-6381.
2. Mir, A., and Golden, B. L. (2016) Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue, Biochemistry 55, 633-636.
Keywords: hammerhead, ribozyme, self-cleaving
97. Title not available online - please see the printed booklet.
Chaitali Misra (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign, IL), Darren J. Parker , Cole Lewis, Sandip G. Chorghade (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign, IL), Jamila Hedhli, (Department of Bioengineering, University of Illinois, Urbana-Champaign, IL), Thomas A. Cooper (Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX), Wawrzyniec L. Dobrucki (Department of Bioengineering, University of Illinois, Urbana-Champaign, IL), Auinash Kalsotra (Department of Biochemistry and Medical Biochemistry, Institute of Genomic Biology, University of Illinois, Urbana-Champaign, IL)
Abstract not available online - please check the printed booklet.
98. Non-canonical 5'-end maturation pathways in Caenorhabditis elegans
Nick Mitchell (The Ohio State University), Jane Jackman (The Ohio State Univeristy)
Abstract not available online - please check the printed booklet.
99. A genome-wide investigation of light intensity effects on the maize RNA structurome
David Mitchell (Department of Chemistry, Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry, Pennsylvania State University), Sarah M. Assmann (Department of Biology, Pennsylvania State University), David H. Mathews (Department of Biochemistry & Biophysics, University of Rochester Medical Center)
Abstract:
Besides being a primary staple food for human consumption and the most important crop in terms of world production, maize is a vital source of animal feed and biofuel. Efforts have been made to increase maize yields by increasing planting density. Dense planting of maize increases the canopy cover and blocks sunlight from reaching the lower leaves, thus stimulating shade avoidance responses in these plants. While shade avoidance increases a plant’s survivability, it does so at a cost to reproductive fitness and potentially yield. As RNAs are involved in a diverse set of essential biological functions, shade avoidance responses may derive from in altered RNA structure. We propose to improve and apply Structure-seq to reveal, genome-wide, in vivo changes in RNA structure and protein interactions caused by low light environments in maize. Structure-seq has been used previously to reveal novel in vivo RNA structural features in Arabidopsis thaliana such as triplet periodicity within the gene coding sequence. However, Structure-seq can only probe adenine and cytosine bases, limiting the amount of information obtained by this method. Thus, we seek to establish capability within Structure-seq to distinguish RNA secondary structure formation from protein binding to RNA by applying new structure-probing chemical reagents to study guanine and uracil bases. Furthermore, we propose the use of in vivo crosslinking via photoactivatable nucleoside analogues to obtain direct proximity information on RNA structure formation and protein binding to RNA. As Structure-seq requires computational structure prediction to infer secondary structure from chemical probing data, we further propose altering the RNA-prediction algorithm to incorporate constraints from proximity information, sequence complementarity, and known protein-binding motifs. Information obtained from the use of Structure-seq can contribute to genetic engineering of maize to improve yield under high planting densities.
References:
Ding, Y., et al., Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq. Nat Protoc, 2015. 10(7): p. 1050-66.
Ding, Y., et al., In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature, 2014. 505(7485): p. 696-700.
Aphalo, P., C. Ballare, and A. Scopel, Plant-plant signalling, the shade-avoidance response and competition. J. Exp. Bot., 1999. 50(340): p. 1629-1634.
Dubois, P.G. and T.P. Brutnell, Topology of a maize field: distinguishing the influence of end-of-day far-red light and shade avoidance syndrome on plant height. Plant Signal Behav, 2011. 6(4): p. 467-70.
Keywords: Structure-seq, RNA Structure, Genome-wide
100. Title not available online - please see the printed booklet.
Nicholas Monteleone (Microbiology, Biochemistry, & Molecular Genetics, Rutgers Biomedical and Health Sciences), Joseph Iacona (Microbiology, Biochemistry, & Molecular Genetics, Rutgers Biomedical and Health Sciences), Carol Lutz (Microbiology, Biochemistry, & Molecular Genetics, Rutgers Biomedical and Health Sciences)
Abstract not available online - please check the printed booklet.
101. Splicing factors SRSF1 and SRSF2 regulation of MDM2 alternative splicing is conserved in mice and humans
Matias Montes (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), Daniel Comiskey (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), John McLaughlin (Center for Molecular and Human Genetics, Nationwide Childrens Hospital), Rehan Hussain (Center for Molecular and Human Genetics, Nationwide Childrens Hospital), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital)
Abstract:
The oncogene MDM2 undergoes alternative splicing and one of its spliced isoforms, MDM2-ALT1, is highly expressed in several cancers such as lung carcinoma, liposarcoma or rhabdomyosarcoma, correlating with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress. Nevertheless, the way that MDM2 splicing is controlled is not entirely understood. Previously we have utilized genotoxic stress to study the induction of the MDM2-ALT1 isoform expression in a minigene system. Using this system, we have seen that the splicing factors SRSF1 and SRSF2 act antagonistically to regulate MDM2 alternative splicing in response to stress. To determine the role of the SRSF1 and SRSF2 binding sites in the context of the endogenous MDM2 gene alternative splicing, we will use genome editing techniques for both the mouse and human MDM2 genes. Our hypothesis is that the cis elements that these factors bind are conserved in both organisms, leading us to propose that the mouse model will therefore act as a model system for testing splice altering therapies.
We have developed a damage-inducible mouse minigene, Mdm2 3-11-12s that recapitulates the splicing of the human MDM2 as well as the mouse endogenous gene by excluding its intervening exon under genotoxic stress (UV-C light). In order to study the conservation of these cis elements we used SELEX-based bioinformatics prediction algorhythms in ESEfinder 3.0 and identified conserved consensus sequences for splicing regulators SRSF1 and SRSF2 in exon 11 of the mouse Mdm2 gene. By using site-directed mutagenesis we generated mutations for the predicted SRSF1 and SRSF2 binding sites. Additionally, using the CRISPR-Cas9 technology we generated specific mutations of the binding sites in exon 11 of the mouse Mdm2 gene, to assess changes in alternative splicing. In the present work we report that regulation of Mdm2 by both SRSF1 and SRSF2 is conserved in humans and mice and that targeting their binding sites might be a mechanism to change p53 activity in specific cancer types.
Keywords: MDM2-ALT1, Alternative Splicing, Gene Editing
102. Applications of 5’-end sequence analysis of small RNA: Nucleotide addition by Thg1/BtTLP and TSS annotations in S. cerevisiae
Blythe Moreland (Department of Physics, Center for RNA Biology, The Ohio State University), Samantha Dodbele (Ohio State Biochemistry Program, Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Spencer Gardner (Center for RNA Biology, The Ohio State University), Jane Jackman (Ohio State Biochemistry Program, Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Ralf Bundschuh (Department of Physics, Department of Chemistry and Biochemistry, Center for RNA Biology, Division of Hematology and Department of Internal Medicine, The Ohio State University)
Abstract not available online - please check the printed booklet.
103. Exploring the function of Cdc5l during zebrafish segmentation
Zachary T. Morrow ( Dept. of Molecular Genetics, The Ohio State University), Thomas L. Gallagher ( Dept. of Molecular Genetics, The Ohio State University), Kiel T. Tietz ( Dept. of Molecular Genetics, The Ohio State University), Jasmine M. McCammon (Department of Molecular & Cell Biology, University of California, Berkeley), Sharon L. Amacher ( Dept. of Molecular Genetics, The Ohio State University)
Abstract:
Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. Oscillating genes include hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression. To achieve and maintain cycling, the segmentation clock is regulated at many levels, including post-transcriptional mechanisms that influence rates of mRNA export, decay, and translation. In a zebrafish screen for genes involved in oscillatory expression, we identified the honu locus as an important component of post-transcriptional regulation. Using genome-wide SNP association mapping, we molecularly identified the honu lesion as a nonsense mutation in the gene encoding the splicing factor Cdc5l. Cdc5l is a core component of the NineTeen Complex (NTC) and plays an important role in pre-mRNA splicing and mitotic progression in mammalian and yeast cells. In honu mutants, cyclic transcripts that are normally cleared rapidly during segmentation accumulate instead, and only about half the normal number of segments form. Using an intron-based probe that distinguishes nascent from mature her1 transcript by in situ hybridization, we find that transcriptional regulation of her1 is normal in honu mutants, and surprisingly, RT-PCR analysis indicates that her1 transcript is spliced normally. Therefore, her1 and likely other cyclic transcripts accumulate post-splicing upon loss of Cdc5l. High resolution imaging of her1 mRNA in honu mutants reveals a pronounced nuclear accumulation defect, suggesting that mRNA export is compromised and may arise due to improper pre-mRNA processing. We are currently investigating the nature and extent of mRNA processing defects in honu mutants to understand the developmental role of Cdc5l and to provide insight into why certain tissues and/or transcripts are particularly sensitive to loss of a core component of the spliceosome.
Keywords: Splicing, Development
104. Invivo expression of helix 69 binding peptides in bacteria
Nisansala Muthunayake (Chemistry Department Wayne State University), Wesley Colangelo (Chemistry Department Wayne State University), Philip R. Cunningham (Chemistry Department Wayne State University), Christine S. Chow (Chemistry Department Wayne State University)
Abstract:
Identifying novel drug targets within the bacterial ribosome is an important approach to overcome the well-known problem of antibiotic resistance. The specific region of the ribosome under investigation in this study is helix 69 (H69). Considering the variety of functions of H69 in protein biosynthesis, as well as differences between the bacterial and human H69 sequences, this RNA is an attractive antibacterial drug target. In a previous study, short peptides that specifically bind to H69 were isolated by using phage-display libraries. In a second approach, modified variants of the selected parent peptide sequence were synthesized to identify more tight binders. The main objective of the current study was to express these H69-targeting peptides in E. coli to investigate the inhibitory effects on protein synthesis. H69-targeting peptides were expressed in vivo as GFP-fusion proteins and their activities were monitored through cellular fluorescence levels. The seven-mer peptide sequence was cloned behind the Tobacco Etch Virus (TEV) protease recognition sequence. Expression of TEV protease from another plasmid in the same cell cleaves the TEV-recognition peptide sequence, and the peptide is then exposed at the N-terminus of GFP. The construct was prepared in which a his tag was placed at the C- terminus of the GFP gene such that the peptide could be purified for further experiments. Expression of two different peptides was shown to have an inhibitory effect on bacterial cell growth. These findings will be helpful for future antimicrobial drug development.
Keywords: helix 69, peptides, ribosome
105. Monitoring htrA RNA thermometer activity using a β-galactosidase assay in Escherichia coli
Yen Anh H. Nguyen (Department of Chemistry and Biochemistry, Denison University), Rachel M. Mitton-Fry (Department of Chemistry and Biochemistry, Denison University)
Abstract not available online - please check the printed booklet.
106. Insight into the requirements for UPF1 association with translating mRNA
Sarah L. Nock (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract:
Nonsense-mediated mRNA decay (NMD) is a cellular process in eukaryotes that acts primarily in the recognition of mRNAs containing premature termination codons (PTCs) and the targeting of these aberrant transcripts for rapid degradation. The core components of the NMD machinery are highly conserved and include the Up-frameshift proteins, UPF1, UPF2, and UPF3. UPF1 is an RNA-dependent ATPase (i.e. ‘RNA helicase’) with known RNA binding activity. We, and others, have demonstrated that UPF1 interacts with both normal and PTC-containing mRNAs, and are interested in better understanding the requirements for the association of UPF1 with mRNA.
In this study, we use sucrose gradient centrifugation (i.e. polysome analysis) to monitor the co-sedimentation of yeast UPF1 with translating mRNA, and to evaluate a role for UPF2, UPF3, and biochemical activities intrinsic to UPF1 for their ability to influence UPF1 association with polysomes. We find that the majority of cellular UPF1 is found on polysomes and that this association of UPF1 with translating RNA does not require either UPF2 or UPF3. We also show that mutations that disrupt the ability of UPF1 to either bind or hydrolyze ATP result in an increase in the amount of UPF1 associated with polysomes. Continuing work will be presented on the analysis of additional UPF1 mutants. Overall, our results provide insight into the requirements for UPF1 to interact with translating mRNA and contribute to our overall understanding of how the NMD machinery surveys and targets substrates to this important quality control pathway.
Keywords: nonsense-mediated mRNA decay, NMD, UPF1, polysomes
107. ADAR3 Inhibits RNA Editing at the Q/R site of GRIA2 Transcripts Though Direct Binding
Eimile Oakes (Genome, Cell and Developmental Biology Program, Indiana University), Ashley Anderson (Medical Sciences Program, Indiana University School of Medicine), Aaron A. Cohen-Gadol (Department of Neurosurgery, Indiana University, Goodman Campbell Brain and Spine), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine)
Abstract:
RNA editing is a cellular process in which RNA-editing enzymes modify transcripts in a site-specific matter. Adenosine to Inosine RNA editing at the Q/R site of a transcript encoding the glutamate receptor subunit B (GRIA2) changes the amino acid encoded from glutamine to arginine; this modification alters the calcium permeability of the glutamate receptor, having profound effects on cellular physiology. 100% of GRIA2 transcripts edited in the human brain; and, loss of editing at this site results in death of the organism. A reduction of GRIA2 editing has been observed in glioblastoma patients and has been shown to contribute to increased tumorigenesis; however, GRIA2 editing is not always correlated with reduction of its primary editing enzyme, ADAR2. The goal of these studies is to determine how editing of GRIA2 is regulated and the mechanism of that regulation. Humans express an editing deficient ADAR family member, ADAR3; we hypothesized that ADAR3 may regulate RNA editing, focusing on editing of GRIA2 mRNA.
We have established that ADAR3 expression inhibits GRIA2 RNA editing in the glioblastoma cell line, U87, and an immortalized normal human astrocyte (NHA) cell line. Furthermore, knockdown of ADAR3 in NHA cells leads to significantly increased editing. To interrogate the biological mechanism of ADAR3 inhibition of RNA editing, I took a two-pronged approach, expressing ADAR3 with mutations in annotated domains to determine its mode of action and immunoprecipitating ADAR3 to elucidate protein and RNA interactions. Our findings indicate that the dsRNA binding domains of ADAR3 are required for inhibition of RNA editing and that ADAR3 binds directly to GRIA2 mRNA transcripts. As the GRIA2 transcript is specifically edited by ADAR2 at the Q/R site, we suggest that ADAR3 directly competes with ADAR2 for binding to GRIA2 transcript, inhibiting RNA editing. To investigate the clinical relevance of these findings, we measure ADAR2 and ADAR3 expression and GRIA2 editing in glioblastoma tumors and matched adjacent brain tissue. We found that GRIA2 editing is reduced and that ADAR3 is overexpressed in a majority of the tumor samples compared to the normal adjacent tissue, suggesting aberrant ADAR protein expression in glioblastoma tumors that is correlated with reduced GRIA2 editing.
Keywords: RNA editing, GRIA2, glioblastoma
108. The effect of 5-Methyl Cytosine on Duplex Stability
Kayla Orr (Biochemistry, Allegheny College ), Abby Dishler (Biochemistry, Allegheny College ), Martin Serra (Biochemistry, Allegheny College )
Abstract:
Thermodynamic parameters for 5-Methylcytosine-guanosine (5MeC-G) pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for RNA duplexes with 5MeC-G pairs to improve nearest neighbor parameters for predicting stabilities of helixes. Duplexes where a 5Me-C-G is substituted for a C-G base pair are on average slightly less stable, by 0.5 kcal/mol on average. Nearest neighbor parameters for 5MeC-G base pairs derived from the data display a wider range of values (0.25-4.0 kcal/mol) than are observed for C-G base pairs (2.11-3.26 kcal/mol). On average, the nearest neighbor values for 5MeC-G base pairs 5’ of a Watson –Crick base pair are 1.7 kcal/mol less stable; while a 3’ 5MeC-G Watson-Crick nearest neighbor pair is 1.4 kcal/mole more stable than the corresponding C-G Watson-Crick nearest neighbor pair. A 5’MeC-G terminal base pair destabilizes a helix by 2.2 kcal/mol while a 3’MeC-G terminal base pair stabilizes a helix by 2.3 kcal/mol. These results should improve predictions of secondary structure for RNAs with modified base pairs.
Keywords: RNA thermodynamic stability, 5-Methyl Cytosine
109. Structural clues about the mechanism of RISC assembly
James A Brackbill (The Ohio State University, Department of Chemistry and Biochemistry.), Raul R. Araya Secchi (The Ohio State University, Department of Chemistry and Biochemistry.), Hong-Duc Phan (The Ohio State University, The Ohio State Biochemistry Program.), Mi Seul Park (The Ohio State University, Department of Chemistry and Biochemistry.), Daniel M Dayeh (The Ohio State University, The Ohio State Biochemistry Program.), Meng Sun, Marcos Sotomayor, Kotaro Nakanishi (The Ohio State University, Department of Chemistry and Biochemistry.)
Abstract not available online - please check the printed booklet.
110. Fluorescent microscopy of transcription factories by BrU and EdU labeled foci
Luke Parks (Department of Radiation Oncology, University of Michigan), Michelle Paulsen (Department of Radiation Oncology, University of Michigan), Mats Ljungman (Departments of Radiation Oncology and Environmental Health Sciences, University of Michigan)
Abstract:
Replication and transcription must often operate simultaneously, though the exact nature of this interplay is not fully delineated. Within the nucleus are thousands of RNA polymerases (RNAPol). It has been theorized that the RNAPol may aggregate to form what is referred to as transcription factories. These factories constitute hundreds of RNAPol complexes and may be fixed in location. Conversely, DNA polymerases (DNAPol) are also thought to be fixed to certain locations in the nucleus. By using high resolution fluorescent microscopy and short pulse-labeling of nascent RNA by bromouridine followed by immunocytochemistry using anti-BrdU antibodies, we visualized active transcription in individual human fibroblasts. Our results show about 200 labeled foci per cell representing active transcription factories. We are currently using EdU Click-iT to label nascent DNA in tandem with bromouridine-labeling of nascent RNA, to get a snapshot of the simultaneous nuclear localization of both DNA and RNA synthesis. We hypothesize that the coordination of transcription and replication is aberrant in cancer cells leading to replication stress and genomic instability.
Keywords: Transcription factories, Nascent RNA, Replication
111. Title not available online - please see the printed booklet.
Krishna J Patel (Dept. of Chemistry and Biochemistry; The Ohio State University), Paul Yourik (Dept. of Chemistry and Biochemistry; The Ohio State University), Jane E Jackman (Dept. of Chemistry and Biochemistry; The Ohio State University)
Abstract not available online - please check the printed booklet.
112. Sequential and dynamic RNA:RNA base-pairing interactions between U6atac and U12 snRNAs predicted to form Helix 1a and Helix 1b
Maitri Patel (Biology Department at Cleveland State University), Jagjit Singh (Biology Department at Cleveland State University), Girish Shukla (Biology Department at Cleveland State University)
Abstract:
In eukaryotes, pre-mRNA splicing is an important step for gene expression. Splicing is a two-step process which is carried out by a multi-megadalton molecular weight ribonucleoprotein (RNP) machinery called spliceosome. Spliceosome converts pre-mRNA to mRNA by removing non-coding sequence (introns) and splice together coding sequence (exons). Mammalian pre-mRNA are spliced by two different class of spliceosomes which are known as U2- and U12- dependent spliceosomes. U12 dependent spliceosome is composed of five small nuclear RNAs (snRNA). As compared to U2-dependent spliceosome, there is very less known about the catalytic process of U12-dependent splicing. U6atac and U12 snRNA are central to U12-dependent splicing. Therefore, to understand importance of U6atac and U12 snRNA interaction during splicing we have created a series of 2nd site nucleotide mutations in both U6atac and U12 snRNA to test for their functionality in in vivo splicing assays. Our work will help to better understand the catalytic process of minor class spliceosome and involvement of these snRNA in mammalian gene expression and genetic disorders.
References:
Singh, J., Sikand, K., Conrad, H., Will, C. L., Komar, A. A., & Shukla, G. C. (2016). U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III. Scientific Reports, 31393. doi:10.1038/srep31393
Patel, A. A., & Steitz, J. A. (2003). Splicing double: insights from the second spliceosome. Nature Reviews. Molecular Cell Biology,4(12), 960-970.
Will, C. L., & Lührmann, R. (2005). Splicing of a rare class of introns by the U12-dependent spliceosome. Biological Chemistry, 386(8), 713-724.
Turunen, J. J., Niemelä, E. H., Verma, B., & Frilander, M. J. (2013). The significant other: splicing by the minor spliceosome. Wiley Interdisciplinary Reviews. RNA, 4(1), 61–76.
Keywords: Minor Class Splicing , snRNAs
113. The eukaryotic RNA helicase Mtr4p is a duplex-sensing translocase
Eric M. Patrick (Department of Physics and Astronomy, Michigan State University, East Lansing MI, U.S.A), Sukanya Srinivasan (Center for RNA Molecular Biology and Department of Biochemistry, Case Western University, Cleveland OH, U.S.A), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western University, Cleveland OH, U.S.A), Matthew J. Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing MI, U.S.A)
Abstract:
The conserved Ski2-like RNA helicase Mtr4p plays essential roles in eukaryotic nuclear RNA processing. RNA helicase activity of Mtr4p is critical for biological functions of the enzyme, but the molecular basis for RNA unwinding by Mtr4p is not understood. Here, single-molecule high-resolution optical trapping measurements reveal that Mtr4p unwinds RNA duplexes by 3′ to 5′ translocation on the loading strand, that strand separation occurs in discrete steps of 6 base pairs and that a single Mtr4p protomer performs consecutive unwinding steps. We further show that RNA unwinding by Mtr4p requires interaction with upstream RNA duplex. Inclusion of Mtr4p within the TRAMP complex increases the rate constant for unwinding initiation, but does not change the characteristics of Mtr4p’s helicase mechanism. Our data indicate that Mtr4p utilizes a previously unknown unwinding mode that combines aspects of canonical translocating helicases and non-canonical, duplex sensing helicases, to restrict directional translocation to duplex regions. We have additionally compared Mtr4p unwinding to the non-translocating DEAD box RNA helicase, Ded1p, which shows limited RNA unwinding followed by RNA rezipping. Finally, we present recent high-resolution fleezers measurements of TRAMP unwinding and polymerase activity observed simultaneously. Polyadenylation of the RNA substrate free 3′ overhang by TRAMP is observed via the hybridization of fluorescently labeled oligonucleotide probes.
Keywords: RNA helicase, Ski2-like, Translocase
114. Differential mass spectrometry analysis of transfer RNA by stable isotope labelling
Mellie June Paulines (University of Cincinnati), Patrick A. Limbach (University of Cincinnati)
Abstract not available online - please check the printed booklet.
115. Single-molecule analysis of backbone branched RNAs and lariat debranching enzyme
John R. Pettersson (Department of Chemistry Carnegie Mellon University ), Sourav K. Dey (Department of Chemistry Carnegie Mellon University ), Linda A. Peteanu (Department of Chemistry Carnegie Mellon University ), Subha R. Das (Department of Chemistry Carnegie Mellon University )
Abstract:
The spliceosome processes pre-mRNA by removing introns and ligating exons together. In this process, the excised intron is in the form of a lariat that includes a 2’-5’ phosphodiester bond. The lariat debranching enzyme (Dbr1p) specifically hydrolyzes the 2’-5’ phosphodiester bond producing a linear RNA that can be further processed into snoRNAs, miRNAs and other biologically active RNAs. Dbr1p has not been fully characterized due to the limited access to substrates for the enzyme. We have developed a method to synthesize backbone branched RNAs (bbRNAs) that include a 2’-5’ phosphodiester linkage which mimics the branchpoint and serve as a substrate for Dbr1p, as well as click-branched RNA lariat analogues (cbRNA) that are not cleaved by the enzyme. This access to bbRNAs, cbRNA, and recombinant Dbr1p has enabled us to combine biochemical bulk studies with single-molecule spectroscopy. We use single-molecule TIRF and confocal microscopy to analyze the conformation and dynamics of the RNAs and to further examine the interactions between bbRNAs, cbRNAs and Dbr1p.
Keywords: RNA, Spectroscopy, Enzyme
116. Structural analysis of the sulforhodamine B binding aptamer (SRB-2)
Kyle Piccolo (Dept. of Biochemistry, University of Waterloo), Thorsten Dieckmann (Dept. of Biochemistry, University of Waterloo)
Abstract:
Aptamers are nucleic acid sequences selected for in vitro that non-covalently bind a ligand. An aptamer for a desired ligand is selected for by systematic evolution of ligands by exponential enrichment (SELEX), yielding a nucleic acid sequence with high affinity and selectivity for its target (1). As a result of these properties, aptamers have several potential applications in therapeutics, drug delivery, biosensing, analytical chemistry and live cell imaging (2,3). The sulforhodamine B binding aptamer (SRB-2) is an RNA aptamer that was created via SELEX and subsequently optimized to a 54nt sequence. The authors also determined possible sites of binding interactions for SRB-2 by phosphorothioate mapping (4). SRB-2 has also been successfully implemented in live cell imaging experiments including co-localization with the dinitroaniline-binding aptamer (DNB) (5). As SRB-2 is a potentially useful model system, we wish to determine a solution structure for the aptamer by NMR spectroscopy. 1H NMR and 2D NOESY experiments were performed on SRB-2 and when titrated with sulforhodamine B, a gradual change in the chemical shift and peak intensity of several hydrogen atoms and NOEs were observed until saturation. This indicates that the ligand is binding and it is therefore reasonable that a solution structure complete with restraints, may be determined through further NMR analysis.
References:
(1) Kenan, D.J. and Keene, J.D. 1999. In vitro selection of aptamers from RNA libraries. Methods Mol. Biol. 118: 217–231.
(2) Keefe, A.D.; Pai, S. and Ellington, A. 2010. Aptamers as therapeutics. Nature Reviews Drug Discovery 9, July 2010. pp. 537-550
(3) Tombelli, S.; Minunni, N. and Mascini, M. 2005. Analytical applications of aptamers. Biosensors and Bioelectronics 20. pp. 2424–2434
(4) Holeman, L. A.; Robinson, S.L.; Szostak, J.W. and Wilson, C. 1998. Isolation and characterization of fluorophore binding RNA aptamers. Folding and Design, 3(6): 423-431.
(5) Arora, A.; Sunbul, M. and Jäschke, A. 2015. Dual-colour imaging of RNAs using quencher and fluorophore-binding aptamers. Nucl. Acids Res. 2015 Volume.
Keywords: Aptamer, SRB-2, NMR NOESY
117. Regulation of cross talk between Wnt/Beta-catenin and AR pathways in Prostate cancer progression
Alexis R Plaga (Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University), Jey Sabith Ebron (Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University), Girish C. Shukla (Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University)
Abstract not available online - please check the printed booklet.
118. Title not available online - please see the printed booklet.
Abigail Bieker (Biology, Lewis University), Elizabeth Przekwas (Biology, Lewis University), Mallory Havens (Biology, Lewis University)
Abstract not available online - please check the printed booklet.
119. Transcription Regulation using CRISPR/Cas9
Victoria Rai (University of Michigan Department of Chemistry), Julia R. Widom (University of Michigan Department of Chemistry), Nils G. Walter (University of Michigan Department of Chemistry)
Abstract:
Our ability to control site-specific gene expression has been expanded by the gene-editing tool CRISPR/Cas9. With immunity and inherent memory capabilities, this system applies to our investigation in understanding the mechanisms of the RNA polymerase (RNAP) complex in bacteria. More specifically, by regulating the movement of RNAP in T7 phages at a particular site of transcription using CRISPR/Cas9, we are able to block and unblock the transcription process altogether. As transcription errors can result in numerous genetic disorders, like fibrotic conditions and cancers, the ability to “traffic” RNAP is crucial. Through the creation of multiple guide RNAs, each of which contain a specific sequence that directs protein Cas9 to a target site, we have demonstrated the ability to block transcription. More recently, the process has been successfully unblocked, though further investigation in necessary. While transcription assays revealed that transcription is restarted upon unblocking, further suggesting that Cas9 is fully removed, the unblocked RNAP does not fully produce its runoff product in proportion to its stalled product. The aim remains to allow already bound RNAP to resume transcription upon unblocking. Using a number of avenues, the investigation has turned to techniques in lowering RNAP to DNA ratios to avoiding “bumping” of the polymerases and utilizing other polymerases including those from E. coli, and the bacteriophages T7, SP6, and T3. In line with the aims of this investigation, understanding the interactions between these RNAPs and the protein Cas9 on DNA pertains to transcription regulation, allowing for further application of these regulation techniques.
Keywords: CRISPRCas9, Transcription, RNAP
120. Regulation of RNA editing by C. elegans ADR-1
Suba Rajendren (Genome, Cell and Developmental Biology Program, Indiana University, Bloomington), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine, Bloomington)
Abstract not available online - please check the printed booklet.
121. Novel protein that optimizes tRNA aminoacylation in Helicobacter pylori
Udumbara M. Rathnayake (Chemistry, Wayne State University), Gayathri Silva (Chemistry, Wayne State University), Tamara L. Hendrickson (Chemistry, Wayne State University)
Abstract:
Helicobacter pylori (H. pylori), the pathogenic bacterium that causes stomach ulcers and gastric cancers, lack genes encoding glutaminyl-tRNA synthetase (GlnRS) and asparaginyl-tRNA synthetase (AsnRS) and consequently rely on an indirect pathway to produce Gln-tRNAGln and Asn-tRNAAsn. The first step for Gln-tRNAGln synthesis involves misacylation of tRNAGln with glutamate to produce Glu-tRNAGln, this reaction is catalyzed by glutamyl-tRNA synthetase 2 (GluRS2). Subsequently, an amidotransferase called AdT converts Glu-tRNAGln to Gln-tRNAGln. An analogous, two step process exists to produce Asn-tRNAAsn. In this case, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) misacylates tRNAAsn to produce Asp-tRNAAsn, which is then converted to Asn-tRNAAsn by AdT.
Mechanism/s for the quick delivery of Asp-tRNAAsn and Glu-tRNAGln, from the two misacylating aaRSs to AdT must exist in order to ensure the fidelity of protein synthesis. For example, Thermus thermophilus (T. thermoplilus) assembles a stable ribonucleoprotein complex from tRNAAsn, AdT and ND-AspRS called the Asn-transamidosome, which prevents premature release of Asp-tRNAAsn prior to repair by AdT. This mechanism increases the efficient production of Asn-tRNAAsn while minimizing translational errors. Unlike T. thermoplilus, H. pylori require another protein partner, Hp0100, to form a stable, tRNA-independent Asn-transamidosome. Hp0100 accelerates Asn-tRNAAsn production by ~35 fold. Our preliminary evidence suggests that Hp0100 contains two mutually exclusive ATP binding domains, which are activated by Glu-tRNAGln and Asp-tRNAAsn respectively. Further, Hp0100 also has a putative metal binding motif that preferentially binds to divalent metal ions. Initial studies indicate that there may be dual regulation of Hp0100 ATPase activity: by metals and misacylated aa-tRNA.
Keywords: Helicobacter pylori, Hp0100, Asn-transamidosome
122. Analysis of the Branchpoint Residue in Backbone Branched RNAs
Timothy Ratterman (Department of Chemistry, Carnegie Mellon University), Stephanie Mack (Department of Chemistry, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University)
Abstract:
In splicing, for intron removal, the phosphodiester of the intron 5ʹ-terminal guanosine is joined to the 2ʹ-position on an adenosine residue within the intron to form a lariat structure. Thus, excised lariat introns invariably contain a branchpoint adenosine with a guanosine as the first 2ʹ-branch residue. These lariat introns are used in myriad processes within the cell; however, first the 2ʹ-5ʹ phosphodiester bond of the lariat intron must be cleaved by lariat debranching enzyme (Dbr1p).
Preliminary data from our lab suggests that Saccharomyces cerevisiae Dbr1p can cleave backbone branched RNAs (bbRNAs) in which the branchpoint is not adenosine. Therefore, we are interested in analyzing the binding and cleavage activity of Dbr1p with such non-adenosine branchpoint containing bbRNAs. Towards this, we are synthesizing phosphoramidites for C, G and U branchpoints for bbRNAs that include dual fluorescent labels. These dual-labeled bbRNAs will enable real time kinetics assays of the Dbr1p cleavage reaction.
Keywords: Dbr1p, Backbone branched
123. Structural Insights into RNA Splicing
Aaron R. Robart (Biochemistry, West Virginia University)
Abstract not available online - please check the printed booklet.
124. RAN translation and upstream open reading frame usage shift with neuronal differentiation to modulate downstream protein production
Caitlin M. Rodriguez (Department of Neurology, University of Michigan, Ann Arbor, MI, 48109, USA), Sang Y. Chun (Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, 48109, USA), Ryan E. Mills (Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Human Genetics, University of Michigan, Ann Arbor, MI, 48109, USA ), Peter K. Todd (Department of Neurology, University of Michigan, Ann Arbor, MI, 48109, USA; Veterans Affairs Medical Center, Ann Arbor, MI, 48105, USA)
Abstract not available online - please check the printed booklet.
125. Title not available online - please see the printed booklet.
Ioulia Rouzina (OSU), Tiffiny Rye-McCurdy (OSU), Erik D. Olson (OSU), Shuohui Liu (OSU), Leslie J. Parent (Penn State College of Medicine, Hershey, PA), Karin Musier-Forsyth (OSU)
Abstract not available online - please check the printed booklet.
126. Role of the unique anticodon stem of initiator tRNA-fMet in start codon selection
Bappaditya Roy (Department of Microbiology, Center for RNA Biology, The Ohio State University, OH 43210, USA), Kurt Fredrick (Department of Microbiology, Center for RNA Biology, The Ohio State University, OH 43210, USA)
Abstract not available online - please check the printed booklet.
127. Automated detection and classification of RNA-protein interactions
Poorna Roy (Center for Photochemical Sciences, Bowling Green State University), Blake A. Sweeney (Department of Biology, Bowling Green State University), Neocles B. Leontis (Center for Photochemical Sciences, Bowling Green State University)
Abstract:
RNA-protein interactions play crucial roles in all stages of transcription, translation and gene regulation. RNA-binding proteins interact specifically with RNA molecules using a variety of non-covalent interactions to achieve exquisite binding selectivity, including: (i) electrostatic interactions between fully ionized groups, (ii) hydrogen bonding, (iii) and hydrophobic interactions, such as aromatic stacking. Very often, the same amino acid makes two or more interactions with a single or neighboring nucleotides, thus increasing sensitivity. Such multiple inter-molecular contacts correspond to recurrent binding motifs, some of which have been previously described (Kondo and Westhof, 2011). For example, base and amino-acid specific “pseudo-pairs” result when a single amino acid forms two or more H-bonds with an RNA base. In the course of this work, new pseudo-pairs involving histidine interacting in the RNA minor groove were identified, as well as bidentate interactions in which a single amino acid H-bonds to two stacked RNA bases. As it is important to systematically detect and annotate these these non-covalent interactions to make them searchable in databases, we have developed programs, integrated into the RNA.BGSU.EDU and NDB data pipeline to provide RNA-protein interaction annotations that can be searched symbolically or geometrically. The interactions that are annotated will be illustrated and statistical surveys of the occurrences of these interactions in current releases of NDB will be presented.
References:
Kondo, J and Westhof, E. (2011) “Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide–protein complexes”. Nucleic Acids Res. 2011, 1-10.
Keywords: RNA-protein, Annotation, Database
128. Title not available online - please see the printed booklet.
Brianne Sanford (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio, 43205), Chelsea Brown, Hemant Bid, Thomas Bebee, Daniel Comiskey Jr. (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio, 43205), Frank Rigo (Ionis Pharmaceuticals, Carlsbad, California, 92010), Peter Houghton (Greehey Childrens Cancer Research Institute, University of Texas Health Science Center, San Antonio, Texas, 78229), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital and Department of Pediatrics, OSU Columbus, Ohio, 43210)
Abstract not available online - please check the printed booklet.
129. Telomerase Regulation in Pathogenic Protozoa
Samantha Sanford (University of Pittsburgh Graduate School of Public Health, Infectious Disease and Microbiology), Priscilla Wong (Carnegie Mellon University, Department of Chemistry, CNAST), Kausik Chakrabarti (Carnegie Mellon University, Department of Chemistry, CNAST)
Abstract:
Telomere maintenance is the major physiological means of unlimited cellular proliferation, especially in rapidly dividing pathogenic eukaryotes such as Trypanosoma brucei and Plasmodium falciparum. Stable telomeres have the ability to protect the chromosome termini from degradation and recombination, which is critical for virulence and proliferation of the pathogen. In order to keep the telomere length stable, the synthesis of telomeric DNA by telomerase is necessary. Telomerase is a ribonucleoprotein complex which adds a telomere repeat sequence to the 3’ end of telomeres. It contains a telomerase reverse transcription enzyme (TERT) and carries its own RNA molecule, (TR), which is used as a template when it elongates. The objective of this project is to determine the structural requirements for TR-TERT interactions. Compared to human and yeast models, T. brucei TR has different RNA-protein interactions and conformational changes which could result in different telomerase activity(1). Using SHAPE, the RNA structure and folding is determined through a comparison of several different mutations of various regions including the template and pseudo knot domain, as well as the 3’ and 5’ CD Box domains. The mutant RNA was in vitro transcribed and chemical foot printing was used to determine the RNA structure by SHAPE analysis (2) To determine if the substrate binding of the RNA is affected by any mutation that is in the template gel shift assays will be done. To explore how this structural component interacts with chromatin, preliminary results in Plasmodium have shown that a histone deacetylase, SIR2A, may have the ability to coordinate telomere, telomerase, and subtelomere. By studying the chemical structure and properties of this domain, we will better understand how it can affect the telomerase enzymatic activity properties.
References:
1. Sandhu R, Sanford S, Basu S, Park M, Pandya UM, Li B, Chakrabarti K. 2013. A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei. Cell Res 23:537-551.
2. Wilkinson KA, Merino EJ, Weeks KM. 2006. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1:1610-1616.
3. Turner R, Shefer K, Ares M, Jr. 2013. Safer one-pot synthesis of the 'SHAPE' reagent 1-methyl-7-nitroisatoic anhydride (1m7). RNA 19:1857-1863.
Keywords: Telomerase, RNA Structure, Histone Deacetylase
130. The study of pseudouridine modification in U5 snRNA loop 1
Natalie Schleper (Department of Chemistry, SIUE), Stephanie Wiechmann (Department of Chemistry, SIUE), Madison Harper (Department of Chemistry, SIUE), Mina Sumita (Department of Chemistry, SIUE)
Abstract not available online - please check the printed booklet.
131. Translational control of cardiac hypertrophy: Poly(A) binding protein C1 at the heart of it
Sandip Chorghade (University of Illinois Urbana-Champaign Department of Biochemistry), Joe Seimetz (University of Illinois Urbana-Champaign Department of Biochemistry), Russel S. Emmons, Michael De Lisio (University of Illinois Urbana-Champaign Department of Kinesiology and Community Health), Stefan M. Bresson, Nicholas K. Conrad (University of Texas Southwestern Medical Center), Auinash Kalsotra (University of Illinois Urbana-Champaign Department of Biochemistry)
Abstract:
Poly(A) binding proteins (PABPs) are multifunctional scaffolds that bind to the poly(A) tail to form ribonucleoproteins determining the post-transcriptional fate of mRNAs including their export, stability, and translation. Based on these central roles, PABPs are considered essential for cell survival and function. Here we report that cytosolic PABPC1 protein expression is post-transcriptionally silenced in the adult human and murine hearts. Strikingly, the silencing is cardiomyocyte-specific, evolutionarily conserved, and reversed with endurance exercise and in heart disease. We demonstrate that PABPC1 silencing is driven by its poly(A) tail length shortening during postnatal heart development, which results in reduced polysome association and translation of Pabpc1 transcripts. Furthermore, we demonstrate that PABPC1-depleted neonatal cardiac myocytes are viable; but refractory to stimulus-induced hypertrophic growth due to their inability to initiate new protein synthesis. Remarkably, forced expression of PABPC1 in the adult myocardium of transgenic mice stimulates physiologic hypertrophy by enhancing global translation, which is dependent on eukaryotic initiation factor (eIF)4G-PABPC1 interactions and mRNA circularization. Supplementing PABPC1-deficient cardiomyocytes with wildtype PABPC1 rescues, whereas a mutant form of PABPC1–engineered by Structure-guided mutations to disrupt eIF4G-PABPC1 interactions–fails to rescue the hypertrophic growth and translation defects in these cells. Taken together, our results uncover a cell-type and developmental stage-specific function for PABPC1 and reveal its upregulation as a central mechanism in activating cardiac hypertrophy in response to physiologic and pathologic stimuli.
Keywords: Translation, Poly(A) Tail, post-transcriptional regulation
132. Title not available online - please see the printed booklet.
Dan Seith (Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University), Jamie Bingaman (Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University), Philip Bevilacqua (Department of Biochemistry, Microbiology, and Molecular Biology, The Pennsylvania State University)
Abstract not available online - please check the printed booklet.
133. ATP hydrolysis by UPF1 promotes translation termination at premature stop codons
Lucas D. Serdar (Center for RNA Molecular Biology, Case Western Reserve University), DaJuan L. Whiteside (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract:
Premature termination of translation at nonsense codons present within mRNA protein coding regions yields C-terminally truncated polypeptides with potentially deleterious functions to the cell. Nonsense-mediated mRNA decay (NMD) represents a quality control pathway that limits production of these aberrant protein products by recognizing the nonsense-containing mRNA and targeting it to accelerated degradation. How the NMD machinery is able to monitor the relative position of the terminating ribosome to determine it is premature, and communicate this information to the mRNA decay machinery remains unclear. The ATPase activity of the RNA helicase UPF1 is essential for NMD, however, the precise role and site of action of ATP hydrolysis by UPF1 during NMD remain unresolved. We show here that in yeast, expression of ATPase-deficient UPF1 results in the accumulation of 3’ mRNA decay fragments harboring ribosomes stalled in the vicinity of the premature termination codon. Moreover, we demonstrate that the ability of UPF1 to impinge upon premature termination requires its RNA binding activity, as well as its ability to associate with NMD co-factors UPF2 and UPF3. Our results reveal a functional interaction between UPF1 and the terminating ribosome that is dependent upon UPF1 ATPase activity and necessary for efficient targeting of NMD substrates to rapid degradation. These data give rise to a refined model of NMD substrate recognition and add to growing evidence that mRNA stability is tightly controlled by events directly impacting mRNA translation.
Keywords: NMD, mRNA Decay, Translation
134. Identification of APOBEC3A as an RNA editing enzyme
Shraddha Sharma (Dept. of Pathology, Roswell Park Cancer Institute), Santosh K. Patnaik (Dept. of Thoracic surgery, Roswell Park Cancer Institute), Robert T. Taggart (Dept. of Pathology, Roswell Park Cancer Institute), Sally M. Enriquez (Dept. of Biological Sciences, SUNY Buffalo), Paul Gollnick (Dept. of Biological Sciences, SUNY Buffalo), Bora E. Baysal (Dept. of Pathology, Roswell Park Cancer Institute)
Abstract:
RNA editing co- or post transcriptionally alters RNA transcripts encoded by DNA. Aberrant RNA editing is implicated in the pathogenesis of cancer and neurodegenerative disorders. A-to-I and C-to-U editing are the two most common types of RNA editing in mammals. RNA-dependent adenosine deaminases ADAR1, ADAR2 and ADAR3, and APOBEC1, a cytidine deaminase, are the only known RNA editing enzymes in mammals. Unlike A-to-I RNA editing, the regulation, extent and enzymatic basis of C-to-U RNA editing is poorly understood.
We previously identified hypoxia-inducible site-specific C-to-U editing in SDHB RNA, which encodes a catalytic subunit of succinate dehydrogenase. To identify additional RNA editing events, we analyzed transcriptome-wide sequence data from primary monocytes exposed to hypoxia. In addition, we analyzed transcriptome-wide sequence data from monocyte-derived macrophages and investigated the correlation of SDHB RNA editing with expression of cytidine deaminase enzymes in certain common tumors in The Cancer Genome Atlas. We found site-specific C-to-U editing in transcripts of hundreds of genes in human monocytes and macrophages, including many that are involved in the pathogenesis of viral diseases. Such editing is induced by low oxygen (hypoxia) and interferon in monocytes and also during M1 macrophage polarization. We demonstrate for the first time that APOBEC3A is responsible for RNA editing.
Our findings significantly expand our knowledge of C-to-U RNA editing events, uncover for the first time a role for micro-environment in inducing RNA editing, and reveals the previously unrecognized RNA editing activity of the APOBEC3A cytidine deaminase in innate immune cells.
References:
Sharma, S. et al. APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages. Nature communications 6 (2015).
Sharma, S., Patnaik, S. K., Kemer, Z. & Baysal, B. E. Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes. RNA biology, 00-00 (2016).
Keywords: RNA editing, APOBEC3A, immune cells
135. Computational molecular docking of T box antitermination model with aminoglycosides
Chobee L. Sheets (School of Mathematics and Natural Sciences, University of Rio Grande), John A. Means (School of Mathematics and Natural Sciences, University of Rio Grande)
Abstract:
Computational molecular docking provides a quicker, less expensive way to screen the binding of molecules than in vitro binding studies. Using this method, many molecules, ligands, and interaction patterns can be analyzed in a brief period of time. In this case, the T box antitermination system of Gram positive bacteria was investigated with respect to seven different aminoglycosides: amikacin, kanamycin A, kanamycin B, neomycin, paromomycin, streptomycin, and tobramycin. The two structures of focus are the “on” and “off” structures of the T box riboswitch, also known as the antiterminator and terminator, respectively. Since the T box riboswitch is responsible for regulating amino acid availability through mRNA-tRNA interactions, the binding of these aminoglycosides to the antiterminator structure of the T box gene is key to this study. Using PyRx, AutoDock, and AutoDock Vina these model antiterminator RNA-aminoglycoside interactions were virtually screened to evaluate binding scores. These scores were then compared to previous in vitro binding data in order to support the claim that this software is suitable for producing accurate results in regards to modeling the binding of aminoglycosides with targeted antiterminator structures of T box genes.
Keywords: T box riboswitch, docking, aminoglycosides
136. Determining the dominant secondary structures of a RNA molecule during contranscriptional folding dynamics by grouping similar dominant structures.
Catharine Shipps (Physics, The Ohio State University), Ralf Bundschuh (Physics, The Ohio State University)
Abstract:
Cotranscriptional folding dynamics of RNA molecules can have important functional consequences, e.g., in the case of riboswitches. Computational methods that predict these cotranscriptional folding dynamics generate a myriad of distinct intermediate RNA structures that are difficult to interpret thus requiring a method that summarizes the most prevalent of these structures to allow mechanistic insights into the essence of the folding process. There are already several computational tools in existence that can be used to determine dominant secondary structures during folding kinetics of shorter RNA molecules. However, during cotranscriptional folding the number of secondary structures accessible to a sequence grows exponentially with the length of the sequence and we found that the large number of secondary structures becomes prohibitively expensive when using these previous methods for molecules longer than 100bp or so. To help solve this computational efficiency problem as well as to make interpretation of the folding pathways more intuitive, we introduced and implemented the idea of further grouping similar dominant secondary structures. Our goal is to determine the ultimately dominant structures during cotranscriptional folding using this grouping method.
Keywords: RNA secondary structures, cotranscriptional folding , folding dynamics
137. Overcoming a “molecular ruler” mechanism: the unusual heterotrimeric tRNA splicing endonuclease of Trypanosoma brucei
Gabriel Silveira dAlmeida (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Christopher Trotta (PTC Therapeutics Inc), Arthur Gnzl (Arthur Gnzl), Juan Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University)
Abstract:
In all three major lines of descent (Bacteria, Archaea and Eukarya) introns interrupt tRNA sequences rendering them non-functional for protein synthesis; intron removal is therefore essential. In eukaryotes, intron cleavage is catalyzed by an evolutionarily conserved heterotetrameric tRNA splicing endonuclease composed of four subunits: Sen 54, 34, 15 and 2. Bioinformatic analysis using previously published eukaryotic Sen sequences led us to the identification of only one homolog of the tRNA splicing endonuclease in Trypanosoma brucei, suggesting that either the other subunits are missing or, as a whole, the enzyme is very divergent in these organisms. Here we present evidence for a highly divergent and unique enzyme composed only of three sub-units; homologs of Sen 34, 15 and 2, while lacking the larger Sen54 subunit. By performing tandem-affinity chromatography followed by mass spectrometry analysis we purified and identified TbSen subunits from a T. brucei S100 fraction. Gel filtration chromatography and in vitro activity assays revealed that the active enzyme had a size within the range of 58 to 72 kDa, consistent with that of a heterotrimer. Furthermore, in contrast with most eukaryotes, immunofluorescence localization assays showed that the enzyme was cytoplasmic. This observation is also in stark contrast with the yeast system where the enzyme localizes to the surface of the mitochondria. Our results demonstrate that TbSen diverges from previously described eukaryotic enzymes, both in structure and localization. Interestingly, in most eukaryotes, Sen54 serves as a “molecular ruler” that carefully measures the distance between the splice sites and the backbone of the folded tRNA, aiding in substrate identification and catalytic site positioning. Our finding of heterotrimeric endonuclease then obviates the need for a Sen54 sub-unit and may remove the substrate recognition restriction set forth by the “molecular ruler” mechanism. These observations have direct implications for both the evolution of the enzyme in trypanosomes and its potential for targeting of additional substrates while not just being limited to tRNAs.
Keywords: trypanosoma, tRNA, splicing
138. High Throughput Sequencing of Partially Edited RNAs Provides Insight into the Roles of the TbRGG2 Subcomplex Proteins in Trypanosome RNA editing.
Rachel M. Simpson (Department of Microbiology & Immunology, University at Buffalo School of Medicine), Andrew E. Bruno (Center for Computational Research, University at Buffalo School of Medicine), Jonathan Bard (Next-gen Sequencing Core , University at Buffalo School of Medicine), Runpu Chen, Yijun Sun (Department of Computer Science and Engineering , University at Buffalo School of Medicine), Michael Buck (Department of Biochemistry, University at Buffalo School of Medicine), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo School of Medicine)
Abstract:
Uridine (U) insertion/deletion RNA editing is an essential process whereby kinetoplastid mitochondrial mRNAs are altered to their final coding forms through sequential utilization of multiple trans-acting gRNAs that act as templates. The enzymes that catalyze U insertion/deletion are part of a multiprotein complex termed the RNA Editing Core Complex (RECC) or editosome. In addition, a large, possibly dynamic and heterogeneous complex termed MRB1 (mitochondrial RNA binding complex 1) is also essential for RNA editing and appears to constitute the platform for editing. Within MRB1 is the incompletely defined TbRGG2 subcomplex, at least some of whose components are essential for the 3’ to 5’ progression of editing. Most RNAs in the steady state population are not completely edited and contain segments of edited RNA between the fully edited and not-yet edited portions of a transcript that match neither and appear to be mis-edited. These segments are termed junction regions, and it is not known whether these regions represent translatable alternative editing, mis-edited sequence that will be corrected, or a terminal error. Using high throughput sequencing, we have analyzed large populations of partially edited sequences for RPS12 and ND7-5’ in parental cells and cells depleted of TbRGG2 subcomplex proteins. We determined that multiple members of the TbRGG2 subcomplex facilitate the progression of editing through a single gRNA and are essential for proper junction formation. Additionally, we found that these proteins have distinct but overlapping roles in facilitating the progression of editing. This study is the first study in which high throughput sequencing is used to elucidate the role of RNA editing factors in Trypanosoma brucei and provides a platform for future research.
Keywords: RNA Editing, trypanosoma, high throughput sequencing
139. Title not available online - please see the printed booklet.
Deepak K. Singh, Omid Gholamalamdari, Arindam Chakraborty, Abid Khan, (Department of Cell & Developmental Biology, UIUC), Xiao L. Li, Ashish Lal (Center for Cancer Research, National Cancer Institute, Bethesda, MD), Saumya Tiwari, Sarah Holton, Rohit Bhargava (Beckman Institute of Advanced Science and Technology, UIUC), Seyedsasan Hashemikhabir, Sarath C. Janga (School of Informatics and Computing, IUPUI), Zhang Yang, Shuomeng Guang, Jian Ma (Department of Bioengineering, UIUC), Supriya G. Prasanth, Kannanganattu V. Prasanth (Department of Cell & Developmental Biology, UIUC)
Abstract not available online - please check the printed booklet.
140. RNA-protein interaction in the U12-dependent spliceosome
Jagjit Singh (Center for Gene Regulation in Health and Disease, Department of Biological Sciences, Cleveland State University, Cleveland, OH-44115,), Kavleen Sikand (Department of Biochemistry, Panjab University, Chandigarh, India.), Heike Conrad (Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Gttingen, Germany.), Cindy L. Will (Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Gttingen, Germany.), Anton A. Komar (Center for Gene Regulation in Health and Disease, Department of Biological Sciences, Cleveland State University, Cleveland, OH-44115,), Girish C. Shukla (Center for Gene Regulation in Health and Disease, Department of Biological Sciences, Cleveland State University, Cleveland, OH-44115,)
Abstract not available online - please check the printed booklet.
141. Regulation of androgen receptor function and tumor suppressive role of miR-149-5p in prostate cancer.
Savita Singh (Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH, 44115, USA), Jey Sabith Ebron (Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH, 44115, USA.), Eswar Shankar (Department of Urology, Case Western Reserve University & University Hospitals Case Medical Center, Cleveland, OH 44106, USA.), Sanjay Gupta (Department of Urology, Case Western Reserve University & University Hospitals Case Medical Center, Cleveland, OH 44106, USA.), Daniel Lindner (Taussig Cancer Center, Learner Research Institute, Cleveland, OH 44195, USA), Girish C. Shukla (Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH, 44115, USA)
Abstract not available online - please check the printed booklet.
142. Title not available online - please see the printed booklet.
Ashlee Smith (MCDB, OSU), Catey Dominguez (MCDB, OSU), Dawn Chandler (MCDB, OSU)
Abstract not available online - please check the printed booklet.
143. Comparative translatomics reveals a class of conserved, stress responsive, non-canonical uORFs
Pieter Spealman (Department of Biological Sciences, 5000 Forbes Avenue, Carnegie Mellon University, Pittsburgh, PA, USA 15213. ), Armaghan W. Naik, Robert F. Murphy (Computational Biology Department, 5000 Forbes Avenue, Carnegie Mellon University, Pittsburgh, PA, USA 15213.), Scott Kuersten, Lindsay Freeberg (Illumina Inc., Madison, WI, USA ), Gemma May, C. Joel McManus (Department of Biological Sciences, 5000 Forbes Avenue, Carnegie Mellon University, Pittsburgh, PA, USA 15213. )
Abstract:
Upstream open reading frames (uORFs) have emerged as major cis-acting elements involved in regulating mRNA turnover and translation (1,2). While the most well-known uORFs initiate using the canonical AUG start codons, an ever increasing number of uORFs are being proposed that initiate at near-cognate codons (NCCs) (3,4). While translation of such NCC uORFs increases under stress (5), their function and evolution remain largely uncharacterized.
We used comparative translatomics to investigate AUG and NCC uORFs in three yeast species. This required mapping both major and minor transcription start sites (TSSs) in S. cerevisiae, S. paradoxus, and S. uvarum. Consistent with prior work, AUG triplets were depleted from the TLS in each species. Surprisingly, we found the majority of NCC codons to be both enriched in the TLS and conserved between species, unlike AUG. This is consistent with the model that NCC triplets are under selective pressure to maintain translation initiation at uORFs. We then searched the TLs for functional uORFs using a novel machine learning algorithm (uORF-seqr) and ribosome profiling data from each species. Our analysis identified both conserved and species-specific AUG- and NCC-uORFs in hundreds of orthologous TLSs. Numerous features, such as RNA-binding protein distribution, the rate of uORF inclusion in transcript isoforms, and the effect each uORF type has on the translation of the downstream gene, suggest these AUG and NCC-uORFs represent distinct regulatory elements. Because NCCs may be preferentially translated under stress, we also performed ribosome profiling in S. cerevisiae and S. paradoxus after acute amino-acid starvation. For both species, the relative AUG-uORF usage decreased under stress, while NCC usage increased dramatically. Notably, several genes with conserved stress-responsive NCC-uORFs are central to transcription and translation. These results suggest that NCC-uORFs may play an important role in the regulation of mRNA levels and translation in response to stress, discrete from that observed for AUG-uORFs.
References:
1. Wethmar, K. The regulatory potential of upstream open reading frames in eukaryotic gene expression. Wiley Interdiscip Rev Rna (2014).
2. Ingolia et al. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell (2014).
3. Zhang et al. An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA. Nucleic Acids Res. (2011).
4. Brar, G. et al. High-resolution view of the yeast meiotic program revealed by ribosome profiling. Science (2011).
5. Ingolia et al. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. (2009)
Keywords: upstream open reading frames, translation regulation, gene expression
144. Alternative polyadenylation in apicomplexans
Ashley Stevens (Plant and Soil Sciences, University of Kentucky), Dr. Arthur Hunt (Plant and Soil Sciences, University of Kentucky), Dr. Daniel Howe (Veterinary Sciences, University of Kentucky)
Abstract:
This project studied the occurrence of alternative polyadenylation (APA) and poly(A) site choice during the growth of three apicomplexan parasites: Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum . This study addresses the hypothesis that alternative polyadenylation contributes to regulated gene expression in S. neurona, T. gondii, and N. caninum.
RNA was isolated from extracellular parasites and parasites growing in cultured host cells. Poly(A) tags (1) were made and the tag libraries were sequenced on a MiSeq instrument. The data was analyzed using programs including CLC Genomics Workbench and Bedtools, as well as others designed specifically for APA analysis to define sites and assess alternative polyadenylation. The results indicate that S. neurona, T. gondii, and N. caninum possesses a distinctive polyadenylation signal that is reminiscent of that seen in higher plants. The results also show multiple instances of APA. The results from this study confirm our hypothesis that there are changes in poly(A) site choice in the developmental stages of S. neurona, T. gondii, and N. caninum.
Supported by NSF Award 1243849
References:
1. Ma L, Pati PK, Liu M, Li QQ, Hunt AG. High throughput characterizations of poly(A) site choice in plants. Methods. 2014;67(1):74-83.
Keywords: Polyadenylation, Apicomplexan
145. Probing the UV-induced effects on RNA and RNA-modifications by LC-MS
Congliang Sun (University of Cincinnati), Zalfa Abdel-Malek (University of Cincinnati), Kazumasa Wakamatsu (University of Cincinnati), Patrick A Limbach (University of Cincinnati), Balasubrahmanyam Addepalli (University of Cincinnati)
Abstract not available online - please check the printed booklet.
146. Title not available online - please see the printed booklet.
Qinyu Sun, Supriya G. Prasanth, Kannanganattu V. Prasanth (University of Illinois, Urbana-Champaign, Department of Cellular and Developmental Biology), Vidisha Tripathi (National Centre for Cell Science, Pune, India), Je-Hyun Yoon (Medical University of South Carolina, Department of Biochemistry and Molecular Biology), Susan M. Freier (Ionis Pharmaceuticals, Inc.), Ashish Lal (National Cancer Institute, Center for Cancer Research ), Sven Diederichs (Deutsches KrebsforschungszentrumDKFZ- RNA Biology & Cancer)
Abstract not available online - please check the printed booklet.
147. Programmed -1 Ribosomal Frameshifting may in part modulate miRNA function and mRNA deadenylation
Isabella Swafford (First-Year Innovation and Research Experience-FIRE, University of Maryland, College Park), Elizabeth DiCioccio (First-Year Innovation and Research Experience-FIRE, University of Maryland, College Park), Roi Turalde (First-Year Innovation and Research Experience-FIRE, University of Maryland, College Park), Adam Kellerman (First-Year Innovation and Research Experience-FIRE, University of Maryland, College Park), Vivek Advani (First-Year Innovation and Research Experience-FIRE, University of Maryland, College Park), Jonathan Dinman (Cell Biology and Molecular Genetics, University of Maryland, College Park)
Abstract:
Gene expression can be controlled at the level of mRNA stability, and recent work suggests that Programmed -1 Ribosomal Frameshifting (-1PRF), a translational recoding mechanism, consistent with this model(1)(2)(3). -1 PRF sequences are cis-acting mRNA motifs that stochastically direct elongating ribosomes to a -1 frame pre-mature termination codon (PTC, thereby destabilizing the mRNA by Nonsense Mediated mRNA Decay (NMD) pathway in eukaryotes(4)(5). There are an overwhelming majority of -1 PRF signals predicted in over 20 eukaryotic genomes, suggesting it to a universal mechanism of gene regulation(6). Here we have identified functional -1 PRF signals mRNAs encoding components of miRNA processing (DROSHA), RISC (Dicer1) and deadenylase (Ccr4-yeast and CNOT1-human) proteins harbor -1 PRF signals This observation leads to the hypothesis that miRNA function and localization and mRNA deadenylation are in part regulated by -1 PRF. Hence, dysregulation of -1 PRF and/or translational fidelity would impact global mRNA stability. These studies establish a paradigm for understanding the linkage between translational fidelity and human disease.
References:
1. Dinman JD. Mechanisms and implications of programmed translational frameshifting. Wiley Interdiscip Rev RNA Jan;3(5):661–73.
2. Belew AT, et al. Cell cycle control (and more) by programmed −1 ribosomal frameshifting: implications for disease and therapeutics. Cell Cycle. 2015
3. Belew AT, et al. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Res [Internet]. 2011 Apr 1; 39(7):2799–808.
4. Jacobs JL, et al. Identification of functional, endogenous programmed -1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae. Nucleic Acids Res. 2007 Jan 12;35(1):165–74.
5. Belew AT, et al. PRFdb: a database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals. BMC Genomics. 2008 Jan;9(1):339.
Keywords: miRNA, mRNA stability, NMD
148. Title not available online - please see the printed booklet.
Keiko Akagi, Christopher Hlynialuk, Andrew ONeil, Chelsea Moherman (Cancer Biology and Genetics, Ohio State University), Hailing He, Wei Li, Albert de la Chapelle (Cancer Biology and Genetics, Ohio State University), Jarnail Singh, Rosemary Dietrich, Richard A. Padgett (Molecular Genetics, Cleveland Clinic Foundation), Alex D. Robertson, Christopher B. Burge (Biology, Massachusetts Institute of Technology), Judith A. Westman (Internal Medicine, Ohio State University), David E. Symer (Cancer Biology and Genetics, Human Cancer Genetics Program, Ohio State University)
Abstract not available online - please check the printed booklet.
149. Suppression screen for the identification of protein interactors of the trp RNA-binding attenuation protein (TRAP)
Courtney E. Szyjka (Department of Biological Sciences, University at Buffalo), Natalie M. McAdams (Department of Microbiology and Immunology, University at Buffalo), Justin Durland (Department of Biological Sciences, University at Buffalo), Eric Strobel, Julius Lucks (Department of Chemical and Biological Engineering, Northwestern University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, University at Buffalo)
Abstract:
Transcriptional regulation of the tryptophan (trp) biosynthetic operon in Bacillus subtilis is controlled by the trp RNA-binding attenuator protein (TRAP). Regulation of this operon was initially described as depending on two competing RNA structures present in the leader region upstream of the first gene in the operon. Formation of these structures, designated as the anti-terminator and terminator, is mutually exclusive due to shared bases. In the presence of excess tryptophan, TRAP binds to 11 (G/U)AG repeats in the trp leader region RNA and prevents anti-terminator formation, allowing formation of the terminator thus halting transcription of the trp genes. In limiting tryptophan conditions, TRAP does not bind the trp leader RNA, the anti-terminator forms and the genes are transcribed and translated to produce the tryptophan biosynthesis enzymes. However, recent work has shown that, 1) formation of the terminator RNA structure isn’t sufficient to induce transcription termination in the absence of TRAP and 2) in vivo TRAP can induce termination in the absence of the terminator RNA structure. Together these observations suggest that TRAP may have a more direct role in transcription attenuation. Consistent with this suggestion, we have recently isolated a TRAP mutant (E60K) that is capable of binding RNA and tryptophan at wild-type (WT) levels, but is deficient in transcription attenuation. We have designed a suppression screen to identify protein interactors of TRAP that are necessary for efficient transcription attenuation in the trp operon. By placing the toxic mazF gene under control of the trp leader region, we seek to identify B. subtilis proteins that will enhance transcription attenuation of the E60K TRAP mutant in vivo.
Keywords: transcription termination, attenuation
150. Title not available online - please see the printed booklet.
Aamira Tariq (Department of Cell and Developmental Biology,UIUC), Deepak Singh, (Department of Cell and Developmental Biology,UIUC), Zhang Yang,Jian Ma (CARL R. WOESE Institute for Genomic Biology,UIUC), Sarah Holton, Rohit Bhargava (Beckman Institute of Advanced Science and Technology,UIUC), Supriya G Prasanth, Prasanth KV (Department of Cell and Developmental Biology,UIUC)
Abstract not available online - please check the printed booklet.
151. The RNA Helicase Ded1p prevents translation initiation from near-cognate codons
Ulf-Peter Guenther (Center for RNA Molecular Biology and Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA), Frank Tedeschi, Brittany Stawicki, Leah Zagore, Donny Licatalosi (Center for RNA Molecular Biology and Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA), David E. Weinberg, Meghan Zubradt, David P. Bartel (Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA,), Jonathan S. Weissman (Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA)
Abstract not available online - please check the printed booklet.
152. Carbohydrate-linked cisplatin analogue: reactivity studies with RNA and DNA
Supuni Thalalla Gamage (Department of Chemistry, Wayne State University), Nisansala Muthunayake (Department of Chemistry, Wayne State University), Amr Sonousi (Department of Chemistry, Wayne State University), David Crich (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)
Abstract not available online - please check the printed booklet.
153. 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, Nicolas L. Derr, Zachary T. Morrow, Sharon L. Amacher (Department of Molecular Genetics, The Ohio State University)
Abstract:
Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of embryonic segments. This molecular oscillator generates cyclic gene expression in the tissue that generates somites and has the same periodicity as somite formation. Molecular components of the clock include the her/Hes family of transcriptional repressors, but additional transcripts also cycle. Maintenance of clock oscillation requires that transcriptional activation and repression, RNA turnover, translation, and protein degradation are rapid (one cycle is 30 minutes in the zebrafish). We previously isolated a zebrafish segmentation clock mutant, tortuga, that has elevated levels of cyclic transcripts. We show that loss of proline-rich nuclear receptor coactivator protein Pnrc2 is responsible for cyclic transcript accumulation in tortuga deletion mutants and that a new pnrc2 loss-of-function mutant displays an identical phenotype. pnrc2 mRNA is maternally provided and zygotically expressed, and in maternal-zygotic pnrc2 mutants, cyclic transcripts perdure even longer. We show that the her1 3’UTR confers instability to otherwise stable transcripts in a Pnrc2-dependent manner, and that tethering Pnrc2 to the 3’UTR of a stable transcript triggers rapid decay, indicating that the 3’UTR of cyclic transcripts is critical for Pnrc2-mediated decay. Knockdown of pnrc2 in a validated transgenic cyclic reporter line, Tg(her1:her1-Venus), causes reporter transcript accumulation, yet reporter protein is expressed normally. Endogenous cyclic protein also does not accumulate in pnrc2 mutants, suggesting that stabilized cyclic transcripts are not efficiently translated and perhaps are controlled by an additional post-transcriptional mechanism. Our work explores these post-transcriptional mechanisms regulating oscillation dynamics during somitogenesis and will further our understanding of other ultradian oscillators in general.
Keywords: Pnrc2, oscillating transcripts, mRNA decay
154. Antagonistic splicing factors hnRNP A1 and SRSF2 compete for binding to snRNA 7SK
Daniel Totten (Biological Sciences at University of Pittsburgh), Andrea Berman (Biological Sciences at University of Pittsburgh)
Abstract not available online - please check the printed booklet.
155. Functional Programmed -1 Ribosomal Frameshifting signals in the mRNA encoding human NSD1 may regulate histone methylation and transcription
Michael Usewick (First-year Innovation and Research Experience-FIRE University of Maryland), Lyra Morina (First-year Innovation and Research Experience-FIRE University of Maryland), Jerry Mi (First-year Innovation and Research Experience-FIRE University of Maryland), Vivek Advani (First-year Innovation and Research Experience-FIRE University of Maryland)
Abstract:
Recent studies have shown that Programmed -1 Ribosomal Frameshifting (-1 PRF) fits within the paradigm of post-transcription gene regulation (1,2,3). Characterization of numerous functional -1 PRF signals in the human genome suggests that it is a universal mechanism for regulating translation. It has also been shown that these -1 PRF signals function as mRNA destabilizing elements via nonsense mediated decay (NMD) (3,4). 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’. Bioinformatic analysis suggests 1,943 functional -1 PRF signals in the human genome (4,5). Through the First-year Innovative and Research Experiences (FIRE) - Found in Translation (FIT) Undergraduate Research Program at the University of Maryland College, we have identified functional -1 PRF signals encoded by over 40 human genes. Here, we have identified functional frameshift signals in mRNA encoding the nuclear receptor binding SET domain protein 1 (NSD1) gene. This gene encodes for histone methyltransferase that regulates chromosome arrangement and transcription. We hypothesize that the functional -1 PRF signal in NSD1 may contribute to regulating NSD1 expression and hence disease pathogenesis associated with NSD1. Dysregulation of NSD1 results in Sotos Syndrome, Acute myeloid leukemia, and Beckwith-Wiedemann Syndrome (6).
References:
1. Harger, J. et. al (2003). An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae. RNA, 1019-1024.
2.Dinman JD. Mechanisms and implications of programmed translational frameshifting. Wiley Interdiscip Rev RNA. 3(5):661-673. doi:10.1002/wrna.1126.
3.Belew, A. T. et. al (2011). Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Research, 39(7), 2799–808.
4.Belew, A. T. et. al (2008). PRFdb: a database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals. BMC Genomics, 9(1).
5.Belew, A. T., et. al (2014). Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway. Nature, 512, 265–269.
6. NSD1 OMIM - http://www.omim.org/entry/606681
Keywords: -1 PRF, NSD1, Disease
156. Determining the regulatory role of C. elegans ADR-2 on neural gene expression
Pranathi Vadlamani (Biotechnology Professional Science Masters Program, Indiana University), Sarah N. Deffit (Medical Sciences Program, Indiana University School of Medicine), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine)
Abstract not available online - please check the printed booklet.
157. Human La binds poly(A) to promote cap-independent translation
Jyotsna Vinayak (York University), Rawaa Hamad Hussain (York University), Leonid Rozenfeld (York University), Karine Solomon (York University), Mark Allan Bayfield (York University)
Abstract not available online - please check the printed booklet.
158. The use of fission yeast to study the roles of eukaryotic tRNA modifications
Thomas Vornheder (Chemistry; Northern Kentucky University), Maggie Thomas (Chemistry; Northern Kentucky University), Jennifer H. Brooks (Chemistry; Northern Kentucky University), Shelby Russell (Chemistry; Northern Kentucky University), Kathleen McElheney (Chemistry; Northern Kentucky University), Michael P. Guy (Chemistry; Northern Kentucky University)
Abstract:
tRNA must be chemically modified by cellular enzymes to function properly, and defects in these modifications are often associated with human disease. For example, mutations in human FTSJ1, an enzyme required for 2’-O-methylation of tRNA residues 32 (Nm32) and 34 (Nm34) cause non-syndromic X-linked intellectual disability, and upregulation of human DUS2, an enzyme required for dihydrouridine modification of tRNA residue 20 (D20) is linked to non-small cell lung cancer. Despite these links to disease, the precise translational roles of many tRNA modifications are not clear. We are using the fission yeast Schizosaccharomyces pombe as a model to study the cellular roles of several important eukaryotic tRNA modifications such as Nm34 and D20. We previously reported that trm734+ deletion mutants (which lack Nm34) grow slowly in fission yeast due to a defect in tRNAPhe. We report here that trm734+ deletion mutants are also sensitive to cycloheximide, indicating that Nm34 may be important for translational elongation. Furthermore, we have isolated suppressors of the trm734+ deletion strain that overcome the slow growth of mutants and alleviate cycloheximide sensitivity. We are currently working to identify the suppressing mutation(s) in these strains, and to determine which hypomodified tRNA(s) is affected by cycloheximide. In addition, to further define the roles of tRNA modifications in fission yeast, we are setting up a reporter system to quantitatively study the effects of modifications on a model suppressor tRNA.
Keywords: tRNA, S pombe, modifications
159. Using patient-derived data to customize smRNA-seq databases
Logan A Walker (The Ohio State University Department of Physics, Department of Astronomy), David Frankhouser (The Ohio State University Biomedical Sciences Graduate Program), Michael B Broe (The Ohio State University Department of Physics, Department of Evolution, Ecology, and Organismal Biology), Jennifer A Muszynski (Nationwide Childrens Hospital Division of Critical Care Medicince, The Research Institute, The Ohio State University Department of Internal Medicine Division of Pulmonary and Critical Care), Pearlly Yan (The Ohio State University Department of Internal Medicine Division of Hematology, The Ohio State University Comprehensive Cancer Center), Ralf Bundshuh (The Ohio State University Department of Physics, Department of Internal Medicine Division of Hematology, Department of Chemistry and Biochemistry, Center for RNA Biology)
Abstract not available online - please check the printed booklet.
160. Gamma Peptide Nucleic Acids as a Platform for Orthogonal Nucleic Acid Recognition Codes
Ashley Wang (Department of Chemistry, Carnegie Mellon University), Sharon Wu (Department of Chemistry, Carnegie Mellon University), Iulia Sacui, Wei-Che Hsieh (Department of Chemistry, Carnegie Mellon University), Arunava Manna (Department of Chemistry, Carnegie Mellon University), Bichismita Sahu (Department of Chemistry, Carnegie Mellon University), Danith H. Ly (Department of Chemistry, Carnegie Mellon University)
Abstract:
Nucleic acids have recently shown great promise in nanotechnology, including advances in molecular computing and assembly. The assembly of nucleic acids can be rationally designed and synthesized through knowledge and programmability of their thermodynamic behaviors and 3D structures. However, enzymatic degradation and cross-hybridization with the host genome present barriers for the further exploitation of nucleic acids in in-vivo studies. Such complications can be addressed with the use of peptide nucleic acid (PNA), a synthetic analogue of DNA and RNA which displays resistance towards enzymatic degradation and shows high sequence specificity. Our previous studies found that a simple backbone modification at the γ-position of the N-(2-aminoethyl) glycine unit preorganized the γ-PNA into a right handed (RH) helical motif, making γ-PNA more thermodynamically favorable for binding to complementary sequences (Bahal et al., 2012, Sahu et al., 2011). In our present work, we found that by switching the stereochemistry of the γ-position substituent, a mirror image of the RH molecule was produced. Circular dichroism studies revealed the γ-PNA adopted a left-handed (LH) helical sense. UV melting and fluorescent studies demonstrated that LH γ-PNA cannot hybridize to RH γ-PNA, DNA or RNA, classifying this new PNA as being orthogonal to the others. Furthermore, UV melting studies show that given the same base sequences but different helical senses, LH and RH γ-PNA have similar binding affinities toward their complementary sequences, allowing us to predict the thermodynamic properties of LH γ-PNA. More interestingly, without the γ-substituent, PNA adopted a nonhelical (NH) motif, allowing the NH PNA to act as an interface between LH γ-PNA and RH γ-PNA. These three moieties constitute a new platform for recognizing and manipulating endogenous genomic material, which can open up to new opportunities for in-vivo molecular self-assembly and computing.
References:
I. Sacui, W.-C. Hsieh, A. Manna, B. Sahu, and D. H. Ly, "Gamma peptide nucleic acids:
As orthogonal nucleic acid recognition codes for organizing molecular self-assembly",
J. Am Chem. Soc. 2015, 137, 8603.
B. Sahu, I. Sacui, S. Rapireddy, K. J. Zanotti, R. Bahal, B. A. Armitage, and D. H. Ly, “Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing γ-peptide nucleic acids with superior hybridization properties and water solubility”, J. Org. Chem. 2011, 76, 5614.
R. Bahal, B. Sahu, S. Rapireddy, C. M. Lee, and D. H. Ly, “Sequence-Unrestricted, Watson–Crick Recognition of Double Helical B-DNA by (R)-MiniPEG-γPNAs”, ChemBioChem. 2012, 13, 56.
Keywords: Peptide nucleic acid, Molecular self-assembly, Molecular computing
161. Repression of LINE-1 retrotransposition through cooperation of Condensin II and GAIT complexes in epithelial cells
Jacqueline R. Ward (Cellular and Molecular Medicine, Cleveland Clinic), Vasu Kommireddy (Cellular and Molecular Medicine, Cleveland Clinic), Dalia Halwani (Cellular and Molecular Medicine, Cleveland Clinic), Peter A. Larson (Department of Human Genetics, University of Michigan Medical School), John V. Moran (Department of Human Genetics, University of Michigan Medical School), Michelle S. Longworth (Cellular and Molecular Medicine, Cleveland Clinic)
Abstract not available online - please check the printed booklet.
162. The relationship between conformational dynamics and catalysis in the lead-dependent ribozyme
Neil A. White (Department of Biochemistry and Molecular Biology, Michigan State University), Minako Sumita (Department of Biochemistry and Molecular Biology, Michigan State University), Charles G. Hoogstraten (Department of Biochemistry and Molecular Biology, Michigan State University)
Abstract:
In both NMR and crystallographic structures of the lead-dependent ribozyme, or leadzyme, a conformational rearrangement is necessary to achieve the in-line geometry appropriate for self-cleavage. Thus, we and others have hypothesized that there is a transition from the inactive, ground-state structure to an active conformation which exists a small percentage of the time. Testing such models requires analyzing which molecular motions exist and also, whether the detected motions are catalytically relevant. In other words, we study dynamics-function relationships, in analogy with the classic structure-function relationship. In this work, we report NMR spin relaxation studies in combination with metabolically-driven specific stable isotope labeling to detect microsecond scale motions of the ribose group at the active site of the leadzyme. To test the functional relevance of this apparent ribose repuckering mode, we supplement our previously-reported application of locked nucleic acid (LNA) nucleotides with a modified structure that retains the nucleophilic 2′-hydroxyl while covalently restricting the ribose conformation to C3′-endo. The combined results are consistent with a dynamic repuckering of the ribose group adjacent to the scissile phosphate being a critical step in the ribozyme catalytic mechanism.
Keywords: leadzyme, conformational dynamics, RNA catalysis
163. Analysis of the RNA binding specificity of UPF1, the core protein involved in targeting aberrant mRNA to nonsense-mediated mRNA decay
Micayla Carafelli (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106, Cuyahoga Community College, Cleveland, OH 44115), DaJuan Whiteside (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106)
Abstract not available online - please check the printed booklet.
164. Title not available online - please see the printed booklet.
Rebecca N Williams-Wagner (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Frank J Grundy (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Tina M Henkin (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract not available online - please check the printed booklet.
165. 5-amino-pentanolylated elongation factor P influences cellular differentiation in Bacillus subtilis
Anne Witzky (Molecular Genetics and Center for RNA Biology, Ohio State University), Andrei Rajkovic (Molecular Cellular and Developmental Biology Program and Center for RNA Biology, Ohio State University), Katherine R. Hummels (Department of Biology, Indiana University), Sarah Erickson (Department of Chemistry, Ohio State University), Daniel B. Kearns (Department of Biology, Indiana University), Michael Ibba (Department of Microbiology, Ohio State University)
Abstract:
Bacillus subtilis is a gram-positive bacteria that exhibits two different types of motility: swimming and swarming. Although both movements require the same basic flagellar components, a number of regulatory factors have been identified that differentially impact swimming and swarming. For example, elongation factor P (EF-P) is a protein that is required for swarming but not swimming motility. EF-P is a universally conserved translation factor that relieves ribosomal pausing at polyproline motifs. In order to perform this function, EF-P requires post-translational modification. Although the position of the modification is conserved, the structure can vary widely between organisms. Here, we identify a novel EF-P post-translational modification, a 5-aminopentanol moiety, on Lys32. Based on the structure of the modification, it would appear to be derived from a polyamine. However, deletion of genes required for polyamine biosynthesis has no effect on modification, indicating that the substrate arises from another pathway. Mutation of Lys32 to an alanine prevents modification, abolishes swarming motility, and severely impairs EF-P dependent translation of a polyproline-GFP reporter construct in vivo. The Lys32Ala mutation also results in reduction in the levels of two polyproline containing flagellar components, likely explaining the requirement of EF-P for proper swarming. These findings not only establish a novel EF-P modification, but they also highlight its biochemical and physiological significance. Future studies will focus on identification of the genes required for EF-P modification in B. subtilis.
References:
Kearns DB, Chu F, Rudner R, Losick R (2004) Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility. Mol Microbiol 52: 357-369.
Lassak J, Wilson DN, Jung K (2016) Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A. Mol Microbiol 99: 219-235.
Keywords: Translation, Elongation Factor P, Bacillus subtilis
166. Exploring role of non-canonical exon junction complexes in human nonsense-mediated mRNA decay
Lauren Woodward (Department of Molecular Genetics, The Ohio State University), Justin Mabin (Department of Molecular Genetics, 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:
The exon junction complex (EJC)—a multi-protein complex composed of eIF4AIII, Y14 and Magoh—is characterized by its deposition ~24nt upstream of exon-exon junctions. However, recent transcriptome-wide analyses of EJC binding in human cells by multiple laboratories has revealed that EJC is frequently detected at locations other than the canonical -24nt position. Such EJC occupancy at the so-called non-canonical (nc) sites is puzzling in light of the prevailing view of EJC occupancy based on in vitro studies, which supports a splicing-dependent and sequence-independent EJC deposition almost exclusively at -24 position. Aside from their differences in position, ncEJCs protect longer stretches of RNA than cEJCs, suggesting that ncEJCs may associate with additional proteins. ncEJCs are widespread, binding the first exon, internal exons, 3’UTRs and even some intronless mRNAs. Analysis of ncEJC footprints located in internal exons has revealed an enrichment of SR-protein binding motifs, suggesting some ncEJCs are represent binding of SR-proteins in tight interactions with EJC. However, some ncEJCs are bona fide eIF4AIII-binding sites as revealed by direct crosslinking of eIF4AIII to these sites via CLIP-seq. Like the numerous roles of cEJCs in post-transcriptional gene regulation, ncEJCs would also be expected to contribute to mRNA processing and turnover. EJCs are well known to trigger nonsense-mediated mRNA degradation (NMD) when present downstream of a stop codon. To begin to address the origin and function of ncEJCs, we are focusing on ncEJCs that occur in 3'UTR regions and hence could activate NMD. We identified many endogenous mRNAs from HEK293 cells that undergo NMD and bear ncEJCs in their 3’UTRs, but lack classical NMD-inducing features. We have validated that some of these NMD targets are indeed up-regulated upon eIF4AIII depletion. We are currently testing the hypothesis that a sub-set of ncEJCs are similar to cEJCs in their composition and biological function.
Keywords: EJC, noncanonical EJC, NMD
167. Gamma Peptide Nucleic Acids as a Platform for Orthogonal Nucleic Acid Recognition Codes
Ashley Wang (Department of Chemistry, Carnegie Mellon University), Sharon Wu (Department of Chemistry, Carnegie Mellon University), Iulia Sacui, Wei-Che Hsieh (Department of Chemistry, Carnegie Mellon University), Arunava Manna (Department of Chemistry, Carnegie Mellon University), Bichismita Sahu (Department of Chemistry, Carnegie Mellon University), Danith H. Ly (Department of Chemistry, Carnegie Mellon University)
Abstract:
Nucleic acids have recently shown great promise in nanotechnology, including advances in molecular computing and assembly. The assembly of nucleic acids can be rationally designed and synthesized through knowledge and programmability of their thermodynamic behaviors and 3D structures. However, enzymatic degradation and cross-hybridization with the host genome present barriers for the further exploitation of nucleic acids in in-vivo studies. Such complications can be addressed with the use of peptide nucleic acid (PNA), a synthetic analogue of DNA and RNA which displays resistance towards enzymatic degradation and shows high sequence specificity. Our previous studies found that a simple backbone modification at the γ-position of the N-(2-aminoethyl) glycine unit preorganized the γ-PNA into a right handed (RH) helical motif, making γ-PNA more thermodynamically favorable for binding to complementary sequences (Bahal et al., 2012, Sahu et al., 2011). In our present work, we found that by switching the stereochemistry of the γ-position substituent, a mirror image of the RH molecule was produced. Circular dichroism studies revealed the γ-PNA adopted a left-handed (LH) helical sense. UV melting and fluorescent studies demonstrated that LH γ-PNA cannot hybridize to RH γ-PNA, DNA or RNA, classifying this new PNA as being orthogonal to the others. Furthermore, UV melting studies show that given the same base sequences but different helical senses, LH and RH γ-PNA have similar binding affinities toward their complementary sequences, allowing us to predict the thermodynamic properties of LH γ-PNA. More interestingly, without the γ-substituent, PNA adopted a nonhelical (NH) motif, allowing the NH PNA to act as an interface between LH γ-PNA and RH γ-PNA. These three moieties constitute a new platform for recognizing and manipulating endogenous genomic material, which can open up to new opportunities for in-vivo molecular self-assembly and computing.
References:
I. Sacui, W.-C. Hsieh, A. Manna, B. Sahu, and D. H. Ly, "Gamma peptide nucleic acids:
As orthogonal nucleic acid recognition codes for organizing molecular self-assembly",
J. Am Chem. Soc. 2015, 137, 8603.
B. Sahu, I. Sacui, S. Rapireddy, K. J. Zanotti, R. Bahal, B. A. Armitage, and D. H. Ly, “Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing γ-peptide nucleic acids with superior hybridization properties and water solubility”, J. Org. Chem. 2011, 76, 5614.
R. Bahal, B. Sahu, S. Rapireddy, C. M. Lee, and D. H. Ly, “Sequence-Unrestricted, Watson–Crick Recognition of Double Helical B-DNA by (R)-MiniPEG-γPNAs”, ChemBioChem. 2012, 13, 56.
Keywords: Peptide nucleic acid, Molecular self-assembly, Molecular computing
168. Title not available online - please see the printed booklet.
Congcong Xu (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute; The Ohio State University), Hui Li (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute; The Ohio State University), Kaiming Zhang (Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine), Daniel W. Binzel (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute; The Ohio State University), Wah Chiu (Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine), 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)
Abstract not available online - please check the printed booklet.
169. Investigation of multiple binding properties of POT1-TPP1 proteins in telomere ssDNA protection
Mengyuan Xu (Department of Pharmacology, Case Western Reserve University), Janna Kiselar (Center for Proteomics and Bioinformatics, Case Western Reserve University), Derek Taylor (Department of Pharmacology, Case Western Reserve University)
Abstract:
Telomeres are specialized nucleoprotein complexes that compose the very ends of linear chromosomes. Telomeres are needed to prevent the illicit induction of DNA damage response and end-to- end chromosome fusions. Telomere DNA consists of thousands of bases of double-strand DNA (dsDNA) before ending with a single strand DNA (ssDNA) overhang at the 3’ end that is 50-200 nucleotides in length. Both dsDNA and ssDNA of the telomere is characterized by a repeating hexameric sequence (TTAGGG in human). POT1 (Protection of Telomeres 1) is a telomere end-binding protein that binds specifically to telomere ssDNA. Together with TPP1 (Telomere Protection Protein 1), POT1 forms a heterodimer to both positively and negatively regulate telomere length. For example, POT1-TPP1 binds with nanomolar affinity to telomere ssDNA to protect it from nucleolytic attack. However, the POT1-TPP1 complex also actively recruits telomerase, the enzyme responsible for telomere extension, to enhance its activity. Most information regarding POT1-TPP1 and telomerase has been performed using short ssDNA oligonucleotides as a substrate. However, the physiological length of G-rich telomeric ssDNA is prone to form stable intermolecular structures called G-quadruplexes, which might additionally play a role in telomere maintenance. Our lab has shown that POT1-TPP1 proteins can help to disrupt G-quadruplex structures by coating long, telomere ssDNA with multiple proteins. We have used hydroxyl radiolytic footprinting coupled with mass spectrometry to better understand the protein-protein interactions that are involved in assembling long tracts of telomere ssDNA coated with multiple POT1-TPP1 proteins. Our data highlight key residues, including H266, an amino acid that is mutated (H266L) in patients suffering from chronic lymphocytic leukemia. We are mutating the H266 in recombinant POT1 protein and will quantitatively determine the consequences it imposes in disrupting G-quadruplex structures and in TPP1- and ssDNA interactions. This information will provide important knowledge for understanding how telomere proteins regulate telomere length and how cancer mutations disrupt these important processes.
Keywords: Telomere, POT1, Footprinting
170. Title not available online - please see the printed booklet.
Ryota Yamagami (Department of Chemistry, Pennsylvania State University.), Philip C. Bevilacqua (Department of Chemistry, Pennsylvania State University. Center for RNA Molecular Biology, Pennsylvania State University. Microbiology and Molecular Biology, Pennsylvania State University.)
Abstract not available online - please check the printed booklet.
171. Title not available online - please see the printed booklet.
Karl Kroll, David Frankhouser, Dimitrios Papaioannou, Paige Stump, (Division of Hematology, Department of Internal Medicine, Ohio State University and James Comprehensive Cancer Center), Alexander Pelletier, Stefano Volinia, James Blachly, Clara Bloomfield (Division of Hematology, Department of Internal Medicine, Ohio State University and James Comprehensive Cancer Center), Ramiro Garzon, John Byrd (Division of Hematology, Department of Internal Medicine, Ohio State University and James Comprehensive Cancer Center), Ralf Bundschuh (Department of Physics, Division of Hematology, Department of Internal Medicine, Ohio State University and James Comprehensive Cancer Center), Pearlly Yan (Division of Hematology, Department of Internal Medicine, Ohio State University and James Comprehensive Cancer Center)
Abstract not available online - please check the printed booklet.
172. Processive RNA degradation by the exonuclease Pop2p
Xuan Ye (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Armend Axhemi (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH )
Abstract not available online - please check the printed booklet.
173. Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding
Lanqing Ying (Department of Microbiology and Center for RNA Biology, The Ohio State University), Kurt Fredrick (Department of Microbiology and Center for RNA Biology, The Ohio State University)
Abstract:
The ribosome actively participates in decoding, with a tRNA-dependent rearrangement of the 30S A site playing a key role. Ribosomal ambiguity (ram) mutations have mapped not only to the A site but also to the h12/S4/S5 region and intersubunit bridge B8, implicating other conformational changes such as 30S shoulder rotation and B8 disruption in the mechanism of decoding. Recent crystallographic data have revealed that mutation G299A in helix h12 allosterically promotes B8 disruption, raising the question of whether G299A and/or other ram mutations act mainly via B8. Here, we compared the effects of each of several ram mutations in the absence and presence of mutation h8Δ2, which effectively takes out bridge B8. The data obtained suggest that a subset of mutations including G299A act in part via B8 but predominantly through another mechanism. We also found that G299A in h12 and G347U in h14 each stabilize tRNA in the A site. Collectively, these data support a model in which rearrangement of the 30S A site, inward shoulder rotation, and bridge B8 disruption are loosely coupled events, all of which promote progression along the productive pathway toward peptide bond formation.
Keywords: translation, aminoacyl-tRNA selection, fidelity
174. Title not available online - please see the printed booklet.
Leah L. Zagore (Center for RNA Molecular Biology, Biochemistry Department, Case Western Reserve University), Thomas J. Sweet (Center for RNA Molecular Biology, Biochemistry Department, Case Western Reserve University), Donny D. Licatalosi (Center for RNA Molecular Biology, Biochemistry Department, Case Western Reserve University)
Abstract not available online - please check the printed booklet.
175. Detection of Post-Transcriptional RNA Modifications in the Radioresistant Bacterium Deinococcus radiodurans R1
Ruoxia Zhao (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), Patrick A. Limbach (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati)
Abstract:
The bacterium Deinococcus radiodurans is one of the most ionizing radiation resistant families of bacteria discovered to date. D. radiodurans has extraordinary resistance to all reactive oxygen species(ROS)-generating agent including desiccation, ionizing radiation, UV radiation, mitomycin C and so on.
In order to understand whether transfer and ribosomal RNAs (tRNAs, rRNAs) are modified differently when D. radiodurans are exposed to ionizing radiation (IR), the modified nucleosides in control D. radiodurans and D. radiodurans with different extent of IR (60Co) have been analyzed by Reverse-Phase High Performance Liquid Chromatography Mass Spectrometry (RP-HPLC-MS/MS). RNA modification mapping is conducted by utilizing a base-specific ribonuclease (RNase T1) to cleave a larger RNA into smaller digestion products (oligonucleotides). The location and identity of nucleotides and modified nucleotides can be determined by using of a DNA-based exclusion list which enhances data dependent RP-HPLC-MS/MS detection method. Determining the modified RNA sequence will increase the understanding of structure-function relationships of RNAs.
References:
Battista, J. R. 1997. Against all odds: the survival strategies of Deinococcus radiodurans. Annu. Rev. Microbiol. 51:203–224.
Battista, J. R., A. M. Earl, and M. J. Park. 1999. Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends Microbiol 7:362-5.
Ross, Robert, et al. "Sequence mapping of transfer RNA chemical modifications by liquid chromatography tandem mass spectrometry." Methods (2016).
Cao, Xiaoyu, and Patrick A. Limbach. "Enhanced detection of post-transcriptional modifications using a mass-exclusion list strategy for RNA modification mapping by LC-MS/MS." Analytical chemistry 87.16 (2015): 8433-8440.
Keywords: Radioresistant, RNA modifications, HPLC-MSMS
176. Regulatory Mechanisms of tRNA Fragments in Ovarian Cancer Cells
Kun Zhou (Department of Biomedical Sciences, University of Minnesota), Juan E. Abrahante (University of Minnesota Informatics Institute, University of Minnesota), Kevin W. Diebel (Department of Biomedical Sciences, University of Minnesota), Brent Schotl (Department of Biomedical Sciences, University of Minnesota), Monique A. Spillman (Texas A&M University Medical School, Baylor University Medical Center), Lynne Bemis (Department of Biomedical Sciences, University of Minnesota)
Abstract not available online - please check the printed booklet.
177. Identification of a conserved motif required for Trm732 function in eukaryotes
Julia Ziebro (Department of Chemistry, Northern Kentucky University), Daisy DiVita (Department of Chemistry, Northern Kentucky University), Justen Mamaril (Department of Chemistry, Northern Kentucky University), Gregory M. OConnor (Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA), Katie L. Weiner (Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA), Michael P. Guy (Department of Chemistry, Northern Kentucky University)
Abstract:
Posttranscriptional tRNA modifications are required for accurate and efficient protein synthesis, and defects in several modifications are associated with human diseases. One of the most common tRNA modifications is 2’-O-methylation (Nm). Lack of this modification on residues 32 (Nm32) and 34 (Nm34) in tRNA causes slow growth in yeast and non-syndromic X-linked intellectual disability in humans. In eukaryotes, formation of Nm32 requires the methyltransferase Trm7 and its partner protein Trm732. Human TRM732 (THADA) has been associated with type 2 diabetes and polycystic ovary syndrome in several genome-wide association studies, but it is not clear why Trm732 is required for Nm32 formation. The goal of this study is to determine the role of Trm732 in Nm32 formation. Using site-directed mutagenesis and a growth assay in yeast we have identified a conserved motif in Trm732 that is required for efficient Nm32 formation. We are currently working to identify the cause of the modification defect in Trm732 variants with changes in this motif. Additionally, to study Nm32 and other 2’-O-methylations in metazoans, we are developing a sensitive primer extension-based technique. By coupling this technique with gene knockdown we hope to significantly enhance our ability to study tRNA modifications in diverse eukaryotes.
Keywords: tRNA, trm732, 2-O-methylation
178. Deep analysis of tails on Trypanosoma brucei mitochondrial transcripts reveals life stage as well as transcript-specific regulation
Sara L. Zimmer (Department of Biomedical Sciences, University of Minnesota Medical School), Vahid H. Gazestani (Institute of Parasitology, McGill University), Marshall Hampton (Department of Mathematics and Statistics, University of Minnesota Duluth), Aubie K. Shaw (Department of Biomedical Sciences, University of Minnesota Medical School), Charles Liggett (Department of Biomedical Sciences, University of Minnesota Medical School), Reza Salavati (Institute of Parasitology, McGill University)
Abstract not available online - please check the printed booklet.