Cellular, Molecular & Biochemical Sciences Training Program/Center for RNA Biology Annual Symposium 2018

Poster abstracts

1. 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, Ohio ), Frank J. Grundy (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus, Ohio ), Tina M. Henkin (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus, Ohio )

Abstract:
The S-box riboswitches regulate the expression of genes involved in methionine and cysteine metabolism. Most S-Box riboswitches operate at the level of transcription attenuation, such that a terminator helix is stabilized when S-adenosylmethionine (SAM) binds the aptamer. In silico analyses have suggested that a rarer class of S-box riboswitches may regulate at the level of translation initiation. We identified a leader RNA, metI from Desulfurispirilum indicum, that has the potential for SAM-dependent sequestration of the Shine-Dalgarno (SD) region which suggests translational regulation. The aptamer domain of the RNA bound SAM with affinity comparable to that of the well-characterized Bacillus subtilis yitJ S-box riboswitch, and with similar selectivity against S-adenosylhomocysteine (SAH). A mutation at a position previously shown to disrupt SAM binding in other S-box RNAs resulted in loss of SAM binding in metI. These results demonstrate that the aptamer domain has SAM binding properties similar to those of previously characterized S-box riboswitches. The SD region of the RNA exhibited SAM-dependent structural rearrangements consistent with the hypothesis that regulation occurs at the level of translation initiation. Additionally, binding of 30S subunits to metI RNA was reduced in the presence of SAM, demonstrating that the riboswitch regulates at the level of translation initiation. Preliminary fluorescence studies with the full-length metI RNA suggest that the riboswitch can perform reversible regulation in response to changing levels of SAM. The metI riboswitches from Desulfurispirilum indicum and Bacillus subtilis will be used as a model to compare the characteristics of transcriptional and translational riboswitches in the same class. We hypothesize that the translational riboswitches can perform multiple reversible regulatory decisions whereas transcriptional riboswitches cannot reverse their regulatory decisions. Such a comparative study will provide insight into the functional role of transcriptional vs. translational regulation of the same gene by the same class of riboswitch in different organisms.

Keywords: S-box, Translation, Riboswitch

2. Thermodynamics and Kinetics of the Three-component Collision of Phi29 pRNA-3WJ Assembly for Nanoparticles Targeting and Therapeutic Delivery to Prostate Cancer

Daniel Binzel (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; College of Medicine, Dorothy M. Davis Heart and Lung Research Institute ), Bin Guo (Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX 77030, USA), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; College of Medicine, Dorothy M. Davis Heart and Lung Research Institute )

Abstract:
The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles, but has been limited by particle instability. Previously, it was found the three-way junction (3WJ) of the Phi29 DNA packaging motor pRNA was found to be ultra-stable and assemble with ease. The three-way junction is composed of three short oligo RNA strands and proven to be thermodynamically stable. Thermodynamic and kinetics studies found that the pRNA 3WJ formed at a rapid rate creating a single-step three component collision with a lack of dimer intermediate formation while being governed by entropy, instead of enthalpy as commonly seen in RNA duplexes. The 3WJ proved to be stable at high temperatures, ultra-low concentrations, and produced a Gibbs free energy of formation well below other studied RNA structures and motifs. With the high stability and folding efficiency of the pRNA 3WJ, it serves as an ideal platform for RNA nanoparticles. RNA nanoparticles were constructed for the targeting of prostate cancer cells expressing Prostate Specific Membrane Antigen (PSMA) through the addition of an RNA apatmer; and the delivery of anti-miRNA sequences. The resulting nanoparticles remained stable while showing specific entry in PSMA positive cells through cell surface receptor endocytosis. The entry of the nanoparticles allowed for specific delivery against onco-miRNAs. Delivery of anti-miRNAs led to the upregulation of pro-apoptotic genes, and down regulation of anti-apoptotic genes in vitro and in vivo. These findings display RNA nanotechnology can result in stable nanoparticles and result in the specific treatment of cancers, specifically prostate cancer.

References:
1. Binzel DW, Khisamutdinov EF, Guo P. Entropy-driven one-step formation of Phi29 pRNA 3WJ from three RNA fragments. Biochemistry. 2014. 53(14):2221-31.
2. Binzel DW, Khisamutdinov E, Vieweger M, Ortega J, Li J, Guo P. Mechanism of three-component collision to produce ultrastable pRNA three-way junction of Phi29 DNA-packaging motor by kinetic assessment. RNA. 2016 22(11):1710-1718.
3. Binzel DW, Shu Y, Li H, Sun M, Zhang Q, Shu D, Guo B, Guo P. Specific delivery of miRNA for high efficient inhibition of prostate cancer by RNA nanotechnology. Molecular Therapy. 2016. 24(7):1267-77.

Keywords: RNA Nanotechnology, Kinetics, Prostate Cancer

3. SnRK1 phosphorylates eIF4E/eIFiso4E to inhibit translation as a component of antiviral defense

Aaron N. Bruns (OSBP), Sizhun Li (The Ohio State University), David M. Bisaro (Department of Molecular Genetics, The Ohio State Uninversity)

Abstract:
SNF1 related kinase 1 (SnRK1) is a central sensor and regulator of energy homeostasis in plants. As the plant homologue of animal AMP-activated protein kinase (AMPK) and yeast sucrose non-fermenting kinase (SNF1), SnRK1 senses the ATP:AMP ratio in the cell, activating catabolic processes when energy levels are low while simultaneously inhibiting anabolic processes. We previously demonstrated that SnRK1 conditions an innate antiviral defense effective against DNA and RNA viruses, including geminiviruses and TMV. We also showed that the geminivirus pathogenicity proteins AL2 and L2 interact with and inactive SnRK1 as a counterdefense. However, because SnRK1 has a multitude of targets, the mechanism by which SnRK1 interferes with viral infectivity was elusive. We have now demonstrated that SnRK1 interacts with and phosphorylates the mRNA cap binding protein eukaryotic initiation factor 4E (eIF4E), and its plant specific homologue eIFiso4E, at two serine and threonine residues that are conserved throughout the plant kingdom as well as invertebrates and yeast. However, these sites are absent in vertebrate eIF4E homologues, suggesting distinct regulatory mechanisms. Furthermore, we discovered that eIF4E/iso4E phosphorylation by SnRK1 inhibits translation in vivo using both plant and complemented yeast systems. This is the first evidence that SnRK1 directly inhibits translation in plant cells. It also suggests that SnRK1 phosphorylation of eIF4E/iso4E is a host defense analogous to PKR-mediated phosphorylation of eIF2α in vertebrate cells. Many plant viruses, including geminiviruses, rely on cap-dependent host translation for protein synthesis. Thus, limiting translation initiation through SnRK1 phosphorylation of eIF4E/eIFiso4E can effectively impede virus spread.

Keywords: SnRK1, eIF4E, Translation

4. Measurements of first- versus multiple- round translation in E. coli argue against a mechanism to ensure coupling of transcription and translation

Menglin Chen (Department of Microbiology Ohio State Biochemistry Program, The Ohio State University), Kurt Fredrick (Department of Microbiology, The Ohio State University)

Abstract:
In prokaryotes, the synthesis of RNA and protein occurs simultaneously in the cytoplasm. A number of studies indicate that translation can strongly impact transcription, and this is believed to be due to coupling between RNA polymerase (RNAP) and the ribosome. Whether there exists a mechanism to ensure or promote RNAP-ribosome coupling remains unclear. Here, we used an efficient hammerhead ribozyme and developed a novel reporter system to measure first- versus multiple-round translation in E. coli. Six pairs of co-transcribed and differentially-translated genes were analyzed. For five of them, the stoichiometry of the two protein products came no closer to unity (1:1) when the rounds of translation were dramatically reduced. These data are consistent with models of stochastic coupling and suggest that RNAP often transcribes without a linked ribosome.

Keywords: ribosome, RNA polymerase, NusG

5. Identification of A Novel Epigenetic Silencing Factor in Arabidopsis Thaliana

Sarah G. Choudury (MCDB, Department of Molecular Genetics), Kaushik Panda (Department of Molecular Genetics), Diego Cuerda Gil (Department of Molecular Genetics), Andrea McCue (Department of Molecular Genetics), Alissa Cullen (Department of Molecular Genetics)

Abstract:
In order to maintain genome integrity, fungi, plants, and animals modify transposable element (TE) chromatin to epigenetically repress TE activity. DNA methylation is critical for this epigenetic repression. Once established at TEs, DNA methylation can be propagated through cell divisions by methyltransferases. However, the mechanism by which DNA methylation and epigenetic silencing are originally targeted to TEs is not well understood. Our lab has identified a pathway in Arabidopsis that acts to direct de novo cytosine DNA methylation to transcriptionally active TEs, and is thus in part responsible for the initiation of silencing at these loci. This pathway utilizes endogenous 21-22 nucleotide (nt) small interfering RNAs (siRNAs) which result from the post-transcriptional degradation of TE mRNAs. The siRNAs direct Argonaute (AGO) proteins to chromatin which then triggers de novo DNA methylation. Previous studies from our lab genetically identified AGO6 as the key effector protein in this pathway. Because of AGO6’s key role in the initiation of silencing, we have focused on it and its interactors to better understand the pathway mechanism. Previous experiments in our lab utilized immunoprecipitation (IP) of AGO6 followed by mass spectrometry (MS) to identify AGO6-interacting proteins. We set out to discover novel silencing factors by screening through AGO6-interactors identified by IP-MS. For those factors that were found to play an important role in silencing, I aimed to understand their mode of action by performing molecular characterizations including whole genome methylation analysis, sRNA production assays, and AGO sRNA-loading assays. Using this approach we have identified a novel factor required for silencing initiation at TEs.

Keywords: Epigenetics, DNA Methylation, Transposable Elements

6. The role of lpr-3 as a cell non-autonomous activator of oncogenic let-60/Ras in C. elegans

Marcos Corchado (Molecular Genetics, The Ohio State University), Komal Rombani (Biomedical Sciences Graduate Program, The Ohio State University), Gustavo Leone (Biochemistry and Molecular Biology Department, Medical University of South Carolina), Helen Chamberlin (Molecular Genetics, The Ohio State University)

Abstract:
Extracellular signals from the stroma are required for the maintenance and progression of cancer cells. However, identifying such factors is particularly challenging due to the complexity of stromal cell types in mammals. A recent screen performed in a simpler system, the nematode C. elegans, identified lipocalin-related 3 (lpr-3) as a candidate for such a signal. lpr-3 is expressed in mesodermal tissue but is required for let-60/Ras-mediated hyperproliferation of a set of epithelial cells known as the vulva precursor cells (VPCs). Moreover, most lipocalins transport hydrophobic ligands and initiate signaling cascades by binding to specific membrane receptors. The first aim of my research is to identify the receptor of LPR-3. It is known that human lipocalins are endocytosed after binding to a receptor to perform their function. To test if LPR-3 functions through a similar mechanism, I inhibited receptor-mediated endocytosis in the VPCs by performing a VPC-specific RNAi knockdown of clathrin on let-60(gf) animals. Results show a significant suppression of hyperproliferation, which supports the hypothesis that an LPR-3 receptor is present. Subsequently, we performed a literature review to identify candidate receptors. Human Lipocalin-2 was found to function similarly to LPR-3 and binds to a member of solute carrier protein family (SLC) of membrane receptors. Thus, I performed VPC-specific RNAi knockdown experiments for 239 out of 364 C. elegans SLC genes on let-60(gf) animals. In particular, the gene pitr-1 was found to partially suppress hyperproliferation. We hypothesize that partial suppression might be due to the presence of a redundant receptor and suggest performing double RNAi knockdown experiments with pitr-1 and candidates as future work. As an alternative approach, I am performing a genome-wide VPC-specific RNAi screen on let-60(gf) animals to identify potential receptors and downstream effectors of LPR-3. These results fit a model in which LPR-3 binds to PITR-1 and is endocytosed to promote let-60(gf) hyperproliferation. A genome-wide screen for LPR-3 receptors and downstream effectors has been completed for chromosome I and yielded 40 candidates thus far.

Keywords: C elegans, Ras, lpr-3

7. RMR12 is a CHD3 nucleosome remodeler required for maintaining paramutations and normal development

Natalie Deans (Department of Molecular Genetics, The Ohio State University, Columbus, OH), Brian Giacopelli (Department of Molecular Genetics, The Ohio State University, Columbus, OH), Daniel Hlavaty, Emily McCormic (Department of Molecular Genetics, The Ohio State University, Columbus, OH), Charles Addo-Quaye (2Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN), Brian Dilkes (Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN), Jay Hollick (Department of Molecular Genetics, Center for Applied Plant Sciences and Center for RNA Biology, The Ohio State University, Columbus, OH)

Abstract:
In maize, paramutations result in meiotically heritable changes in the regulation of certain alleles of purple plant1 ¹, a gene encoding a transcription factor required for anthocyanin production². A strongly expressed Pl1-Rhoades allele is suppressed in trans when combined with a transcriptionally and post-transcriptionally repressed Pl1-Rhoades allele, and both alleles are transmitted in a repressed (denoted Pl´ ) state. At least sixteen loci whose functions are required to maintain repression (rmr) of Pl´ have been identified by ethyl methanesulfonate mutagenesis³. All known RMR proteins mediate 24 nucleotide (24nt) RNA biogenesis^{4, 5, 6, 7, 8}, and four are putative orthologs of Arabidopsis proteins central to an RNA-directed DNA Methylation (RdDM) pathway facilitating repressive chromatin modifications. Here we describe four recessive alleles defining the rmr12 locus. Unlike other rmr-type mutations found to date^{4, 5, 7, 8, 9, 10}, rmr12 mutants display a unique set of defects, including male gametophyte dysfunction, that highlight a novel mechanistic connection between paramutation and developmental gene control. We used positional cloning to discover that rmr12 encodes a Chromodomain Helicase-DNA Binding3 (CHD3) protein whose presumed Arabidopsis ortholog, PICKLE, alters nucleosome positions in vitro¹¹ and affects both development and chromatin modifications specified by RdDM^{12, 13}. Maize CHD3 represents the first RMR protein not having a predicted role in 24nt RNA biogenesis and thus might facilitate paramutations by converting 24nt RNA effectors into meiotically-heritable nucleosome alterations.

References:
1. Hollick et al. 1995 Genetics 141, 709. | 2. Cone et al. 1993 Plant Cell 5, 1795 | 3. Hollick and Chandler 2001 Genetics 157, 369 | 4. Erhard et al. 2009 Science 323, 1201 | 5. Hale et al. 2007 PLoS Biol. 5, 2156 | 6. Nobuta et al. 2008 PNAS 105, 14958 | 7. Stonaker et al. 2009 PLoS Genet. 5, e1000706. | 8. Barbour et al. 2012 Plant Cell 24, 1761 | 9. Dorweiler et al. 2000 Plant Cell 12, 2101. | 10. Parkinson et al. 2007 Dev. Biol. 308, 462. | 11. Ho et al. 2013 Biochim. Biophys. Acta 1829, 199 | 12. Ogas et al. 1997 Science 277, 91 | 13. Yang et al. 2017 Genome Biol. 18, 103

Keywords: Development, Gene regulation, Chromatin

8. Characterization of the Function of an Orphan 3'-5' RNA Polymerase Involved in Noncoding RNA Processing.

Samantha Dodbele (Ohio State Biochemistry Program, The Ohio State University OSU; Center for RNA Biology, OSU), Blythe Moreland (Center for RNA Biology, OSU; Department of Physics, OSU), Yicheng Long (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU), Ralf Bundschuh (Center for RNA Biology, OSU; Department of Physics, OSU; Division of Hematology and Department of Internal Medicine, OSU), Jane Jackman (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU; Department of Chemistry and Biochemistry, OSU)

Abstract:
Until the discovery of tRNAHis guanylyltransferase (Thg1) from Saccharomyces cerevisiae (Sc), nucleotide polymerization was believed to exclusively occur in the 5'-3' direction. Thg1 shifts this paradigm by catalyzing the non-templated addition of a required guanosine to the 5' end of tRNAHis in a 3'-5' direction. Enzymes exhibiting similarity to ScThg1, called Thg1-like proteins (TLPs) catalyze a Watson-Crick dependent 3'-5' polymerization. TLPs have been found in all three domains of life, including eukaryotic organisms such as Dictyostelium discoideum (Ddi). However, the roles and mechanisms of TLPs compared to their relatively more well-studied Thg1 counterparts are less understood.

Previous work by our group has demonstrated the functions of two TLPs in the model eukaryote D. discoideum. These TLP enzymes, DdiTLP2, and DdiTLP3 catalyze a Watson-Crick dependent 3'-5' polymerization, and are responsible for mitochondrial tRNAHis maturation, and mitochondrial tRNA 5'-editing, respectively. However the biological function of a third TLP enzyme encoded in D. discoideum, DdiTLP4, remains unknown. In vitro studies suggest DdiTLP4 can act on several small, noncoding RNAs (ncRNA) in addition to tRNAs. Moreover, depletion of DdiTLP4 causes a severe growth defect in D. discoideum. Now we have the first evidence to suggest that the essential function of DdiTLP4 is due to its role in small RNA processing and its activity on specific ncRNA substrates. Depletion of DdiTLP4 followed by RNA-Seq was used to identify in vivo substrates of DdiTLP4 and enabling the identification of any type of RNA whose 5'-end sequence is altered in the absence of DdiTLP4 activity. This work comprises the first comprehensive insight into 3'-5' polymerase substrate specificity, including into non-tRNA related activities associated with these enzymes. Furthermore, investigation into the biological function of DdiTLP4 has provided greater understanding of the 5'-end maintenance machinery of eukaryotes and into diverse biological roles for 3'-5' polymerization.

Keywords: 3 to 5 polymerization, ncRNA, RNA-Seq

9. Reb1 is a pioneer factor that dynamically regulates nucleosomal DNA accessibility

Ben Donovan (The Ohio State University), Caroline Jipa (The Ohio State University ), Chao Yan (The Pennsylvania State University), Lu Bai (The Pennsylvania State University), Michael Poirier (The Ohio State University )

Abstract:
Packaging the eukaryotic genome into nucleosomes greatly limits transcription factor (TF) occupancy by restricting binding site accessibility and accelerating TF off-rates. Because of this, nucleosomes are precisely positioned to regulate TF activation of transcription. Reb1 is a TF from S. cerevisiae that can recruit chromatin remodelers and generate nucleosome-depleted regions. Previous studies also suggest that Reb1 preferentially targets nucleosomes at the DNA entry/exit region. To probe Reb1-nucleosome interactions, we inserted a Reb1 binding site in increments of 5 bp throughout the entry/exit region and monitored Reb1 binding and Reb1-induced nucleosome structural changes. Gel shift measurements reveal Reb1 binds nucleosomes with a similar affinity to naked DNA, which is in stark contrast to other TFs such as Gal4 that binds nucleosomal binding sites with drastically lower affinities. This property is shared with eukaryotic pioneer factors Sox2 and Oct4 suggesting Reb1 functions as a pioneer factor. Ensemble FRET measurements show that Reb1 binding to its site within the entry-exit region traps nucleosomes in a partially unwrapped state without evicting histones. This indicates that Reb1 functions to expose nucleosomal DNA and reduce nucleosome stability. Single molecule fluorescence measurements show that despite similar equilibrium affinities, exchange kinetics are 50-fold slower at nucleosomal DNA sites relative to naked DNA, highlighting another distinct nucleosome binding behavior as compared with other TFs that have dramatically increased exchange kinetics at nucleosomal binding sites. From these results, we propose that Reb1 can function as a pioneer factor that induces nucleosome unwrapping and resides at nucleosomal DNA entry/exit sites for minutes to facilitate the recruitment of chromatin remodelers.

References:
Y. Luo, J. A. North, S. D. Rose, and M. G. Poirier, «Nucleosomes accelerate transcription factor dissociation», Nucleic Acids Res., vol 42, no 5, pp 3017–3027, 2014.
C. Jiang and B. F. Pugh, «Nucleosome positioning and gene regulation: advances through genomics.», Nat. Rev. Genet., vol 10, no 3, pp 161–172, 2009.
N. Krietenstein et al., «Genomic Nucleosome Organization Reconstituted with Pure Proteins», Cell, vol 167, no 3, p 709–721.e12, 2016.
R. T. Koerber, H. S. Rhee, C. Jiang, and B. F. Pugh, «Interaction of Transcriptional Regulators with Specific Nucleosomes across the Saccharomyces Genome», Mol. Cell, vol 35, no 6, pp 889–902, 2009.
A. Soufi, M. F. Garcia, A. Jaroszewicz, N. Osman, M. Pellegrini, and K. S. Zaret, «Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming», Cell, vol 161, no 3, pp 555–568, 2014.

Keywords: chromatin, pioneer factor, single molecule

10. The mRNA methyltransferase, METTL3, regulates cardiac hypertrophy

Lisa E. Dorn (Department of Physiology and Cell Biology, The Ohio State University), Jop H. van Berlo (Cardiovascular Division, University of Minnesota), Chuan He (Department of Chemistry, University of Chicago), Federica Accornero (Department of Physiology and Cell Biology, The Ohio State University)

Abstract:
Cardiac hypertrophy is a risk factor for developing heart failure, and cardiac hypertrophy is mediated by increased synthesis of specific proteins in cardiomyocytes. Although significant progress has been made in understanding the transcriptional changes occurring in the remodeling heart, very little is known about how post-transcriptional events control the synthesis of maladaptive proteins in the stressed myocardium. The most abundant mRNA post-transcriptional modification is N6-methyladenosine (m6A), catalyzed by Methyltransferase-like 3 (METTL3). m6A levels are increased in human failing hearts, as well as in isolated neonatal rat cardiomyocytes (NRCMs) subjected to hypertrophic stimuli. To determine m6A’s role in cardiac remodeling, we generated a mouse model for cardiomyocyte-specific METTL3 overexpression and found that increasing m6A methylation is sufficient to promote cardiomyocyte hypertrophy. Specifically, mice overexpressing METTL3 have increased heart weight to body weight and tibia length ratios under baseline conditions. Analyses of cardiac cross-sections in these mice demonstrate increased cardiomyocyte area without a concurrent increase in fibrosis or other degenerative remodeling. METTL3-mediated hypertrophy suggests that, conversely, cardiac METTL3 knockdown may prevent hypertrophy. We adopted a loss of function approach through siRNA-mediated METTL3 silencing in NRCMs as well as a cardiomyocyte-specific METTL3 knockout mouse line. Reducing METTL3 prevents cardiomyocyte hypertrophy and blunts expression of pro-hypertrophic markers in isolated cells exposed to hypertrophic stimuli, and METTL3 knockout mice demonstrate cardiac structural and functional changes with aging consistent with heart failure and an inability to undergo physiological hypertrophy. Our recent findings support a role of METTL3 in modulating the cardiac hypertrophic response, and suggest METTL3 as a potential therapeutic target in the management of hypertrophy in heart failure.

Keywords: hypertrophy, m6A

11. Construction of AFM Imaging of Hexameric pRNA of phi29 DNA Packaging Motor by RNA Nanotechnology

Dana Driver (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry), Yi Shu, Peixuan Guo

Abstract:
The packaging of bacteriophage phi29 DNA genome is accomplished through the use of six RNA molecules called pRNA. pRNA serves as an interface between the gp16 ATPase and the connector protein. Without pRNA, gp16 does not bind to the procapsid and packaging does not occur. The pRNA contains upper and lower loops via a hand-in-hand integration mechanism in inter-RNA interactions. The right loop is delineated with a capital letter, while the left loop is the lower case letter. pRNA A-b’ or B-a’ with a mutation in only one hand will lead to an inactive pRNA unsuitable for packaging; however, mixing of pRNA A-b’ and B-a’ in equal concentrations to form a dimer by hand-in-hand interaction will result in active pRNA. We report the use of hand-in-hand interaction of the pRNA that allows for the construction of hexamer. The pRNA functionality for DNA packaging was assayed by DNAase protection viral assembly. Up to 8 x 10^7 PFU of infectious virions per mL were assembled in vitro, with the addition of 11 proteins derived from cloned genes and nucleic acids synthesized separately. The ultimate goal of this project is to image six pRNA strands assembled into a hexamer. A RNA triangle was fused to each pRNA strands at the 3’ end. Each RNA was produced from the PCR product using unmodified RNA transcription. Bottom-up assembly was achieved by annealing the pRNA strands and running them in 4% native gel. Through application of RNA nanotechnology, we have assembled the hexameric pRNA complex with six purified RNA molecules. Sequential addition of each RNA strands was detected by gel as evidence by the presence of RNA oligomers with gradual reduction of migration rate in native gel. Formation of RNA hexamer was confirmed by AFM imaging.

Keywords: pRNA, phi29 bacteriophage, hexamer RNA

12. New tRNA interaction sites in the US T-box riboswitch

Jane K Frandsen (Ohio State Biochemistry Program, Center for RNA Biology), Anna V. Sherwood (Molecular Cellular and Developmental Biology Graduate Program, Center for RNA Biology), Frank J. Grundy (Department of Microbiology, Center for RNA Biology), Tina M. Henkin (Department of Microbiology, Ohio State Biochemistry Program, Molecular Cellular and Developmental Biology Graduate Program, Center for RNA Biology)

Abstract:
T-box riboswitches regulate the expression of essential amino acid-related genes in Firmicutes and Actinobacteria by monitoring the aminoacylation status of a cognate tRNA (1). In T-box RNAs with a canonical Stem I, the Specifier Loop interacts with the anticodon of the cognate tRNA and the terminal loop and AG bulge form a loop-loop structure that interacts with the tRNA elbow. The Ultrashort (US) Stem I class of ileS T-box riboswitches, which regulate isoleucyl-tRNA synthetase genes, have an alternate Specifier Loop structure and lack the elements necessary for interaction with the tRNA elbow, but contain the highly conserved Stem II and Stem IIA/B pseudoknot, whose role in tRNA recognition remained unknown (2). We have now demonstrated that both Stem II and Stem IIA/B of the US ileS RNA contribute to tRNA^Ile affinity (3). Additionally, using selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE), crosslinking, and mutational studies, we identified two new interaction sites, one between the S-turn element in Stem II and the tRNA T arm and the other between the pseudoknot and the tRNA D loop. This is the first example of tRNA recognition by an S-turn motif or a pseudoknot element, revealing new types of RNA-RNA interactions. These data provide the first biochemical evidence for the functional role of Stem II and the pseudoknot, which are present in the majority of T-box RNAs but absent in the glyQS RNAs that have been used for most of the current biochemical and structural analyses. We hypothesize that these interactions are important for the recognition and binding of the cognate tRNA and discrimination against non-cognate tRNA in those T-box RNAs in which they are present, and propose that structural variability in T-box riboswitches indicates alternate solutions to the tRNA recognition problem.

References:
1. Henkin TM. Biochim Biophys Acta 1839:959-63 (2014).
2. Sherwood AV, Grundy FJ, Henkin TM. Proc Natl Acad Sci USA 112:1113-18 (2015).
3. Sherwood AV, Frandsen JK, Grundy FJ, Henkin TM. Proc Natl Acad Sci USA 115:3894-99 (2018).

Keywords: Riboswitch, tRNA, S-turn, Pseudoknot, SHAPE

13. Proximal 3'UTR introns elicit EJC-dependent NMD during zebrafish embryonic development

Pooja Gangras (Department of Molecular Genetics), Thomas L. Gallagher (Department of Molecular Genetics), Kiel T. Tietz (Department of Molecular Genetics), Natalie C. Deans (Department of Molecular Genetics), Sharon L. Amacher (Department of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics)

Abstract:
Post-transcriptional control of gene expression is essential for proper development, and is achieved largely by RNA-binding proteins. One such protein complex, the Exon Junction Complex (EJC), is deposited 24 nts upstream of exon-exon junctions during pre-mRNA splicing. The EJC influences many aspects of post-transcriptional regulation, including Nonsense Mediated mRNA Decay (NMD). NMD is a surveillance system that degrades aberrant mRNAs and non-aberrant mRNAs containing ‘NMD-inducing features’ such as 3’UTR introns (3’UIs). Post-splicing, a 3’UI leads to an EJC bound downstream of the stop codon – if the distance between the two is ≥50 nts, the mRNA is targeted for NMD by the NMD-regulator Upf1. To study EJC function during development, we generated zebrafish mutants in EJC core protein genes rbm8a and magoh. Homozygous rbm8a and magoh mutants are paralyzed and have muscle and neural defects. As expected, RNA profiling reveals that annotated aberrant and natural NMD targets are significantly upregulated in EJC mutants. Surprisingly, some upregulated natural transcripts contain a conserved proximal 3’UI (<50 nts downstream of the stop codon). These ‘proximal 3’UI-containing NMD targets’ are similarly upregulated in Upf1-deficient and NMD inhibitor-treated embryos, suggesting that this subset of rbm8a- and magoh-regulated transcripts is regulated via NMD. The same trend is observed in Upf1-deficient mammalian cells. One proximal 3’UI-containing NMD target transcript encodes Foxo3b, which is known to play a role in autophagy, muscle atrophy and Wnt signaling. Consistent with our hypothesis that rbm8a and magoh mutant defects are due, at least in part, to increased Foxo3b activity, we show that heterozygous knockout of foxo3b in EJC mutants leads to partial phenotypic rescue. Our findings show that proximal 3'UTR introns are a new, atypical NMD-inducing feature that may be critical for regulating gene expression during embryogenesis.

Keywords: EJC-dependent NMD, zebrafish, development

14. Role of the conserved GTPase BipA in assembly of the 50S subunit of the ribosome

Michelle R. Gibbs (Department of Microbiology and Center for RNA Biology, The Ohio State University), Kyung-Mee Moon (Department of Biochemistry and Molecular Biology, University of British Columbia), Leonard J. Foster (Department of Biochemistry and Molecular Biology, University of British Columbia), Kurt Fredrick (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract:
BipA is a conserved translational GTPase that resembles elongation factor EF-G and 30S assembly factor LepA. Recent evidence from the Flower group suggests that BipA functions in 50S subunit assembly, but the precise role of the factor remains unclear. Here, we use stable isotope labeling of amino acids in culture and mass spectrometry (SILAC / MS) to examine the function of BipA in ribosome biogenesis. During growth at suboptimal temperature, loss of BipA leads to accumulation of immature large subunit particles (~40S) that lack several proteins. These include L2, L14, L16, L17, L19, and L32. Parallel analysis of the control (wild-type) strain shows accumulation of virtually identical intermediate particles, although at much lower levels. These data suggest that BipA acts in some way to destabilize this ~40S intermediate that accumulates in the cold. Loss of BipA causes no apparent defect in 30S subunit assembly. In fact, the proportion of 30S assembly intermediates decreases in the mutant strain, presumably due to an increase in free mature 30S subunits unable to enter the translation pool because they have no functional 50S partner. Notably, LepA and BipA bind similarly to the ribosome, and the GTP hydrolysis activity of each factor depends on the intact 70S ribosome. Based on these observations, we propose that, for each subunit, part of the assembly process occurs in the context of the 70S ribosome.

Keywords: ribosome assembly, GTPase, BipA

15. Using Molecular Dynamics to Characterize Mutants in the Connector Region of Rho

Max Gilliland, Nicholas Sunday, Marcos Sotomayor, Irina Artsimovich

Abstract:
Transcription termination factors are necessary to silence synthesis of aberrant RNAs. Bacterial Rho protein is an archetype of factor-dependent termination. In Escherichia coli, Rho inhibits expression of anti-sense, corrupted, and horizontally-acquired RNA messages. E. coli Rho is a hexamer made up of 419 amino acids per subunit. Rho is an ATP-dependent, RecA-family hexameric helicase composed of an N-terminal RNA-binding and a C-terminal ATPase/helicase domains separated by a flexible 30-residue long connector region. A key step in the Rho mechanism is a switch from an open, RNA-loading state into a closed, translocation-competent state in which the RNA is captured inside the inner pore; this switch is activated by Rho binding to canonical RNA sequences yet Rho also has to act on non-canonical sites. Our genetic data suggest that the flexible connector region may modulate the transition between Rho's inactive open and active closed states. We have identified substitutions that confer defects in Rho-dependent termination and are predicted to reduce connector flexibility (e.g., Gly→Asp). We hypothesize that the connector region is involved allosterically in binding to of divergent RNA sequences and is a potential target for factors that control Rho activity. Using molecular dynamic simulation software NAMD, we can develop a model of the connector region by mutating residues in silico. By modeling the closure of Rho mutants, we can assess the contributions of individual amino acid residues to Rho function. Quantitative analysis of root-mean-square deviation of atomic positions and inner pore diameter along with viewing the mechanism with VMD modeling software can give insights to how the selected substitutions affect ring closure. Additionally, elucidating the molecular details of Rho action will lead to understanding of other motor proteins that couple ATP hydrolysis to translocation on polymer substrates.

Keywords: Transcription, Termination Factor, Molecular Dynamics

16. Size, shape, and sequence-dependent of immunogenicity of RNA nanoparticles

Sijin Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; The Ohio State University, Columbus, OH 43210, USA), Hui Li (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; The Ohio State University, Columbus, OH 43210, USA), Mengshi Ma (Center for Research on Environmental Disease; College of Medicine, Department of Toxicology and Cancer Biology; University of Kentucky, Lexington, KY 40506, USA), Jian Fu (Center for Research on Environmental Disease; College of Medicine, Department of Toxicology and Cancer Biology; University of Kentucky, Lexington, KY 40506, USA), Yizhou Dong (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; The Ohio State University, Columbus, OH 43210, USA), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy; College of Medicine; Dorothy M. Davis Heart and Lung Research Institute; NCI Comprehensive Cancer; The Ohio State University)

Abstract:
RNA molecules have emerged as promising therapeutics. Like all other drugs, the safety profile and immune response are important criteria for drug evaluation. However, literatures on RNA immunogenicity have been controversial. Here, we used the approach of RNA nanotechnology to demonstrate the immune response of RNA nanoparticles is size, shape, and sequence-dependent. RNA triangle, square, pentagon and tetrahedron with same shape but different sizes, or same size but different shapes were used as models to investigate the immune response. The levels of pro-inflammatory cytokines induced by these RNA nanoarchitectures were assessed in macrophage-like cells and animals. It was found that RNA polygons without extension at the vertexes were immune-inert. However, when single-stranded RNA with a specific sequence was extended from the vertexes of RNA polygons, strong immune responses were detected. These immunostimulations are sequence-specific, since some other extended sequences induced little or no immune response. Additionally, larger size RNA square induced stronger cytokine secretion. 3D RNA tetrahedron showed stronger immunostimulation than planar triangular RNA. These results suggest that the immunogenicity of RNA nanoparticles is tunable to produce either a minimal immune response that can serve as safe therapeutic vectors, or a strong immune response for cancer immunotherapy or vaccine adjuvants.

References:
Guo S, Li H, Ma M, Fu J, Dong Y, Guo P. Size, Shape and Sequence-dependent Immunogenicity of RNA Nanoparticles. Mol Ther Nucleic Acids. 2017 Dec; 9:399-408.

Keywords: RNA nanotechnology, RNA nanoparticle, RNA nanostructure

17. Interrogating the conformation of HIV-1 Gag and its stoichiometry of binding to genomic RNAs

Samantha Hinckley Sarni (Ohio State Biochemistry Program), Erik Olson (Ohio State Biochemistry Program), Karin Musier-Forsyth (Ohio State Biochemistry Program), Vicki Wysocki (Ohio State Biochemistry Program)

Abstract:
Gag, the main structural protein in HIV-1 virions, can bind nucleic acids and form virus-like particles.^1-5 This promiscuous RNA-binding protein nonetheless selectively and efficiently packages the dimeric HIV-1 genome from amongst a vast excess of host RNAs. Gag recognizes and binds with high specificity to the Psi element within the 5’-untranslated region of genomic RNA.⁶ At least three motifs within Psi contribute to specific HIV-1 Gag interactions: two G-rich bulges within stem-loop 1 (SL1) and single-stranded G residues that flank SL3.⁷ It was determined via fluorescence anisotropy that as the concentration of ammonium acetate increased from 50 mM to 500 mM, the KD of GagΔp6 for non-Psi RNA (TARpolyA(A34U)) increased from 340 nM to 9 μM. In contrast, under identical conditions, the KD of GagΔp6 for PsiΔDIS (a monomeric form of the Psi element) increased from 8 to 39 nM. These data indicate that electrostatic interactions play a more dominant role in Gag:non-Psi interactions than in Gag-Psi interactions, consistent with previous studies using NaCl.^7,8 Native mass spectrometry analysis of GagΔp6 alone on a QE+EMR Orbitrap platform revealed that multiple molecular weight species were present near the theoretical molecular weights of GagΔp6 monomer and dimer. This was further investigated by ion mobility MS on a G1 QTOF platform. The ion mobility data from the G1 indicated that the full-length GagΔp6 occupies two distinct drift time distributions, consistent with the multiple conformations Gag has previously been shown to adopt. The protein-RNA complexes were analyzed on the QE+EMR. The GagΔp6-PsiΔDIS complex presented 2:1 and 3:1 GagΔp6:PsiΔDIS stoichiometry. In contrast, the Gag:TARpolyA(A34U) complex mixture showed an abundance of monomeric TARpolyA(A34U) and some 1:1 GagΔp6:TARpolyA(A34U) complex. These data are consistent with a model where Psi RNA supports a greater Gag binding stoichiometry than non-psi RNA, which could nucleate virion formation and ensure packaging of genomic RNA.

References:
1. Campbell et al., Journal of Virology 1999, 73 (3), 2270-2279.
2. Campbell et al., Proc Natl Acad Sci U S A 2001, 98 (19), 10875-9.
3. Campbell, et al. J Virol 1995, 69 (10), 6487-97.
4. Levin et al., J Virol 1974, 14 (1), 152-61.
5. Muriaux et al., Proc Natl Acad Sci U S A 2001, 98 (9), 5246-51.
6. Liu et al., J Mol Biol 2017, 429 (16), 2542-2555.
7. Rye-McCurdy et al., Viruses 2016, 8 (9).
8. Jones et al., Proc Natl Acad Sci U S A 2014, 111 (9), 3395-400.
9. Webb et al., RNA 2013, 19 (8), 1078-88.

Keywords: HIV-1, Native Mass Spectrometry, Viral Assembly

18. Elucidating the allosteric mechanism of ligand binding to TRAP using native MS

Melody Pepsi Holmquist (Department of Chemistry and Biochemistry, The Ohio State University), Elihu C. Ihms (Department of Chemistry and Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Cooperativity in ligand binding is crucial for regulation of biological pathways. However, it is challenging to quantify cooperativity due to the multiple identical binding sites and the symmetry of homo-oligomeric proteins. In addition, there is difficulty of measuring accurately and unambiguously the populations of protein-ligand species in order to determine microscopic equilibrium constants. Native MS can quantify homo-oligomeric protein species with different numbers of bound ligands, provided the populations can be directly obtained from the ion counts, and that MS-friendly native buffers do not alter the thermodynamics. By using native mass spectrometry, we measured homotropic binding of tryptophan (Trp) to Bacilllus stearothermophilus TRAP, a homo-oligomeric protein with 11 identical binding sites. To obtain the cooperative microscopic thermodynamics such as coupling free energies and binding affinities of Trp binding to TRAP, we used a nearest-neighbor thermodynamic model. The populations of TRAP species were determined by mass spectra obtained on a high-resolution Orbitrap mass spectrometer under native conditions to quantify the Trp-TRAP states populated at different Trp concentrations. The effect of MS buffers on the structure of TRAP was explored by ion mobility MS experiments, yielding experimental collision cross section (CCS) measurements for various TRAP species. SID-MS was implemented to study the fragments of TRAP with bound Trp. Native MS results were compared to those from solution ITC and NMR experiments in phosphate, AmAc and EDDA buffers. The ITC and NMR solution experiments showed that TRAP retains its native-like structure in MS solutions and that the Trp binding was not inhibited in MS solutions, validating our native MS titration experiments. Using native MS allows us to obtain microscopic thermodynamic constants such as K_d and free energies of the neighboring binding sites, helping us better understand the mechanism of homotropic allostery in ligand binding to TRAP.

Keywords: microscopic thermodynamics, Native MS, ion mobility

19. Distinct substrate specificities of human tRNA methyltransferases TRMT10A and TRMT10B

Nathan Howell (Ohio State University), Jane Jackman (Ohio State University)

Abstract:
Substantial biological resources are invested into producing functional tRNA molecules, including an extensive system of post-transcriptional nucleotide modifications. In this universal process, chemical functional groups are changed, rearranged, and added to individual nucleotides in a specific pattern for each target tRNA. One such modification is the addition of a methyl group to the N-1 atom of ninth-position purines (m1N9), which occurs in archaea and eukarya and is catalyzed by the Trm10 family of enzymes. We sought to explain the unusual presence of two predicted cytosolic Trm10 homologs (TRMT10A and TRMT10B) in humans and other metazoa. A yeast genetic system established in our lab suggested that TRMT10A and TRMT10B are not functionally redundant, leading to the use of in vitro approaches to investigate the substrate specificities of each enzyme. Using a combination of kinetic and binding assays, we demonstrate that human TRMT10A and TRMT10B exhibit distinct tRNA substrate specificities that are consistent with known modification patterns in human tRNA. Ongoing experiments suggest a new model for control of Trm10 substrate recognition based on interactions between Trm10 and other tRNA modification enzymes, which had not been previously observed for this enzyme family and has important implications for the behavior of Trm10 enzymes in diverse biological systems.

Keywords: tRNA modifications, enzymology, Trm10

21. Uncovering variations in conserved signal transduction pathways: the case of the EGFR pathway in Caenorhabditis vulval development

Leanne H. Kelley (Molecular Genetics, Ohio State University), Marcos Corchado (Molecular Genetics, Ohio State University), Abdulrahman M. Jama (Molecular Genetics, Ohio State University), Helen M. Chamberlin (Molecular Genetics, Ohio State University)

Abstract:
With conserved signal transduction pathways serving as the framework for evolution, it is the modifications to these conserved networks that give rise to morphological diversity across species (Mahalak et al. 2017). One such conserved signal transduction network is the EGFR pathway, which regulates vulval development in Caenorhabditis. C. elegans and C. briggsae share much of their morphology and development, despite diverging ~30 million years ago (Sharanya et al. 2015). While the phenotypic vulval cell patterning remains invariant between C. elegans and C. briggsae, genetic variation is evident when the EGFR pathway is perturbed. For example, Cel-sur-2/MED23 mutants are vulvaless, while Cbr-sur-2/MED23 mutants can exhibit normal vulval development (Mahalak et al. 2017). This suggests a compensatory process in C. briggsae that can promote vulval cell patterning when the EGFR pathway is blocked. To identify this compensatory mechanism, we aim to investigate differences in vulval development between C. elegans and C. briggsae by creating mirror mutations based on known gain- and loss-of-function mutations in the EGFR pathway. To date, differences in phenotype between the species have been identified in two genes, spr-4 and htz-1, with C. briggsae mutants exhibiting vulval cell hyperproliferation, while C. elegans mutants show normal vulval cell differentiation (Sharanya et al. 2015). Rescue experiments suggest that Cbr-htz-1 functions cell non-autonomously to regulate vulval cell fate, suggesting that these genes may increase lin-3/EGF transcription. RNA-seq data shows that lin-3/EGF is upregulated in Cbr-htz-1 mutants, but is downregulated in Cel-htz-1 mutants. htz-1’s influence on lin-3/EFG regulation and its role in transcription suggest a role as a synthetic multivulvae (SynMuv) gene, a group of genes that negatively regulate vulval cell patterning in a redundant manner. Uncovering genetic variations within the EGFR pathway between C. elegans and C. briggsae will ultimately broaden our understanding of flexibility in signal transduction pathways over the course of evolution.

References:
Mahalak, K.K., Jama, A.M., Billups, S.J., Dawes, A.T., and Chamberlin, H.M. 2017. Differing roles for sur-2/MED23 in C. elegans and C. briggsae vulval development. Development Genes and Evolution 227, 213-218.

Sharanya, D., Fillis, C.J., Kim, J., Zitnik E.M. Jr,. Ward, K.A., Gallagher, M.E., Chamberlin, H.M., and Gutpa, B.P. 2015. Mutations in Caenorhabditis briggsae identify new genes important for limiting the response to EGF signaling during vulval development. Evolution and Development 17, 34-48.

Keywords: Caenorhabditis, signal transduction pathway, evolution

22. ASO screen uncovers splicing as a therapeutic vulnerability in the insulin-like growth factor (IGF) signaling pathway

SAFIYA KHURSHID (Nationwide Childrens Hospital, Columbus Ohio), Matias Montes (Nationwide Childrens Hospital, Columbus Ohio), Andy Goodwin (Nationwide Childrens Hospital,Columbus Ohio), 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 (Nationwide Childrens Hospital; Department of Pediatrics, The Ohio State University, Columbus, Ohio, 43210)

Abstract:
The insulin receptor (INR) undergoes alternative splicing to produce 2 isoforms: the full-length INRB and exon 11 skipped INRA isoform. The expression of these isoforms is tightly regulated during development, however there is an aberrant increase in INRA expression in cancer. INRA encodes for a receptor which has high affinity for both insulin & IGF2 growth hormones and it exploits the IGF pathway to accelerate the onset of tumors. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and there is inadequacy of less toxic therapies.
We found a significant increase in the expression of INRA levels in a cohort of 40 RMS patients as well as multiple RMS cell lines as compared to the controls. We went on to show that cellular stress such as hypoxia increases alternative splicing to produce more INRA. To get a mechanistic insight into this phenomenon, we utilized this hypoxia-inducible splicing system & performed an antisense-oligonucleotide (ASO) screen to characterize sequence elements & splicing factors involved in the regulation of INR splicing. We found that sequence elements flanking exon 11 are critical to the increased alternative splicing we see under hypoxia. We performed a refined ASO walk to target the regions important for exon inclusion or exclusion and identified a region known to be a binding site for the splicing factor CUGBP1. RMS derived cell lines exclusively express INRA but when treated with our lead ASO compound, they show a decrease in the INRA expression. ASO treated cells exhibit a significant reduction in cell proliferation, migration & angiogenesis.
Our data shows promising insight into how we can impede the IGF2 pathway by causing the splicing shift & reducing INRA expression to consequently mitigate tumor hallmarks like cell-proliferation, migration & angiogenesis. The goal is to use these ASO compounds as therapeutic interventions in conjunction with already established anti IGF1 receptor therapies to treat RMS.

Keywords: Alternative splicing, Insulin receptor, Pediatric cancers

23. Probing 5′UTR Conformational Dynamics by FRET

Jonathan P Kitzrow (Chemistry and Biochemistry at The Ohio State University), Benjamin Brigham (Molecular Biology & Microbiology at Tufts University), James Munro (Molecular Biology & Microbiology at Tufts University), Karin Musier-Forsyth (Chemistry and Biochemistry at The Ohio State University)

Abstract:
The full-length HIV-1 transcript can act as either mRNA, encoding the Gag and Gag-Pol polyproteins, or as genomic RNA (gRNA) that is selected by Gag during virion assembly. The HIV-1 5′UTR, which is part of all full-length HIV-1 transcripts, is known to regulate many important processes during the viral lifecycle, including RNA splicing, translation, dimerization, gRNA packaging, tRNA primer annealing and initiation of reverse transcription. The HIV-1 5′UTR is believed to exist on a structural tipping point between two predominant conformations, one in which the dimer initiation sequence (DIS) is exposed and available to dimerize and a second conformation in which the DIS is sequestered via interactions with upstream sequences. We have designed a FRET-based assay to investigate the gRNA 5′UTR conformation and dynamics using ensemble and single-molecule approaches. Our studies support the conclusion that the 5′UTR predominantly adopts two stable conformational states. Individual FRET traces showed that 29% of the molecules transitioned between states during the ~150 sec observation window. Inter-molecular FRET observed between an immobilized donor-labeled 5′UTR construct and excess acceptor-labeled 5′UTR was consistent with formation of an extended dimer. Interestingly, heat-annealing tRNALys3 to the primer binding site of an immobilized 5′UTR both in the absence and presence of excess free 5′UTR promoted the DIS sequestered monomeric conformation. In contrast, the presence of nucleocapsid protein (NC) in the absence of tRNA stabilizes high-FRET dimeric states. Overall, these data are consistent with high intrinsic conformational dynamics in the 5′UTR that can be regulated by tRNA primer annealing and NC.

Keywords: HIV-1 5UTR, RNA, FRET

24. Quality control by trans-editing factor prevents global mistranslation of non-protein amino acids

Alexandra B. Kuzmishin (OSBP, Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Jo Marie Bacusmo, William A. Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Yuki Goto, Hiroaki Suga (Department of Chemistry, Graduate School of Science, The University of Tokyo), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)

Abstract:
Accuracy in protein synthesis is maintained through multiple pathways, with a critical checkpoint occurring at the tRNA aminoacylation step catalyzed by aminoacyl-tRNA synthetases (AARSs). In addition to Ala-tRNA^Pro editing mediated by the insertion (INS) domain present in most bacterial prolyl-tRNA synthetases (ProRS), single-domain trans-editing factors structurally homologous to INS are present in some organisms. To date, INS-like editing proteins have been shown to act on specific tRNAs mischarged with genetically encoded amino acids. However, structurally related non-protein amino acids are ubiquitous in cells and threaten the proteome. Here, we show that Rhodopseudomonas palustris ProXp-x, a previously uncharacterized INS homolog, edits a known ProRS aminoacylation error, Ala-tRNA^Pro, but displays even more robust editing of tRNAs misaminoacylated with the non-protein amino acid α-aminobutyrate (Abu) in vitro and in vivo. Additionally, ProXp-x robustly edits D-Ala-tRNA^Pro and D-Thr-tRNA^Val, but shows no activity towards L-Thr in vitro. Abu is the product of the transamination of oxobutyrate and is a precursor metabolite in Thr biosynthesis, but D-amino acids are synthesized from their L-isomers or are acquired from the environment or surrounding organisms. Abu is mischarged by E. coli ValRS and ProRS in vitro, and in vivo experiments showed that Abu is toxic to an E. coli strain encoding an editing-defective ValRS (1). However, expression of R. palustris ProXp-x rescues cell growth, presumably by removing Abu that has been mischarged onto tRNA^Val by the editing-deficient ValRS. Our results indicate that editing by ProXp-x may offer advantages to cells, especially under environmental conditions where concentrations of non-protein amino acids may challenge the substrate specificity of AARSs. Cell-based experiments are underway to explore phenotypic differences between wild-type R. palustris and a ProXp-x null R. palustris strain under a variety of growth conditions.

References:
1. Döring V, Mootz HD, Nangle LA, Hendrickson TL, de Crécy-Lagard V, Schimmel P, Marlière P. Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway. Science. 2001 April 20; 292:501-4.

Keywords: trans-editing, ProXp-x, INS

25. A structured antisense RNA motif can filter out RNA:RNA hybridization mismatches

Samuel D. Stimple, Hopen Yang (Dept. of Chemical and Biomolecular Engineering), Ashwin Lahiry (Dept. of Microbiology), David Wood (Dept. of Chemical and Biomolecular Engineering; Dept. of Microbiology), Richard A. Lease (Dept. of Chemical and Biomolecular Engineering; Dept. of Chemistry and Biochemistry; The Ohio State University, Columbus OH 43210)

Abstract:
Programmed RNA self-assembly can be used to target regulation of gene expression for metabolic engineering or medical applications. When designing an RNA to be implemented in a cell or organism, it will be increasingly important to avoid mismatched RNA base pairing to prevent off-target interactions and undesired side effects. Bacteria produce antisense regulatory small RNAs (sRNAs) that act by forming specific sRNA:mRNA base pairing interactions to post-transcriptionally regulate mRNA fates. Here we describe fingerloops, structured synthetic antisense motifs that mimic native regulatory RNA structures and strongly reduce off-target RNA pairing interactions. Fingerloop antisense sequences are encoded in a hairpin stem-loop within one strand of the RNA helix and into the single-stranded loop. We demonstrate that synthetic fingerloop helices derived from E. coli DsrA sRNA antisense structural motifs encode a target-discrimination function in vivo that is superior to single-stranded antisense regions commonly found in sRNAs. These antisense motifs can be retargeted to bind non-native target mRNA leader sequences. The optimized fingerloop appears to sharply decrease pairing between the sRNA and a mismatched target mRNA with single-base discrimination, as assessed by changes in target reporter gene translation. We hypothesize that RNA loop sequences constrain a helix-nucleation step to filter out mismatches in sRNA:mRNA base pairing. The fingerloop structures may enhance the specificity of E. coli DsrA for two of its mRNA targets (hns, rpoS) that are global transcriptional stress-response regulators. Fingerloops may also represent the simplest and smallest RNA nanotechnological devices, and constitute a new RNA structural motif.

References:
Lahiry, A., Stimple, S.D., Wood, D.W., and Lease, R.A. (2017) Retargeting a dual-acting sRNA for multiple mRNA transcript regulation. ACS Synth. Biol. 6:648-658. DOI: 10.1021/acssynbio.6b00261

Stimple, S.D., Lahiry, A., Taris, J.E., Wood, D.W. and Lease, R.A. (2018) A Modular Genetic System for High-Throughput Profiling and Engineering of Multi-Target Small RNAs In Arluison, V. and Valverde, C. (Eds.), Bacterial Regulatory RNA: Methods and Protocols. Meth. Mol. Biol. 1737:373-391 (ISBN 978-1-4939-7633-1; DOI: 10.1007/978-1-4939-7634-8_21).

Keywords: RNA structure, antisense RNA regulation, RNA-RNA interactions

26. Global Local Folding of the Human Transcriptome

James Li (The Institute for Genomic Medicine at Nationwide Childrens Hospital and The Ohio State University Department of Pediatrics), Jeffrey Gaither, Grant Lammi, David Gordon, Harkness Kuck, Benjamin Kelly, James Fitch, Peter White (The Institute for Genomic Medicine at Nationwide Childrens Hospital and The Ohio State University Department of Pediatrics)

Abstract:
Analyzing sequence variants for disease has largely relied upon the predicted effects missense mutations have on protein function. Previous in silico RNA folding studies suggest that selection in humans and mammals may have been influenced by mRNA secondary structure in certain genes. However, the connection between RNA folding and genetic diseases has yet to be established at the level of an entire transcriptome. Therefore, we performed whole transcriptome analysis to ascertain the effects of single nucleotide polymorphisms (SNPs) on local RNA folding. We aimed to (1) build a cloud-based big data pipeline to procure RNA folding statistics for every possible SNP in the known human transcriptome (~0.5 billion variants), (2) utilize population allele frequencies from 138,632 patients and mammalian conservation scores to determine if there was constraint on SNPs causing large RNA disruptions, thereby supporting our hypothesis that RNA stability/structure may play a role in disease and (3) develop a tool and composite score to analyze patient genomes for highly disruptive SNPs. For every position in all known RefSeq mRNA transcript sequences, we generated 101 nucleotide flanking sequences corresponding to the reference allele and the three possible alternate alleles. Next, we used the ViennaRNA Package to obtain 10 RNA folding disruption metrics for each possible variant (445,740,246 total SNPs). For each of the 10 RNA folding metrics we sorted the SNPs and then divided them into ten equally sized bins. Metric bins with higher RNA disruption values had a lower proportion of SNPs with non-zero allele frequencies compared to bins with lower RNA disruption values. Similarly, median and mean GERP++ scores were greater for higher disruption bins. The correlation of increased RNA disruption values with constrained allele frequencies and GERP++ scores at the level of the whole human transcriptome, suggests that RNA folding plays an important role in human health and disease.

Keywords: sequence variant analysis, vienna, population constraint

27. The host protein SAMHD1 interacts with the HIV-1 promoter to suppress viral transcription

Tai-Wei Li (Molecular, Cellular and Developmental Biology Graduate Program,Ohio State University), Jenna M. Antonucci (Department of Microbiology, Ohio State University), Corine St. Gelais (Center for Retrovirus Research,Ohio State University), Li Wu (Center for RNA Biology, The Ohio State University,)

Abstract:
SAM domain and HD domain-containing protein 1 (SAMHD1) is a novel intracellular deoxynucleotide triphosphohydrolase (dNTPase). Through its dNTPase function, SAMHD1 maintains low dNTP levels in non-dividing immune cells to restrict reverse transcription of human immunodeficiency virus type 1 (HIV-1). However, in activated CD4+ T-cells, where SAMHD1 dNTPase is less active during cell proliferation, HIV-1 can establish productive infection and integrate its proviral genome into the chromosome. Transcriptionally silent HIV-1 provirus is maintained in CD4+ T-cells, which differentiate into memory T-cells and serve as the major latent reservoir of HIV-1. We have shown that SAMHD1 suppresses NF-κB signaling during HIV-1 infection. NF-κB signaling is important to reactivate the latent HIV-1 provirus. During reactivation, the HIV-1 long term repeat (LTR) promoter binds host transcription factors such as NF-κB to initiate transcription of viral mRNA for viral protein synthesis. We hypothesize that SAMHD1 suppresses HIV-1 LTR-driven gene transcription by disrupting binding of NF-κB (p100/p52/p50) to the LTR. SAMHD1 may also suppress the translocation of additional transcription factors from the cytoplasm or directly disrupt the transcription initiation of RNA polymerase II complex in the nucleus. To investigate how SAMHD1 suppresses the HIV-1 LTR function, we will analyze the activity of LTR-driven luciferase reporter constructs in 293T cells with co-expression of nuclear-localized wild-type (WT) SAMHD1 or cytoplasmic mutant SAMHD1 with deletion of nuclear localization signal (ΔNLS). To further delineate the mechanism in CD4+ T cells, WT or ΔNLS SAMHD1 will be expressed in an HIV-1 latently infected J-Lat cell line for reactivation assays. J-Lat cells contain the HIV-1 provirus harboring a green fluorescent protein (GFP) gene that is expressed after treatment with latency reversing agents. We will analyze HIV-1 reactivation by measuring GFP and HIV-1 gag mRNA levels. Through this study, we will better understand SAMHD1’s contribution to maintenance of viral latency in infected cells.

Keywords: SAMHD1, HIV-1, viral transcription

28. Insights into the Homotropic Allostery of the Oligomeric Trp- and RNA-binding protein TRAP via Mechanistic Modelling and Protein Engineering

Weicheng Li, Elihu C. Ihms, Ian R. Kleckner, Melody L. Holmquist (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY14260), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210)

Abstract:
Allostery, the process of conveying the effect of binding at one site to another functional site in macromolecules, regulates virtually every biological process, such as transport, transcription, and enzyme activity. However, the limited understanding about how allostery is realized at a microscopic level—the thermodynamic consequences of the allosteric propagations—prevents the understanding of the macroscopic currents. TRAP (trp RNA-binding Attenuation Protein) is a homo-undecamer whose RNA binding activity is regulated by binding of L-tryptophan (Trp) to its eleven identical binding sites. The Trp binding events are thought to stabilize the RNA-binding state of TRAP and favor RNA binding. Trp binding events show homotropic allostery and cooperativity clearly informed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments. By fitting temperature-dependent ITC data using a nearest-neighbor (NN) statistical thermodynamic model, Trp binding to Bst TRAP A28I, we quantify the microscopic contributions of Trp binding to the affinity at neighboring sites. The NN model provides the microscopic descriptions of how Trp-binding events are energetically affected by the occupancy of neighboring sites. The microscopic descriptions can be extrapolated to predict macroscopic binding behavior. However, the parameters of the NN model are all obtained by global fitting. To directly determine the parameters experimentally, we are engineering the protein to eliminate the effect of nearest neighbor interactions, which would allow the direct determination of intrinsic binding affinity of Trp to TRAP protein, optimizing the NN model. It is expected to accurately describe the energetic interactions of homotropic allostery in the RNA-binding protein TRAP and it may give the hint of how does Trp stabilize the RNA-binding.

Keywords: Allostery, statistical thermodynamic model, Protein Engineering

29. Nanoparticle orientation to control RNA loading and ligand display on exosome for cancer therapy

Zhefeng Li, Fengmei Pi, Daniel Binzel, Hui Li, Farzin Haque, Shaoying Wang (College of Pharmacy; Center for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH 43210, USA. ), Tae Jin Lee, Carlo M. Croce (Comprehensive Cancer Center, Department of Cancer Biology and Genetics, College of Medicine; The Ohio State University, Columbus, OH 43210, USA. ), Meiyan Sun, Bin Guo (Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy; University of Houston, Houston, TX 77030, USA.), Piotr Rychahou, B. Mark Evers (Markey Cancer Center; Department of Surgery; University of Kentucky, Lexington, KY 40536, USA. ), Peixuan Guo (College of Pharmacy; Center for RNA Nanobiotechnology and Nanomedicine; Comprehensive Cancer Center, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210)

Abstract:
Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology to engineer exosome, an extracellular vesicle, for cancer specific delivery of small interfering RNA (siRNA) in vivo. The orientation of arrow-shaped RNA was altered to control ligand display on exosome membranes for specific cell targeting, or cargo loading into exosome. Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the exosome. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the exosomes. RNA interference (RNAi) has powerful therapeutic potential for gene therapy, however, delivery is the major barrier for the clinical translation of RNAi. Exosome hold high efficiency in cell fusion and endosome escape, but the lack of specific cell targeting has led to side effect. Taking the advantage of RNA nanotechnology and exosome, this study provides a novel in vivo delivery platform for cancer therapy. Survivin siRNA was used as therapeutic cargo to verify the delivery efficacy on prostate cancer, breast cancer and patient derived colorectal cancer xenograft model. Taking advantage of the RNA ligand for specific targeting and exosome for efficient membrane fusion and endosome escape, the resulting ligand-displaying exosomes were capable of specific delivery of siRNA to cells, and efficiently blocked tumor growth in breast cancer, brain cancer, and prostate cancer models. Exosomes displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same exosomes instead displaying epidermal growth-factor receptor aptamer inhibited orthotropic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

References:
Pi F, Binzel D, Lee TJ, Li Z, Sun M, Rychahou P, Li H, Haque F, Wang S, Croce CM, Guo B, Evers BM,Guo P. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression. Nature Nanotechnology. 2018 Jan; 13(1):82-89.

Keywords: RNA nanotechnology, Exosome, siRNA delivery

30. Melamine functionalized lipid particles for selective siRNA loading and delivery

Yufeng Liang (Department of Chemistry and Biochemistry), Zhun Zhou (Department of Chemistry and Biochemistry), Sarah Rundell (Department of Chemistry and Biochemistry)

Abstract:
We’ve developed a bifacial polymer nucleic acid (bP_oNA) binding pattern through uridine-melamine-uridine base tripling recognition. bP_oNA has been demonstrated to deliver uridine-rich siRNA into HELA and HepG2 cells through receptor-mediated endocytosis. 40% of luciferase in HELA cells is knocked down by 100 nM of siRNA with bP_oNA and the IC₅₀ is 5 nM for the siRNA-bP_oNA complex which targets the ApoB gene in HepG2 cells. Since melamine-uridine binding pattern shows great potential in siRNA delivery, herein we propose another siRNA delivery approach—melamine functionalized lipid particle for uridine-rich siRNA delivery. Compared to stable nucleic acid lipid particle (SNALP), which contains considerable cationic lipid for electrostatic interaction towards RNA phosphate backbone, the melamine functionalized lipid particle might selectively bind to unpaired uridines in siRNA sequence. We’ve synthesized melamine functionalized phosphatidylethanolamine (PE) lipids and tested their encapsulation efficiency towards uridine-rich RNA by measuring the concentration of the remaining RNA in the supernatant after a lipid pulldown assay. The preliminary data shows that melamine modified lipids selectively encapsulate about 30% of uridine modified RNAs from the surrounding buffers, while no unmodified RNA is bound. To further investigate the binding property of melamine lipid, we propose to test lipid particle assembly under various pHs and with rational lipid compositions. If the melamine lipids show significant encapsulation of uridine-rich RNA, the lipid can be further modified with ligands for cell surface receptor. The neutral or zwitterionic lipid particles without intense positive charges can be specifically targeted to cells through ligand-receptor recognition. Therefore, the target gene in a specific cell might be silenced by this melamine lipid approach.

References:
Zhou, Z., Xia, X., and Bong, D. (2015) Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function. J. Am. Chem. Soc. 137, 8920–8923.

Keywords: siRNA delivery, lipid particle, uridine-melamine-uridine base tripling recognition

31. Heart-centric regulation of cellular and systemic metabolism by the m6A methyltransferase METTL3

Jacob Z. Longenecker (Department of Physiology and Cell Biology, College of Medicine, The Ohio State University; Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University), Jennifer M. Petrosino, Lisa E. Dorn, Federica Accornero

Abstract:
A critical and well-known characteristic of the heart is its ability to alter its metabolic activity in response to a variety of stressors, including variation of caloric availability and physical activity. Perturbation of this characteristic has been shown to have profound effects on cardiac function and importantly, systemic energy homeostasis. While most studies have focused on gene transcription, our laboratory has focused on post-transcriptional gene regulation and the study of N⁶-methyladenosine (m⁶A), the most abundant mRNA modification, in the heart. We have generated mouse models with heart-specific deletion or overexpression of methyltransferase-like 3 (METTL3), the enzyme responsible for the addition of m⁶A to mRNAs. We have found that in these mice, METTL3 deletion results in a significant decrease of cardiometabolic fitness at 8 months of age, as measured using exercise testing and represented as VO₂ max. Strikingly, at 1 year of age mice lacking METTL3 in the heart become obese. A significant difference in total body weight, fat mass %, and lean mass % is not seen until after these mice have shown a cardiometabolic defect, indicating that a metabolic change in the heart precedes a systemic effect on these metrics. Our results suggest that this systemic change in metabolism is controlled via post-transcriptional modulation of heart-specific mRNAs, and provide a novel mechanism of heart-dependent regulation of obesity.

Keywords: Heart, METTL3, Metabolism

32. How LPS structure affects its transport by LptB2FG

Emily Lundstedt (Department of Microbiology), Rebecca Davis (Department of Microbiology), Natividad Ruiz (Department of Microbiology)

Abstract:
The cell envelope is what defines a bacterium from its environment and protects it from the outside world. Gram-negative bacteria are diderms, and thus have a cell envelope composed of both an inner membrane and an outer membrane. The outermost leaflet of the outer membrane is made up of lipopolysaccharide (LPS). Tight packing of LPS molecules makes the outer membrane a potent permeability barrier, protecting the cell from entry of harmful compounds such as antibiotics. LPS is synthesized at the inner membrane and is transported to the outer leaflet of the outer membrane by the LPS transport (Lpt) machinery. The movement of LPS through this machinery is powered by the ATP-binding cassette (ABC) transporter LptB₂FG. Although it is clear that ATP hydrolysis is required for LPS transport, it is not known how the ATP-hydrolysis cycle is coupled to the extraction of LPS. To elucidate this mechanism, we performed structure-function analysis of mutants that alter residues along the interaction interface between the ATPase subunits LptB₂ and transmembrane subunits LptFG. We identified a new essential residue within LptB that is critical for connecting ATP hydrolysis and LPS transport. The lethality of a mutation that alters this residue in LptB could be suppressed by deletion of an LPS biosynthesis gene, lpxM. Since the loss of LpxM results in an altered LPS structure, these findings suggest that this residue in LptB is responsible for conferring conformational changes associated with ATP hydrolysis to drive transport of LPS by lptFG.

Keywords: Antibiotic resistance, Cell envelope, Lipopolysaccharide

33. Selectivity and editing mechanisms of an aminoacyl-tRNA trans-editing factor

Xiao Ma (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Eric M. Danhart, Marina Bakhtina, William A. Cantara, (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Alexandra B. Kuzmishin, Brianne L. Sanford (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Marija Kosutic, Ronald Micura (Institute of Organic Chemistry, Center for Molecular Biosciences, Leopold Franzens University), Karin Musier-Forsyth, Kotaro Nakanishi, Mark P. Foster (Department of Chemistry, Graduate School of Science, The University of Tokyo)

Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for charging amino acids onto their cognate tRNAs. The structural features of the aaRSs catalytic domains provide a high degree of selectivity for attachment of the correct amino acid; however, given the similar stereochemical properties of many of the amino acids, errors in tRNA charging can occur, especially for the smaller and isometric amino acids. Many organisms possess editing enzymes that function in trans to hydrolyze mischarged tRNAs to maintain high fidelity translation. ProXp-ala, a structural homolog of the editing domain from ProRS, can deacylate the mischarged tRNA, Ala-tRNA^Pro. Foster and Musier-Forsyth groups collaborated and have provided evidence that ProXp-ala recognizes features on the acceptor stem of tRNA^Pro, while three overlapping mechanisms contribute to discrimination between Ala-tRNA^Pro and Pro-tRNA^Pro: conformational selection, size exclusion and chemical selection. To validate these hypotheses we plan to use X-ray crystallography, NMR spectroscopy and enzymatic assays to: (1) Determine the high-resolution three-dimensional structure of ProXp-ala in complex with tRNA^Pro to detail recognition between ProXp-ala and the acceptor stem of tRNA^Pro. (2) Determine the structure of ProXp-ala bound to a non-hydrolyzable Ala-tRNA^Pro analog in order to reveal the mechanism of Ala/Pro discrimination. (3) We will perform deacylation experiments using native and model substrates to test the catalytic role played by the 2'OH of the terminal 76A in tRNA^Pro. The high-resolution structures and deacylation assays will facilitate our understanding of both substrate recognition and editing mechanism of ProXp-ala.

References:
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. Danhart, E. M., Bakhtina, M., Cantara, W. A., Kuzmishin, A. B., Ma, X., Sanford, B. L., Košutić, M., Goto, Y., Suga, H., Nakanishi, K., et al. (2017) Conformational and chemical selection by a trans - acting editing domain. Proc. Natl. Acad. Sci. 114, 6774-6783.

Keywords: trans-editing, NMR, RNA-protein interaction

34. Regulation of translational quality control in Salmonella Typhimurium

Rebecca Mann (Ohio State Biochemistry Program ), Michael Ibba (Department of Microbiology)

Abstract:
Translational quality control ensures accurate protein synthesis which is an essential cellular process. Aminoacyl tRNA synthetases are critical enzymes that function during translation to attach a corresponding tRNA to the appropriate amino acid. Some of these enzymes, including phenylalanine tRNA synthetase (PheRS), have editing mechanisms to prevent misaminoacylation of non-cognate amino acids to tRNA(1). Oxidative stress has been shown to diminish translational quality control by oxidizing a critical cysteine in the editing domain of threonyl-tRNA synthetase, thus inhibiting the hydrolysis of Ser-tRNA^Thr(2). Treating cells with hydrogen peroxide has been shown to alter the amino acid pools by oxidizing phenylalanine into tyrosine and its isomers, increasing the pool of non-cognate substrates and therefore increasing the possibility of misacylation of tRNA(3). Due to the increased non-cognate substrate availability, PheRS must maintain accuracy during periods of oxidative stress; however, how PheRS changes structurally and enzymatically during oxidative stress has yet to be elucidated. Upon treatment of PheRS with hydrogen peroxide, structural analysis revealed differential oxidation patterns between protein subunits, paired with a change in the overall secondary structure. Interestingly, this treatment did not impact the cognate amino acid aminoacylation activity, but did lead to increased proofreading activity. These results suggest a possible evolutionary relationship between the effect of oxidative stress on the amino acid pool and the enzyme. Further examination of the structural changes of PheRS will provide new insight into the mechanism of PheRS proofreading and its independence from the active site domain.

References:
1. Ling J, Reynolds N, Ibba M. Aminoacyl-tRNA synthesis and translational quality control. Annu Rev Microbiol. 2009;63:61-78. doi: 10.1146/annurev.micro.091208.073210. PubMed PMID: 19379069.
2. Ling J, Soll D. Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site. Proc Natl Acad Sci U S A. 2010;107(9):4028-33. doi: 10.1073/pnas.1000315107. PubMed PMID: 20160114; PMCID: PMC2840151.
3. Bullwinkle TJ, Reynolds NM, Raina M, Moghal A, Matsa E, Rajkovic A, Kayadibi H, Fazlollahi F, Ryan C, Howitz N, Faull KF, Lazazzera BA, Ibba M. Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code. Elife. 2014;3. doi: 10.7554/eLife.02501. PubMed PMID: 24891238; PMCID: PMC4066437.

Keywords: tRNA, translation, Salmonella

35. Role of EPRS and BEX1 in the post-transcriptional control of pro-inflammatory genes in the heart

Colton R. Martens (Department of Physiology and Cell Biology, College of Medicine, The Ohio State University; Molecular, Cellular, and Developmental Biology, The Ohio State University), Lisa E. Dorn, Federica Accornero

Abstract:
Given the well-established role of chronic inflammation in the onset of many diseases, it is essential that regulators of inflammatory genes are characterized. Aside from its role in charging tRNAs, Glutamyl-prolyl-tRNA synthetase (EPRS) has non-canonical functions in regulating select inflammatory mRNAs. Upon stimulation by inflammatory signals, EPRS associates with the GAIT complex and inhibits the translation of inflammatory genes. A 12-16 hour lag time between the inflammatory signal and the GAIT-mediated attenuation of the inflammatory response suggests that this is a mechanism to prevent maladaptive prolonged inflammation. Our lab has identified BEX1 as a novel binding partner of EPRS in the heart. BEX1 is increased in failing hearts, where it stabilizes pro-inflammatory mRNAs. Consistent with excessive inflammation as a cause of heart failure, mice overexpressing BEX1 are more susceptible to chronic stress-induced heart failure while mice lacking BEX1 are protected. We are currently investigating the significance of the EPRS/BEX1 interaction in the regulation of inflammation. Additionally, we are studying the role of BEX1 in regulating pathogen-induced inflammatory responses. Specifically, we are looking at BEX1-mediated immune response in hearts infected with a cardiotropic virus. Preliminary results suggest that BEX1 serves a cardioprotective role in the context of infection. The maladaptive effect of BEX1 in response to chronic stress and the apparent adaptive role of BEX1 in response to a transient immune stress fit the paradigm in which acute inflammatory responses are essential for maintenance of homeostasis while chronic inflammation is maladaptive.

Keywords: Inflammation, EPRS, BEX1

36. Using GRO-Seq as a tool to understand transcriptional regulation in maize

Allison McClish (Department of Molecular Genetics), Jay Hollick (Department of Molecular Genetics; Centers for RNA Biology and Applied Plant Sciences)

Abstract:
Global Run-On Sequencing (GRO-Seq) reads identify nascent transcription occurring across the entire genome¹. In maize, GRO-Seq has been used to define regions where transcription rates are dependent on RNA polymerase IV (Pol IV) function². In Arabidopsis, Pol IV RNAs are processed into 24 nucleotide (24nt) sizes that facilitate RNA-directed DNA methylation (RdDM)³. It remains unclear in either maize or Arabidopsis whether Pol IV itself or Pol IV-dependent cytosine methylation affects Pol II function⁴. Comparisons of GRO-Seq profiles from dicer-like3 mutants having virtually no 24nt RNAs and rpd1 mutants having neither Pol IVa nor Pol IVb function should distinguish these two potential regulatory mechanisms. Current efforts to characterize and optimize the nuclei isolations and in vitro transcription reactions needed to create GRO-Seq libraries will be presented. Using hybridization of radiolabeled nascent RNAs with single-gene riboprobes, the effects of Sarkosyl (reported transcription initiation inhibitor⁵) are being evaluated and optimal elongation times are being determined. Nuclei isolated from various tissue types and developmental stages are also being evaluated for in vitro transcriptional competency and thus suitability for GRO-Seq library production. GRO-Seq profiles generated from nuclei treated with alpha-amanitin (Pol II-specific inhibitor) will potentially identify nascent Pol IV and/or Pol V transcripts. GRO-Seq profiles of specific mutants will also be compared to distinguish Pol II transcription specifically affected by Pol IVa or Pol IVb^{6, 7}. Ultra-deep coverage of GRO-Seq reads will assist future genome annotations, including gene model validations, enhancer calls, defining 3’ pretermination transcription and other intergenic regions of non-coding transcription.

References:
¹ Core et al. 2008 Science
² Erhard et al. 2015 Genetics
³ Matzke and Mosher. 2014 Nature Reviews Genetics
⁴ Hale et al. 2009 PLoS Genetics
⁵ Gariglio et al. 1974 FEBS Letters
⁶ Stonaker et al. 2009 PLoS Genetics
⁷ Sidorenko et al. 2009 PLoS Genetics

Keywords: transcriptional regulation, RNA polymerase IV, global run-on sequencing

37. Identifying motor proteins that function in male germ unit movement in Arabidopsis pollen tubes

Tyler Mendes (Department of Molecular Genetics, The Ohio State University, Columbus, Ohio), Iris Meier (Department of Molecular Genetics, The Ohio State University, Columbus, Ohio)

Abstract:
When plant fertilization occurs, pollen is deposited on the stigma of a flower and subsequently germinates, forming a pollen tube that grows towards the ovules. The nucleus of this cell, termed the vegetative nucleus (VN), is transported down the pollen tube as it grows, along with two male generative cells (GCs). Together, the two GCs and the VN are termed the male germ unit (MGU). In wild type Arabidopsis pollen, the VN travels in front of the GCs down the growing pollen tube¹. The exact mechanism of the movement of the MGU is not understood, but previous research has shown that LINC complexes play a role². The LINC complex spans the nuclear membrane and connects the nucleus to the cytoskeleton, either directly or via motor proteins². Null mutants in the genes that encode two component proteins of this complex, WIP and WIT, result in a VN movement defect that manifests as nuclear order switching, wherein the GCs lead while the VN trails behind¹. This, in turn, leads to a male fertility defect. It is likely that WIT acts here as an adapter between the nuclear membrane and cytoskeletal motor proteins, as it has a similar function in root hairs³. However, the motor protein(s) that act in VN movement in Arabidopsis have not been identified. In plants, there are two kinds of cytoskeletal motor proteins: kinesins (microtubule-associated) and myosins (actin-associated). To determine which motors are involved in VN movement, we have bioinformatically identified 17 pollen-expressed kinesins (PEK1-17) and 6 pollen-expressed myosins. T-DNA insertional mutants in each gene are being screened for male fertility and VN movement defects. Of these, we have so far identified a mutant in the gene encoding PEK14 that exhibits a fertility defect consistent with that of a WIT null mutant (wit12). DAPI staining revealed that pek14 pollen exhibits an intermediate nuclear order switching phenotype, with order being reversed in 50% of pollen tubes (95% in wit12 pollen tubes). Future experiments will establish if PEK14 physically interacts with WIP and WIT and further characterize the role of PEK14 in MGU transport. Additionally, we are continuing to screen for additional motor proteins that may play a role in GC delivery.

References:
1) Zhou, X., and Meier, I. (2014). “Efficient Plant Male Fertility Depends on Vegetative Nuclear Movement Mediated by Two Families of Plant Outer Nuclear Membrane Proteins.” Proc. Natl. Acad. Sci. USA. 111:32, 11900-11905.
2) Zhou, X., et al. (2012). “Novel Plant SUN-KASH Bridges Are Involved in RanGAP Anchoring and Nuclear Shape Determination.” J Cell Biol. 196:2, 203-211.
3) Zhou, X., et al. (2015). “Plant Nuclear Shape Is Independently Determined by the SUN-WIP-WIT2-Myosin XI-i Complex and CRWN1.” Nucleus. 6:2, 144-153.

Keywords: Pollen , Plant cell biology, Motor proteins

38. A novel structural probe: fluorescent-labeled bPNA(+)

Shiqin Miao (Department of Chemistry and Biochemistry), Jie Mao (Department of Chemistry and Biochemistry), Yufeng Liang (Department of Chemistry and Biochemistry), Christopher DeSantis (Department of Chemistry and Biochemistry)

Abstract:
We offer a unique labeling method, using fluorescent-labeled bPNA(+) as a probe for monitoring long-range RNA interactions. We employ a novel compact DNA recognition element, t2M, which comprises two melamines on a linear, 5-atom, sp³-hybridized motif displayed on various scaffolds and forms a base triple with two thymine or uracil bases. This work explores the recognition of nucleic acids by the t2M motif for versatile molecular targeting applications. First, we confirmed the binding affinity and stoichiometry of t2M and t4M with unstructured, T-rich DNAs to form hairpin-like structures. Second, we exploited the ability of t2M and t4M to act as functional switches in deactivated hammerhead ribozymes. We found that hammerhead ribozymes mutated by stem or loop replacement with a U-rich sequence in stems II and III could have bond scission function restored by t2M/t4M. Third, we tested the utility of the t2M motif in multivalent recognition by displaying this motif on a native peptide backbone (bPNA(+)). We accomplished scalable synthesis of bPNA(+) and observed sub-nanomolar K_d. Finally, we applied fluorescent-labeled bPNA(+) as a probe for monitoring long-range RNA interactions. We use a hexapeptide and decapeptide bPNA(+), labeled with a FRET pair, respectively, to label RNA secondary structures to provide FRET reporters of RNA-RNA interactions. Initially, we confirmed the site-selectivity of bPNA(+) as driven by length-matching using U-sites of different sizes and bPNA(+) of the corresponding lengths in the ColE1 kissing loop system by FRET. Next, we designed an RNA host containing the well-studied GAAA tetraloop-receptor interaction as a proof-of-concept system. This novel probe has been successfully applied to RNAse P and HIV-1 systems in RNA-center collaborations. Our work will show that the t2M motif can recognize T/U rich sequences as well as act as a structural probe.

References:
Mao J, DeSantis C, Bong D. Small Molecule Recognition Triggers Secondary and Tertiary Interactions in DNA Folding and Hammerhead Ribozyme Catalysis. J Am Chem Soc 2017;139:9815–8.

Keywords: RNA recognition motif , Structural probe, Functional switch

39. The role of the nuclear matrix protein Matrin3 as a regulator of MDM2 alternative splicing

Matias Montes (MCDB, The Ohio State University), Aishwarya Jacob (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital)

Abstract:
MDM2, a well-known negative regulator of the tumor suppressor protein p53 undergoes complex alternative splicing. One of its spliced isoforms, MDM2-ALT1 comprised of exons 3 and 12, is highly expressed in several cancers such as lung carcinoma, liposarcoma or rhabdomyosarcoma, and its expression correlates with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress. The way that MDM2 splicing is controlled is not well understood.

Both published and unpublished data from our lab indicate that the factors SRSF1 (ASF/SF1), SRSF2 (SC35), FUBP-1 and PTBP1 act as either positive or negative regulators of this process. Interestingly, PTBP1 (Polypyrimidine Tract Binding Protein 1) is commonly known as a repressor of exon inclusion, but our results show that, in case of MDM2 alternative splicing it acts a positive regulator. This factor has been shown to interact with the nuclear matrix protein Matrin3, a recently identified splicing factor. Interestingly Matrin3 has also been shown to be implicated in the DNA damage response induced by genotoxic stress. RNA affinity chromatography experiments coupled with mass spectrometry, indicate that Matrin3 binds to intronic regions of the MDM2 pre-mRNA, thus supporting the hypothesis. In this work we investigate the role of Matrin3 as regulator of MDM2 alternative splicing and its interaction with the splicing factor PTBP1 in said regulation. Elucidating the participation of this factor in the alternative splicing of MDM2 could bring us one step closer in understanding how this is regulated under genotoxic stress and eventually also in cancer such as rhabdomyosarcoma.

Keywords: Splicing, MDM2, Matrin3

40. Model of pulldown alignments from SssI-treated DNA can improve methylation quantification from MBD enrichment-capture experiments

Blythe S. Moreland (Department of Physics, The Ohio State University), Kenji M. Oman (Fred Hutchinson Cancer Research Center), Ralf A. Bundschuh (Department of Physics, Department of Internal Medicine, Division of Hematology, Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Pulldown experiments that use methyl-binding domain (MBD) proteins to preferentially bind to methylated CpGs and enrich a sample for highly methylated DNA remain a popular tool for probing a cell’s methylome. The conversion from the number of aligned reads at a genomic window to an absolute methylation level has been improved in the past by the computational tool Baymeth, which draws additional information from pulldown experiments done on an SssI-treated control sample. We circumvent the need for this additional sample by building a model for the pulldown probability at each genomic location. Using empirical data on the dependence of fragment length and CpG number to pulldown probability, we can model an SssI-control pulldown data set well enough to improve DNA methylation predictions in the Baymeth framework beyond those that do not use an SssI-treated control sample, and in many cases beyond those that do.

Keywords: DNA Methylation, MBD pulldown

41. Transcriptional control and biogenesis of intronic RNase P RNA in Drosophila

Geeta Palsule (Molecular Genetics, The Ohio State University ), Lien B. Lai (Chemistry and Biochemistry, The Ohio State University ), Venkat Gopalan (Chemistry and Biochemistry, The Ohio State University ), Amanda Simcox (Molecular Genetics, The Ohio State University )

Abstract:
RNase P, an essential ribonuclease, is present in all domains of life and is required for the removal of 5’ leader sequences from pre-tRNAs. The ribonucleoprotein form of the enzyme is comprised of a catalytic RNA (RNase P RNA, RPR) and a variable number of protein subunits (RNase P Proteins, RPPs). Although RPR is a typical Pol III-regulated gene in most organisms, we found the Drosophila gene is inserted into a recipient gene intron and transcribed by Pol II. By analyzing genomes of other insects and crustaceans, we discovered the genetic change in the regulation of RPR originated in an ancestor of this major group of animals some 500 million years ago. Clearly this dependence on recipient gene transcription and processing from an intron requires a distinct mode of biogenesis for the inserted RPR. I am using a genetic approach to find the factors involved in RPR biogenesis and to test the functional consequence, if any, of the change in transcriptional control. Using reporter genes assayed in Drosophila S2 tissue-culture cells, we found both a splicing-dependent and splicing-independent mode of producing mature RPR. The 5’ end of the pre-RPR is processed by XRN2 and I am currently investigating the exosome and Rexo5 as 3’ end-processing candidates. To investigate the involvement of the nine protein subunits (RPPs) in RPR maturation, I depleted individual RPPs and assayed for mature RPR by northern blot analysis. This indicated several RPPs are required for RPR maturation or stability. Interestingly, however, when I switch RPR transcription back to the ancestral, Pol III-mode, RPR is stable and unaffected when RPPs are depleted. I am using CRISPR/Cas9 to engineer flies with an ancestral, Pol III-regulated gene, so that I can analyze any functional consequences. Overall my work is expected to provide insights into small RNA biogenesis and how the switch in gene transcription contributed to the evolutionary history of RPR.

Keywords: RNase P RNA, Transcriptional control, RNA processing

42. Human Argonaute3 can be uniquely activated to become “mature slicer”

Mi Seul Park (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Hong-Duc Phan (Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA), Florian Busch (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Samantha H. Hinckley (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Kotaro Nakanishi (1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA. 2 Ohio State Biochemistry Program, 3 Center for RNA Biology)

Abstract:
Of the four human Argonaute (AGO) paralogs, only AGO2 has been shown to have slicer activity. The others (AGO1, AGO3, and AGO4) have been thought to assemble with microRNAs to form slicer-independent effector complexes that bind target mRNAs and silence gene expression through translational repression and deadenylation but not cleavage. Here, we report that recombinant AGO3 loaded with miR-20a cleaves complementary target RNAs, whereas AGO3 loaded with let-7a, miR-19b or miR-16 do not, indicating that AGO3 has slicer activity but that this activity depends on the guide RNA. Our cleavage assays using chimeric guides revealed the significance of seed sequence for AGO3 activity, which depends specifically on the sequence of the post-seed. Unlike AGO2, target cleavage by AGO3 requires both 5'- and 3'-flanking regions. Our 3.28 Å crystal structure shows that AGO3 forms a complete active site mirroring that of AGO2, but not a well-defined nucleic acid-binding channel. These results demonstrating that AGO3 also has slicer activity but with more intricate substrate requirements, explain the observation that AGO3 has retained the necessary catalytic residues throughout its evolution. In addition, our structure inspires the idea that the substrate-binding channel of AGO3 and consequently its cellular function, may be modulated by accessory proteins.

References:
Park M.S., Phan H.D., Busch F., Hinckley S.H., Brackbill J.A., Wysocki V.H., Nakanishi K. Human Argonaute3 has slicer activity. Nucleic Acids Res. 2017; 45:11867–11877.

Keywords: Argonaute, miRNA

43. BEX1 mediated translational control of muscle growth

Jennifer M. Petrosino (Biomedical Sciences Graduate Program; Department of Physiology and Cell Biology, College of Medicine), Colin D. Angell (Department of Physiology and Cell Biology, College of Medicine ), Mike Adam (Cincinnati Childrens Hospital Medical Center), Steve S. Potter (Cincinnati Childrens Hospital Medical Center), Federica Accornero (Department of Physiology and Cell Biology, College of Medicine )

Abstract:
In adult skeletal muscle, myogenesis counteracts damage by promoting muscle regeneration. When muscles regenerate following injury or stress, mRNA translation becomes de-repressed, thus allowing for muscle repair. The cumulative effect of activating myogenic translation to promote myofiber repair is enhanced global rates of protein synthesis, which can consequently result in increases in muscle fiber cross-sectional area (hypertrophy). Using single and repeated intramuscular injections of barium chloride to model conditions of acute and chronic injury, we identified that Brain Expressed X-Linked Protein 1 (BEX1), a protein highly induced during myogenesis, is required for muscle repair and growth. Genetic ablation of BEX1 resulted in impaired growth and repair mechanisms in injured skeletal muscle. To gain mechanistic insights into how regeneration is affected by the loss of BEX1, we performed single-cell droplet sequencing on injured wild-type and BEX1 knockout muscles and identified that in the absence of BEX1, myogenic cells specifically fail to upregulate translation-specific pathways required for repair. Without upregulation of these pathways, BEX1 KO muscles failed to repair back to baseline size, demonstrating that the loss of BEX1 in regenerating muscles has anti-hypertrophic consequences. To begin to understand the molecular details of how BEX1 functions in regulating translation and muscle size, we undertook an extensive analysis of the BEX1 protein interactome by proteomic screening. This approach and follow-up experiments revealed that BEX1 is part of the multi-tRNA synthetase complex that enhances the efficiency of translation. To see if the ability of BEX1 to target protein translation affects the potential for muscles to undergo overload-induced hypertrophy, we utilized a model of synergistic muscle ablation and found that the loss of BEX1 was sufficient to completely abrogate increases in muscle growth. Additionally, delivery of adeno-associated virus BEX1 (AAV2-BEX1) to wild-type mice, and a dystrophic model with impaired protein synthesis, resulted in increases in muscle size and functional hypertrophy. Together with the finding that BEX1 is required for, and promotes, the hypertrophic response of skeletal muscle, our data implicate BEX1 as a regulator of muscle growth.

Keywords: translation , tRNA synthetases, skeletal muscle growth

44. The effect of chemical modifications on thermal and in vivo stability of phi29 bacteriophage RNA three-way junction

Xijun Piao (College of Pharmacy & Center for RNA Nanobiotechnology and Nanomedicine), Hongzhi Wang (College of Pharmacy & Center for RNA Nanobiotechnology and Nanomedicine), Daniel W. Binzel (College of Pharmacy & Center for RNA Nanobiotechnology and Nanomedicine), Peixuan Guo (College of Pharmacy; Center for RNA Nanobiotechnology and Nanomedicine; College of Medicine; Dorothy M. Davis Heart and Lung Research Institute; James Comprehensive Cancer Center)

Abstract:
The RNA three-way junction (3WJ) derived from phi29 bacteriophage DNA packaging motor has recently been used as a vehicle for targeted drug delivery. The high thermal stability and strong resistance to in vivo degradation of this nucleic acid-based platform are crucial to retain hybridized and intact 3WJ. Here we report the effects of different chemical modifications on the thermal and in vivo stability of this 3WJ. It was found that the thermal stability gradually increased by chemical modification in the order of phosphorothioate DNA < DNA < RNA < 2'-F < LNA. This is supported by the studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically-modified oligonucleotides, the thermal stability of this 3WJ can be tuned to cover a wide range of melting temperatures (Tm's) from 21.2 ^oC to over 95 ^oC. The LNA 3WJ was found resistant to boiling temperature denaturation, 8 M urea denaturation and 50% serum degradation. Interveinal injection of fluorescent LNA/2’-F hybrid 3WJs into mice and analysis of urine revealed its exceptional in vivo stability. It is thus concluded that incorporation of LNA into RNA nanoparticles derived from phi29 3WJ can extend the in vivo half-life of the RNA nanoparticles and it is thus expected to improve the pharmacokinetics profile of phi29 3WJ.

References:
Xijun Piao, Hongzhi Wang, Daniel W Binzel, and Peixuan Guo*, “Assessment and Comparison of Thermal Stability and In vivo stability of Phosphorothioate-DNA, DNA, RNA, 2’-F RNA and LNA in the Context of Phi29 pRNA 3WJ”, RNA, 2018, 24, 67-76.

Keywords: Three-way junction, thermal stability, in vivo stability

45. Using Proximity-Dependent Biotinylation to Identify Proteins Proximal to Histone Acetyltransferase 1 in vivo

Liudmila Popova (MCDB), Miranda Gardner (OSBP), Michael Freitas (Cancer Biology and Genetics), Mark Parthun (Biological Chemistry and Pharmacology)

Abstract:
Histone Acetyltransferase 1 (Hat1) is an evolutionarily conserved enzyme known to acetylate lysines 5 and 12 in the tail of the newly synthesized histone H4. Besides playing this role in replication-coupled chromatin assembly, Hat1 has been shown to contribute to DNA damage repair in a variety of organisms. However, much remains unknown about Hat1 functions in vivo. In order to gain detailed insight into cellular roles of Hat1 in mammalian cells we utilized a proximity-dependent biotinylation approach (BioID), which relies on fusing a mutant biotin ligase BirA (R118G) to the protein of interest (Hat1) in order to allow for biotinylation of proteins vicinal to the protein of interest in vivo. For this experiment, a triplicate of HEK 293 cells was transfected i with Hat1-BirA (R118G) construct and treated with exogenous biotin for 24 hours. Biotinylated proteins were isolated, and the pull-downs were analyzed by mass spectrometry. Statistical analysis yielded a list of ~60 proteins enriched in the Hat1-BirA (R118G) transfected-exogenous biotin treated sample compared to the control. The list of enriched proteins included several actin-related proteins, transcriptional regulator KAISO, and other proteins.

References:
Lambert J.P. et al. Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes. (2015) J Proteomics. 118, 81-94.
Parthun, M.R. (2012) Histone acetyltransferase 1: More than just an enzyme? Biochim Biophys Acta, 1819, 256-63.

Keywords: Hat1 , BioID

46. Reconstitution of diphtheria toxin catalytic domain in cancer cells

Vedud Purde (The Ohio State Biochemistry Program), Elena Kudryashova (Department of Chemistry and Biochemistry), David Heisler (Department of Chemistry and Biochemistry)

Abstract:
Diphtheria toxin (DT) is one of the deadliest compounds on earth. Toxicity of (DT) produced by Corynebacterium diphtheriae is defined by the catalytic domain (DTA), which transfers an ADP-ribosyl group from NAD+ to an unconventional diphthamide residue uniquely found in ribosomal elongation factor 2 (eEF2). ADP-ribosylation of diphthamide inactivates eEF2 and blocks ribosomal protein synthesis in the affected cell leading to cell death. DTA has been widely used in targeted therapeutics to construct immunotoxins - artificial proteins that contain a toxin conjugated to a targeting element with a purpose of delivering toxicity to cancer cells. A major limitation of this approach is a lack of true cancer-specific cell surface markers, leading to a non-specific side toxicity of immunotoxins towards non-transformed healthy cells.

We sought to employ split-inteins to develop a novel tool with increased selectivity towards cancer cells. Split-inteins are enzymes that are produced as part of different proteins, but can recognize and excise each other from a precursor protein resulting in the ligation of their flanking sequences. We designed, produced, and characterized different pairs of catalytically inactive fragments of DTA fused to split-intein domains (Split-DTA).

Our in vitro trans- splicing experiments showed that each of the five produced pairs is capable of reconstituting the full length DTA toxin. To deliver split toxins to target cells, they were fused to the N-terminal domain of the anthrax lethal factor (LFN). Products of in vitro trans-splicing reactions of 5 different split-DTA pairs resulted in protein synthesis inhibition in the treated mammalian cells confirming reconstitution of the catalytically active full length DTA toxin. To determine if trans- splicing and formation of active toxins occur in the cytosol, we sequentially delivered individual split constructs to HeLa cells, but inhibition in protein synthesis was not observed. However, when U2OS osteosarcoma cells were stably transfected with a gene encoding one part of a split pair while a corresponding counterpart was delivered as a protein, significant toxicity was observed. Particularly, a decrease in protein synthesis and the subsequent cell death were comparable to those caused by intact DTA

Keywords: Diphtheria toxin, cancer, split-inten

47. The HD domain of SAMHD1 is required for its suppression of interferon regulatory factor 7-mediated type I interferon induction

Zhihua Qin (Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University), Shuliang Chen (Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University), Corine St. Gelais (Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University), Sun Hee Kim (Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University), Serena Bonifati (Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University), Li Wu (Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University)

Abstract:
Homozygous mutations of the gene encoding sterile alpha motif and HD domain containing protein 1 (SAMHD1) can cause Aicardi-Goutières syndrome characterized by the induction of type I interferon (IFN-I), indicating that SAMHD1 is a negative regulator of the innate immune responses. Our recent studies identified that SAMHD1 interacts with IRF7 and inhibits IRF7-mediated IFN-I induction, resulting in suppression of innate immune responses to HIV-1 or Sendai virus infection. However, the domain of SAMHD1 responsible for the IRF7 interaction and IFN-I suppression have not been identified. We hypothesize that SAMHD1 and IRF7 interaction is required for the suppression of IRF7-mediated IFN-I induction by SAMHD1. To map the site of SAMHD1-IRF7 interaction, we generated a series of truncated SAMHD1 mutants and tested their interactions with full-length IRF7 through co-immunoprecipitation (co-IP) in HEK293T cells. We found that mutants lacking the HD domain did not interact with IRF7, suggesting that HD domain of SAMHD1 is important for IRF7 interaction. We then determined the contribution of the HD domain to suppression of IRF7-mediated IFN-I induction using an IFN-sensitive response element (ISRE) reporter assay. Our data showed that the HD domain of SAMHD1 is necessary and sufficient for IRF7 interaction and suppression of IFN-I induction. Our ongoing work is to examine the exact aa within HD domain responsible for the interaction with IRF7 and the suppression of IRF7-mediated ISRE activation using additional SAMHD1 mutants through co-IP assay and dual luciferase assay. Overall, our findings reveal the domain of SAMHD1 contributing to IFN-I inhibition, which help define the mechanisms of SAMHD1 in suppressing the innate immunity.

Keywords: SAMHD1, type I interferon induction, IRF7

48. Regulation of a geminivirus late gene promoter by PRC2

Elizabeth Regedanz (Department of Molecular Genetics, Center for Applied Plant Sciences, Center for RNA Biology, and Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210), Mary Berger (Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas-San Antonio, San Antonio, TX 78249), Garry Sunter (Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas-San Antonio, San Antonio, TX 78249), David M. Bisaro (Department of Molecular Genetics, Center for Applied Plant Sciences, Center for RNA Biology, and Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210)

Abstract:
Geminiviruses are small ssDNA viruses that cause significant yield loss in many agriculturally important crops. Upon entry into the nucleus, the viral genome is converted by host enzymes into a dsDNA replicative form (RF). The RF associates with histones to form non-integrating episomes that both facilitate virus replication and transcription and serve as targets of host defense pathways. As viral chromatin is formed de novo in infected cells, geminiviruses are unique models for examining mechanisms that target and establish epigenetic modifications. Polycomb Repressive Complex 2 (PRC2) is an important repressive regulator of developmental processes via its ability to deposit histone H3 lysine 27 trimethylation (H3K27me3), but how PRC2 activity is regulated at target genes is unclear. We propose that geminivirus gene expression is a model for PRC2 control, as we have found that H3K27me3 is localized to the viral coat protein (CP) promoter, which is repressed early in infection. We have also found that the CP promoter is bound by the plant-specific transcription factor TCP24, which has been implicated in PRC2 recruitment. Importantly, TCP24 mRNA levels decrease in geminivirus-infected cells. Thus, we hypothesize that TCP24 recruits PRC2 to inhibit premature CP expression and permit viral genome amplification. Once a threshold of genomes is produced, reduced TCP24 levels allow CP expression, leading to virion assembly. Thus, our studies offer insight into the temporal control of the geminivirus infection cycle, as well as principles underlying developmental gene regulation.

Keywords: Geminivirus Coat Protein Expression, PRC2, H3K27me3

49. Recognition of RNA substrates by Thg1 family proteins

Tracy M. Roach (Ohio State Biochemistry Program)

Abstract:
tRNAHis guanylytransferase (Thg1) is responsible for adding a G-1 to tRNAHis across from an A73 discriminator nucleotide. This step, and the addition of CCA to the 3'-end of tRNAHis, is required for histidyl tRNA synthetase to recognize and aminoacylate tRNAHis. Because both Thg1 and CCA adding enzyme localize to the cytosol of S. cerevisiae, these two essential enzymes would likely compete for the end-matured tRNAHis substrate, and therefore it is of interest to understand whether there is a preferred pathway in terms of which enzyme is acting on tRNAHis first. Identification of any obligate processing order will define the biologically relevant substrates for each enzyme. To evaluate these possibilities, kinetic studies were used to determine rates of nucleotide incorporation to the 5'-end of tRNA substrates containing and lacking the CCA 3'-end sequence. Here we report that the in vitro studies support a preferred pathway in which CCA addition precedes 5'-end maturation by Thg1 in yeast. To further understand substrate recognition by eukaryotic Thg1 enzymes, an unusual eukaryotic Thg1 enzyme is being explored that appears capable of forming an alternative quaternary structure to the homotetrameric enzyme that has been observed in all other species to date. Efforts to define the catalytically active composition of the Thg1 enzyme in these species will be used as the basis for individual manipulation of one domain of an active Thg1 protein complex at a time to understand the contributions of different subunits to catalysis.

Keywords: Thg1, tRNA processing, enzymology

50. Identifying RIN4 amino acid motifs necessary for its function in plant immunity

Priscila M. Rodriguez Garcia (Department of Molecular Genetics, College of Arts & Sciences, The Ohio State University, Columbus, Ohio, US), Hasung Kim (Plant Immunity Laboratory, Postech Biotech Center, Department of Life Sciences, Pohang University of Science and Technology, Republic of Korea), Keehoon Sohn, Ph.D. (Plant Immunity Laboratory, Postech Biotech Center, Department of Life Sciences, Pohang University of Science and Technology, Republic of Korea), David Mackey, Ph.D. (Department of Horticulture and Crop Science, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Columbus, Ohio, US)

Abstract:
RPM1-interacting protein 4 (RIN4) is a central player in plant immunity. In Arabidopsis thaliana, AtRIN4 connects two major plant defense pathways – pattern- and effector-triggered immunity (PTI and ETI, respectively). In PTI, conserved molecules of pathogens contain pathogen-associated molecular patterns (PAMPs) that are recognized by cell surface receptors to trigger plant basal defenses. In the absence of pathogen, overexpression or lack of AtRIN4 suppresses or enhances defense outputs associated with PTI, respectively. Pathogens have virulence-promoting effector proteins that they inject into the plant cell to suppress PTI. Modification of AtRIN4 by at least 4 different effectors from the pathogen Pseudomonas syringae enhances its suppression of PTI. In ETI, resistant plants activate strong defenses upon recognition of effectors through resistance (R)-proteins. R-protein-induced defenses often include localized cell death, termed the hypersensitive response (HR), thought to contribute to the prevention of pathogen spread. Effector-induced modifications of AtRIN4 induce ETI through activation of two Arabidopsis R-proteins, RPM1 and RPS2, that associate with AtRIN4. In this study, I aim to identify amino acid (aa) motifs necessary for three different aspects of AtRIN4 function: 1) its suppression of PTI, 2) its regulation of ETI, and 3) effector-induced modifications of it. Other studies have attempted to identify RIN4 aa motifs important for its various functions by using AtRIN4 derivatives. However, this approach interferes with other functions of AtRIN4, like its subcellular localization. To overcome this problem, I will use a collection of 27 RIN4 homologs from different plant species developed by our collaborators in Korea. These RIN4 homologs will allow for the identification of aa motifs with putative functions based on phenotypic and sequence differences between these homologs.

Keywords: RIN4, Plant immunity, Arabidopsis

51. Identification of Deletion Junctions and Variants that Affect Genotype-Phenotype Prediction in Spinal Muscular Atrophy

Corey Ruhno (Department of Biochemistry and Chemical Pharmacology, The Ohio State University, Columbus OH), Vicki McGovern (Department of Biochemistry and Chemical Pharmacology, The Ohio State University, Columbus OH), Matthew Avenarius, Pamela J. Snyder, Thomas W. Prior (Department of Pathology, The Ohio State University, Columbus OH), Flavia Nery, Kathryn Swoboda ( Department of Neurology, MassGeneral Hospital for Children, Boston MA), Jennifer Rogenbuck, John T. Kissel (Department of Neurology, The Ohio State University, Columbus OH), Arthur H.M. Burghes (Department of Biochemistry and Chemical Pharmacology, The Ohio State University, Columbus OH)

Abstract:
Spinal muscular atrophy is a degenerative motor neuron disease that is caused by low levels of the SMN protein. SMA severity is inversely correlated with SMN2 copy number, however exceptions to this are not uncommon. The region of the genome that contains SMN1 and SMN2 is very unstable and contains numerous repeats, making it prone to genomic rearrangements like inversions and deletions. Mismatches between the copy number of SMN2 when measured at the 5' marker AG1-CA and exon 7 indicate possible partial deletions of SMN2. Despite this, no deletion breakpoints have been determined in the region. We have sequenced the SMN2 genes of nearly 200 patients and developed a bioinformatic pipeline for identifying deletions, inversions, and variants that could modify SMN2 and thus the SMA phenotype. We have identified two different deletions, both of which result in loss of exons 7 and 8, rendering the gene non-functional. We have screened for the prevalence of one of these deletions amongst populations of individuals with varying copy number of SMN1 and SMN2. It was most prevalent in individuals with 0 copies of SMN2 and 1 copy of SMN1, with a prevalence of 63%. Other genotypes with a high prevalence of the deletion include 2 SMN1/0 SMN2 at 46%, 0 SMN1/1 SMN2 at 35%, and 2 SMN1/1 SMN2 at 34%. While this deletion is more common in SMN2 it clearly does occur in SMN1. We also have identified exonic variants that lie in the SMN2 gene that alter SMN2 function. PLS3 was also captured but no association was found between milder than expected patients and variants known to increase PLS3 expression. In order to identify variants outside of SMN2 that alter severity of SMA we have performed whole genome sequencing of 3 families with discordant sibling phenotypes and 4 with concordant phenotypes as controls. We have found 23 exonic variants that segregate with the milder than expected phenotypes and many intronic variants as well. We are also performing pathway analysis with this data set to identify the critical genes. Candidate genes/pathways that are expressed in motor neurons are tested in additional families.

References:
Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet. 1978;15:409-413. doi:10.1136/jmg.15.6.409.
McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, Prior TW, Burghes a H. Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am J Hum Genet. 1997;60(6):1411-22. doi:10.1086/515465.
Coovert DD, Le TT, Mcandrew PE, Strasswimmer J, Crawford TO, Mendell JR, Coulson SE, Androphy EJ, Prior TW, Burghes AHM. The survival motor neuron protein in spinal muscular atrophy. 1997;6(8):21-22.
Lefebvre S, Burlet P, Liu Q. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet. 1997;16.

Keywords: spinal muscular atrophy, modifier, bioinformatics

52. Characterizing a Novel Method of Human Plastin Regulation

Christopher S. Schwebach (Department of Chemistry and Biochemistry, Ohio State), Jonathan C. Wright (Department of Chemistry and Biochemistry, Ohio State), Elena Kudryashova (Department of Chemistry and Biochemistry, Ohio State)

Abstract:
The actin cytoskeleton is a vast network intricately regulated by numerous actin binding proteins. Plastins are one family of such proteins that non-covalently crosslink actin into bundles and contribute to cellular processes including cell migration and invasion. Bundling is achieved upon binding of two actin binding domains (ABD1 and ABD2) to actin filaments. Our recent findings show that the ABDs are not functionally equal. ABD1 acts as the primary, unregulated and constitutively interacting, but low affinity (~3uM) domain. In contrast, we found using fluorescence anisotropy (FA) that ABD2 alone binds actin with an affinity of ~15nM, i.e., much higher than those of full-length plastin or ABD1 alone. By pyrene-actin polymerization and TIRF microscopy we found that ABD2 is also able to nucleate actin, a property not previously described for full-length plastins. These results imply inhibition of ABD2 in the full protein context. Next, we found that ABD1 and 2 bind each other with high affinity (~20 nM) when expressed in trans and that ABD1 inhibits ABD2’s ability to nucleate actin filaments. Mutations that increase PLS association with actin in yeast also lead to dramatically enhanced bundling capabilities when introduced to human plastins. Interestingly, these mutations also decrease the thermal stability of full-length plastin to the level of ABD1 and ABD2 suggesting weaker association between the domains. Importantly, this same shift in thermal stability is seen in wild-type plastin in the presence of actin. These data support a novel mode of plastin regulation where ABD1 allosterically inhibits ABD2. Upon ABD1-actin binding, ABD2 inhibition is released allowing bundling. Understanding these complex mechanisms will lead to targeted therapies treating cancer metastases, which are known to be stimulated by ectopic expression of PLS2.

Keywords: Actin, Biochemistry, Plastin

53. Tipping the epigenetic scales between genes and transposable elements

Meredith J. Sigman (Molecular Genetics, OSU), Diego Cuerda Gil (Molecular Genetics, OSU), R. Keith Slotkin (Molecular Genetics, OSU)

Abstract:
Transposable elements (TEs) are mobile fragments of DNA that cause double-stranded DNA breaks upon transposition and are thus inherently mutagenic. To prevent DNA damage, multicellular organisms such as the model plant Arabidopsis thaliana transcriptionally silence TEs via heterochromatin formation enacted through epigenetic modifications such as DNA and histone methylation. Cytosine methylation is added to a locus through a small RNA-guided pathway termed RNA-directed DNA Methylation (RdDM). My research examines which RdDM associated factors are sufficient to tip a locus into a methylated “TE-like” state. I have successfully recruited RdDM to a genic locus through the addition of homologous sRNAs. Additionally previous work shows that RdDM factors are associated with double-strand break (DSB) repair. The dual role of canonical RdDM proteins in DSB repair begs the question of whether this pathway can also serve as a preliminary surveillance mechanism capable of targeting TE and exogenous DNA at the genome integration step. I assayed whether a DSB could trigger RdDM at a broken locus through the use of CRISPR-Cas9. Finally, the methylation of DNA and histones can be removed by demethylases to protect a locus from RdDM. We hypothesize that the histone demethylase IBM1 is a key factor in determining whether a locus creating sRNAs will transition into RdDM.

References:
Wei et al. (2012) A role for sRNAs in double strand break repair. Cell , Volume 149 , Issue 1 , 101 - 112

Keywords: Epigenetic, Transposable Element, RNA-directed DNA Methylation

54. 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 (School of Medicine, University of Connecticut), Juan Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University)

Abstract:
Introns interrupt tRNA sequences in all major lines of descent (Bacteria, Archaea and Eukarya), rendering them nonfunctional for protein synthesis. Intron removal is therefore, essential. In all known eukaryotes, intron cleavage, the first step in the tRNA splicing pathway, is catalyzed by a conserved heterotetrameric tRNA splicing endonuclease (Sen) composed of four subunits: Sen54, 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 (Sen34), suggesting that either the other subunits are missing or, as a whole, the enzyme is highly divergent in these organisms. In this work, we present evidence for a divergent and unique enzyme composed of three subunits: homologs of Sen34, 15 and 2. 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, immunofluorescence localization assays showed that the enzyme was cytoplasmic, in stark contrast to the nuclear localization of Sen in most eukaryotes. The results presented here demonstrate that TbSen greatly diverges from previously described eukaryotic enzymes in both 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 a heterotrimeric endonuclease then obviates the need for a Sen54 subunit 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: tRNA, splicing, Trypanosoma

55. Investigating the Role of the Connector Region in E. coli Termination Factor Rho

Nicholas Sunday (Department of Microbiology), Max Gilliland (Department of Microbiology), Marcos Sotomayor (Department of Chemistry & Biochemistry), Irina Artsimovitch (Department of Microbiology)

Abstract:
Transcription termination factor Rho is an ancient hexameric protein that is present in most bacteria. Rho monomer is composed of two domains separated by a flexible connector. The N-terminal domain (NTD) contains a primary RNA-binding site, the C-terminal domain - the secondary RNA-binding site, ATPase and helicase modules. In a textbook model, Rho-NTD binds to a pyrimidine-rich rut site in the RNA, triggering a switch from an open state to an active, closed-ring state in which the nascent RNA is trapped within Rho-CTD. ATP turnover then promotes Rho translocation along the nascent RNA until Rho encounters and releases RNA polymerase. This mechanism is supported by in vitro studies on model substrates but fails to account for how Rho works on myriad RNAs that lack rut sequences, such as xenogenes, antisense transcripts, and R-loops. Using a selection for Escherichia coli mutants defective in termination at suboptimal sites, we identified novel substitutions in the Rho connector that are predicted to reduce its flexibility. We and others also showed that the transcription elongation factor NusG cooperates with Rho to induce termination at non-canonical sites. We hypothesize that the connector mediates allosteric communications between the primary and secondary RNA-binding sites that are necessary for ring closure. We plan to combine in silico analyses of Rho dynamics with in vitro and in vivo assays of Rho-dependent termination to test if the connector flexibility facilitates the formation of the catalytically-active state of Rho and is particularly critical at suboptimal sites.

Keywords: Rho, Termination, Regulation

56. EF-P functions as a growth-rate dependent translation elongation factor

Rodney Tollerson II (Department of Microbiology), Anne Witzky (Department of Molecular Genetics), Michael Ibba (Department of Microbiology)

Abstract:
Elongation Factor P (EF-P) is a universally conserved translation factor that alleviates ribosome pausing at polyproline motifs by facilitating peptide bond formation. Without EF-P, translation elongation becomes the rate-limiting step of protein synthesis, leading to a wide range of phenotypes. In this study, we observe that phenotypes resulting from the loss of EF-P are dependent on growth rate in Escherichia coli. In rapid growth conditions, Δefp E. coli displays many defects, including increased doubling time, sensitivity to antimicrobial agents, and global ribosome pausing compared to the wildtype. When growth is slowed under conditions such as low temperature or nutrient deprivation, these defects are abolished. Using polyproline translation reporter assays, we observe the requirement for EF-P in translation of polyproline motifs is greatly diminished under slow growth conditions. These global changes in EF-P dependence for translation of specific motifs in slow growth conditions may be attributed to decreased ribosome queuing, similar to effects previously observed at the single transcript level. These results highlight the growth rate-dependent role for EF-P in E. coli and its role in adaptation to environmental changes.

Keywords: translation elongation, physiology

57. Protein interactome analysis reveals stabilization of mammalian capping enzyme by heat shock protein 90

Jackson B. Trotman (Department of Biological Chemistry and Pharmacology, The Ohio State University), Bernice Agana (Department of Chemistry and Biochemistry, The Ohio State University), Andrew J. Giltmier (Department of Biological Chemistry and Pharmacology, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Daniel R. Schoenberg (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
A defining feature of eukaryotic messenger RNAs (mRNAs) is the presence of a 5′ N7-methylguanosine cap, which is essential for proper RNA processing, localization, and translation. Beyond the canonical nuclear cap synthesis pathway, mammalian cells also possess cytoplasmic machinery capable of restoring a cap structure onto uncapped and/or partially degraded RNA 5′ ends. Central to both pathways is capping enzyme (CE), a bifunctional triphosphatase-guanylyltransferase that localizes to both the nucleus and the cytoplasm. To gain broader insight into the cellular context of cytoplasmic recapping, we sought to characterize the protein interactome of cytoplasmic CE (cCE). Stable U2OS cell lines were generated to inducibly express a tagged form of cCE or negative control EGFP, and these bait proteins were affinity purified and analyzed by bottom-up proteomics to identify interacting partners. Several cCE-interacting proteins with broad cellular functions were identified and validated, including the 90-kDa heat shock protein (HSP90). Specifically blocking HSP90 function with three different inhibitory drugs (geldanamycin, radicicol, and onalespib) caused levels of both nuclear and cytoplasmic CE to destabilize, establishing CE as a novel HSP90 client protein.

Keywords: capping enzyme, proteomics, HSP90

58. Pax/EGL-38 cell-specific functions in the C. elegans uterine ventral cells

Allison Webb (OSBP), Ryan Johnson (MG), Helen Chamberlin (MG)

Abstract:
Paired-box (PAX) transcription factors are known regulators of coordinated development. Despite the broad biological requirements for PAX, its activities in coordinating organogenesis between neighboring, disparate cells are not well understood. To investigate PAX function, we are utilizing a Caenorhabditis elegans ortholog, EGL-38. In C. elegans, EGL-38 is required both in the epidermal, vulval vulF cells and the mesodermal, uterine uv1 cells. EGL-38 is known to function in the vulF cells to specify uv1 cell identity, and we believe that EGL-38 is functioning within the uv1 cells to activate a battery of neuropeptide genes required for egg-laying. We have identified two uv1 neuropeptides genes, nlp-2 and nlp-7, whose expression is dependent on EGL-38 activity in the uv1 cells. An nlp-2 PAX binding element (nre1) has been shown to be required for Pnlp-2 reporter expression and to be bound by the EGL-38 DNA binding domain in vitro; we are currently using site-directed mutagenesis to investigate a similar region of DNA in the nlp-7 promoter as an EGL-38 binding site. We are also investigating the role of EGL-38 in egg-laying by examining the impact on egg retention in nlp-2, nlp-7, and flp-11 (an additional neuropeptide) mutants. A role for EGL-38 to activate targets which participate directly in a biological function such as egg-laying has not previously been identified, and we hope to expand the understanding of PAX protein function in general through this work.

Keywords: Developmental Biology, Genetic program, PAX protein

59. A subpopulation of Exon Junction Complexes (EJC) containing CASC3 represents a late stage of EJC composition and is displaced during translation to the 3’UTR

Lauren Woodward (Department of Molecular Genetics), Justin Mabin (Department of Molecular Genetics), Robert Patton (Department of Physics), Mengxuan Jia (Department of Chemistry and Biochemistry), Vicki Wysocki (Department of Chemistry and Biochemistry), Ralf Bundschuh (Department of Physics)

Abstract:
Proteins bind nascent mRNAs cotranscriptionally to form ribonucleoprotein complexes (mRNPs). An integral component of mRNPs is the exon junction complex (EJC), which assembles 24nt upstream of exon junctions during splicing and remains bound until its co-translational removal. The EJC is composed of a trimeric core (consisting of EIF4AIII, MAGOH, and RBM8A) and serves as a binding platform for other proteins that influence mRNP fate. Our proteomic analyses in mammalian cells depict two mutually exclusive EJC compositions, which we characterize as either RNPS1- or CASC3-containing EJCs. Both RNPS1 and CASC3 shuttle between the nucleus and cytoplasm; however, RNPS1 is primarily nuclear, while CASC3 localizes to the cytoplasm and cytoplasmic granules. Sequencing of the footprints of these distinct EJCs shows that RNPS1- and CASC3- containing EJCs bind the same site on mRNAs, suggesting EJCs undergo a constitutive compositional switch from RNPS1 to CASC3. Change in EJC protein composition is accompanied by transition from the previously described multimeric structure to a monomeric conformation. The CASC3-containing EJC occupancy is highly sensitive to translating ribosomes resulting in a dramatic shift away from the -24nt position. Additionally, we observe that CASC3-EJCs are more enriched on inefficiently translated mRNAs. This demonstrates a pre-translational switch in mRNP composition where the monomeric CASC3-EJCs bind mRNPs that interact directly with the translation machinery. Surprisingly, concomitant with displacement of the CASC3-EJCs away from the -24nt position, we see an accumulation of CASC3-EJC footprints at the 3’end of actively translating mRNAs. We show EJCs are moved to the 3’ end of mRNAs by ribosomes before disassembly, and these 3’ localized EJCs influence the stability of some mRNAs. This study reveals new insights into the changes that EJCs undergo in the cytosol prior to and during translation and how these changes can influence mRNA fate.

Keywords: EJC, translation, mRNP

60. Investigating UPF3B-independent nonsense-mediated mRNA decay

Zhongxia Yi (Department of Molecular Genetics), Lauren A. Woodward (Department of Molecular Genetics), Justin W. Mabin (Department of Molecular Genetics)

Abstract:
Nonsense-mediated mRNA decay (NMD) rapidly degrades aberrant transcripts with premature termination codon (PTC) to limit the production of truncated polypeptides. NMD also regulates around 10-20% of the normal transcripts, and is essential for development, differentiation and stress response [1]. Intriguingly, while in yeast all three NMD factors, Upf1p, Upf2p and Upf3p, are essential for NMD, several NMD substrates can still undergo efficient NMD after depleting UPF3B in human cells [2]. In human, recognition of NMD substrates is largely assisted by exon junction complexes (EJCs), while yeasts do not contain EJCs. EJCs are deposited at exon-exon junctions by spliceosome after splicing and they facilitate the mRNA export to the cytoplasm, where EJCs will be removed by the first translating ribosome. An EJC downstream of a terminated ribosome signals a premature termination event in which this EJC recruits NMD factors UPF3B, UPF2 and UPF1 sequentially that finally leads to the degradation of the transcript [3]. Recently, our lab has discovered the heterogenous EJC compositions and we have identified at least two mutually exclusive EJCs. We hypothesize that the alternative EJC composition might help bypass the UPF3B need. Here, our work shows that EJCs with distinct compositions preferentially associate with different NMD factors. An alternative EJC, characterized by containing EJC component RNPS1, preferentially associates with UPF2 over UPF3B. We are creating knockout cell lines using CRISPR-Cas9 to test the effect of RNPS1 and UPF3B on the association between UPF1 and EJCs. We also plan to characterize transcriptome-wide substrates of UPF3B-independent NMD. The parallel NMD branches could help explain how NMD regulates subsets of transcripts differentially in biological processes.

References:
[1] Karousis ED, Nasif S, Mühlemann O. Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact. Wiley Interdiscip Rev RNA, 2016, 7: 661-682.
[2] Chan WK, Huang L, Gudikote JP, Chang YF, Imam JS, MacLean JA et al. An alternative branch of the nonsense-mediated decay pathway. EMBO J, 2007, 26: 1820-1830.
[3] Woodward LA, Mabin JW, Gangras P, Singh G. The exon junction complex: a lifelong guardian of mRNA fate. Wiley Interdiscip Rev RNA, 2017, 8: e1411.

Keywords: Nonsense-mediated mRNA decay, Exon junction complexes, UPF3B

61. RNA-based micelles: A novel platform for paclitaxel loading and delivery

Hongran Yin (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; The Ohio State University, Columbus, OH 43210, United States), Yi Shu (Nanobiotechnology Center, Markey Cancer Center and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, United States), Mehdi Rajabi, Hui Li (Nanobiotechnology Center, Markey Cancer Center and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, United States), Mario Vieweger (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; The Ohio State University, Columbus, OH 43210, United States), Sijin Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; The Ohio State University, Columbus, OH 43210, United States)

Abstract:
RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures for biotechnological and biomedical applications. In addition to current self-assembly strategies utilizing base pairing, motif piling and tertiary interactions, we reported for the first time to build RNA based micellar nanoconstruct with a cholesterol molecule conjugated onto one helical end of a branched pRNA three-way junction (3WJ) motif. The resulting amphiphilic RNA micelles consist of a hydrophilic RNA head and a covalently linked hydrophobic lipid tail that can spontaneously assemble in aqueous solution via hydrophobic interaction. Taking advantage of the feature of pRNA 3WJ branched structure, the assembled RNA micelles are capable of escorting multiple functional modules. As a proof of concept for delivery for therapeutics, Paclitaxel was loaded into the RNA micelles with significantly improved water solubility. The successful construction of the drug loaded RNA micelles was confirmed and characterized by agarose gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence Nile Red encapsulation assay. The estimate critical micelle formation concentration ranges from 39nM to 78nM. The Paclitaxel loaded RNA micelles can internalize into cancer cells and inhibit their proliferation. Further studies showed that the Paclitaxel loaded RNA micelles induced cancer cell apoptosis in a Caspase-3 dependent manner but RNA micelles alone exhibited low cytotoxicity. Finally, the Paclitaxel loaded RNA micelles targeted to tumor in vivo without accumulation in healthy tissues and organs. There is also no or very low induction of pro-inflammatory response. Therefore, multivalence, cancer cell permeability, combined with controllable assembly, low or nontoxicity nature, and tumor targeting are all promising features that make our pRNA micelles a suitable platform for potential drug delivery.

Keywords: pRNA three way junction, RNA nanotechnology, RNA micelles

62. Global analysis of translation processivity in E. coli

Lanqing Ying (Department of Microbiology and Center for RNA Biology, The Ohio State University), William Baez (Department of Physics and Center for RNA Biology, The Ohio State University), Ralf Bundschuh (Department of Physics and Center for RNA Biology, The Ohio State University), Kurt Fredrick (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract:
During translation, the ribosome sometimes fails to complete synthesis of full-length protein product. Based on previous reporter gene studies, mutations that slow down elongation reduce translation processivity. A possible explanation is that longer decoding dwell time causes a higher frequency of peptidyl-tRNA drop-off in these mutant ribosomes. Here, we used ribosome profiling to globally measure the translation processivity in wild-type and various mutant strains. In contrast to the earlier predictions, we have found that translation processivity is generally independent of decoding rate. Based on our findings, we infer that processivity errors mainly occur during the translocation step of elongation.

Keywords: protein synthesis, decoding , tRNA

63. Assessing the biological function of cytoplasmic tRNA methyltransferase (Trm10) enzymes in Danio rerio

Ben Jepson (Dept. of Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Dept. of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Transfer RNAs (tRNAs) are highly modified across all domains of life. The m1R9 tRNA methyltransferase (Trm10) catalyzes the methylation of the N-1 atom of the purine nucleotides Adenine and Guanine at position nine of substrate tRNAs. Trm10 family enzymes are conserved across all archaeal and eukaryotic organisms. In Saccharomyces cerevisiae, there is only one Trm10 enzyme. However, in higher eukaryotes, there are multiple Trm10 homologs in individual organisms. Humans, for example, have one mitochondrial (TRMT10C) and two cytoplasmic (TRMT10A and TRMT10B) Trm10 enzymes. Mutations in one of the cytoplasmic paralogs, TRMT10A, have been implicated in diseases that include glucose metabolic and neurodegenerative disorders. This observation suggests that these two cytoplasmic enzymes have non-redundant roles in the cell, and previous work in the lab has shown that TRMT10A and TRMT10B have different enzymatic activities and tRNA substrate specificities. Here we use Danio rerio (zebrafish), which also encodes two cytoplasmic Trm10 enzymes as a model organism in order to investigate their biological function. We use in vitro biochemical assays to show that zebrafish trmt10a and trmt10b show similar enzymatic activities and substrate specificities. However, only zebrafish trmt10a is capable of complementing the 5-FU hypersensitivity phenotype of a trm10∆ S. cerevisiae strain, consistent with the proposed non-redundant roles for these enzymes in vivo. Future work will attempt to fill the gap in knowledge of substrate specificity from two directions. The first approach will utilize an RNA-seq based method to analyze the modification status of zebrafish tRNAs in an attempt to identify potential substrates for zebrafish trmt10a/b; these findings will be used in further experiments using recombinant zebrafish Trm10 proteins. The second approach will take advantage of existing zebrafish trmt10a/b mutant strains, where total RNA will be isolated and analyzed using primer extension. Completion of this work will leave us with a greater understanding of the differing substrate specificities of the cytoplasmic Trm10 homologs in zebrafish and will help to shed some light on the distinct functionalities of the Trm10 family as a whole.

Keywords: tRNA modification, tRNA methylation, Trm10

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