Cellular, Molecular & Biochemical Sciences Training Program/Center for RNA Biology Annual Symposium 2021

Poster abstracts

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1. Immune cells direct signaling and disease progression in a zebrafish model of Duchenne Muscular Dystrophy

Natalie Aloi (MCDB), Geremy Lerma (Molecular Genetics), Joseph Beljan (MCDB), Jared Talbot (University of Maryland School of Biology and Ecology)

Abstract:
Duchenne Muscular Dystrophy (DMD) is a devastating, universally fatal disease affecting 1 in 3000 males. Although the standard of care for DMD is treatment with immunosuppressive drugs, the immunopathology of DMD is poorly understood. Understanding the behavior of immune cells and immune-directed signaling in DMD is critical to the development of more effective therapies. Macrophages, which comprise a large portion of the immune infiltrate in DMD, can be polarized towards a pro-inflammatory M1 or an anti-inflammatory M2 phenotype. M2 polarized macrophages secrete TGF-β, a pro-fibrotic cytokine implicated in dystrophic muscle damage. However, promoting M2 polarization improves muscle pathology in models of DMD. It is unclear how M2 macrophages provide a protective effect in DMD despite producing TGF-β. Ease of imaging and amenability to drug treatment make zebrafish larvae an ideal model to study macrophages in vivo. Our confocal analysis of dystrophic larvae reveals that macrophages are actively recruited to dystrophic lesions and clear from the site of injury during lesion resolution. Staining for the phosphorylated SMAD3 protein (pSMAD3), which marks cells responding to TGF-β, shows an elevated level of TGF-β-responsive cells and macrophages near dystrophic lesions. These data suggest that macrophages may indeed play a central role in the regulation of TGF-β signaling in dystrophic tissues.

References:
References
1.) McDouall RM, Dunn MJ, Dubowitz V. Nature of the mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy. J Neurol Sci. 1990;99(2-3):199‐217.
2.) Kharraz Y, Guerra J, Mann CJ, Serrano AL, Muñoz-Cánoves P. Macrophage plasticity and the role of inflammation in skeletal muscle repair. Mediators Inflamm. 2013;2013:491497.
3.) Nguyen-Chi, M., Laplace-Builhe, B., Travnikova, J., et al. Identification of polarized macrophage subsets in zebrafish. ELife. 2015;4:e07288.
4.) Ceco E, McNally EM. Modifying muscular dystrophy through transforming growth factor-β. FEBS J. 2013;280(17):4198‐4209.
5.) Berger J, Currie PD. Zebrafish models flex their muscles to shed light on muscular dystrophies. Dis Model Mech. 2012;5(6):726‐732.

Keywords: Duchenne Muscular Dystrophy, Macrophage, Zebrafish

2. Investigating Regulators of Nonsense Mediated mRNA Decay in Zebrafish Neuromuscular Development

Rene Arvola (Department of Molecular Genetics, OSU), Pooja Gangras (Department of Molecular Genetics, OSU), Zhongxia Yi (Department of Molecular Genetics, OSU), Thomas Gallagher (Department of Molecular Genetics, OSU), Sharon Amacher (Department of Molecular Genetics, Department of Biological Chemistry and Pharmacology, OSU), Guramrit Singh (Department of Molecular Genetics, OSU)

Abstract:
Embryonic development requires highly specific spatiotemporal control of gene expression achieved, in part, by RNA binding proteins that regulate the stability and expression of mRNAs. The Exon Junction Complex (EJC) is a conserved multiprotein complex that is deposited on spliced mRNAs and is critical for many aspects of post-transcriptional gene regulation. In higher eukaryotes, the EJC serves as a regulator of mRNA quality control through stimulating Nonsense-Mediated Decay (NMD) of mRNAs bearing premature termination codons (PTCs). The EJC is necessary for murine brain development through supporting the proliferation of neural progenitors [1]; moreover EJC core components Rbm8a and Magoh are crucial for proper development of motor neurons and muscle during zebrafish embryogenesis [2]. The Cancer Susceptibility Candidate 3 (Casc3) protein is a peripheral EJC component that potentiates decay of a subset of transcripts [3]. Moreover, Casc3 has documented roles in mRNA localization, including in the murine brain [4]. Using human cell-based assays, we have demonstrated that CASC3 can stimulate decay of a PTC-bearing reporter and regulates endogenous PTC-containing mRNAs. To address how Casc3 modulates EJC function during development in vivo using zebrafish, we have generated casc3 mutants via CRISPR. Unlike rbm8a and magoh mutant zebrafish, casc3 homozygous mutants are viable; furthermore, preliminary studies suggest that several EJC-dependent NMD targets are not dysregulated in these mutants. Ongoing work includes investigating possible perturbations in motor neuron development in casc3 mutants. Future work will investigate whether Casc3 stimulates decay and/or localization of a subset of EJC-dependent targets, and whether this contributes to neuromuscular development or other tissue-specific roles.

References:
[1] Silver et al. (2010). The exon junction complex component Magoh controls brain size by regulating neural stem cell division. Nat Neurosci. 13(5):551-8
[2] Gangras et al. (2020). Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3′UTR intron-containing NMD targets. PLOS Genetics 16(6): e1008830
[3] Gerbracht et al. (2020). CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex, Nucleic Acids Research 48(15):8626-8644
[4] Fritzsche et al. (2013). Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons. Cell Rep. 5(6):1749-62

Keywords: mRNA decay, embryonic development

3. Identification of a tRNA-specific function for the tRNA methyltransferase Trm10 in Saccharomyces cerevisiae

Isobel Bowles (OSBP)

Abstract:
tRNA methyltransferase 10 (Trm10) methylates N1 of guanosine at the 9th position of tRNA molecules using methyl donor S-adenosyl methionine (SAM). Upon deletion of TRM10, Saccharomyces cerevisiae strains exhibit growth defects in the presence of antitumor drug 5-fluorouracil (5FU). We hypothesized that tRNA stability decreases with the lack of the m¹G₉ modification in trm10Δ strains and that certain tRNA species are more reliant upon the presence of the methylated G₉ nucleotide, as evident from different growth phenotypes observed upon tRNA overexpression. When Trm10 substrate tRNA^Trp is overexpressed in trm10Δ strains, growth hypersensitivity to 5FU is rescued, while overexpression of other tRNA species in S. cerevisiae do not display a growth rescue. We demonstrated that levels of tRNA^Trp decrease in trm10Δ strains and that overexpression of tRNA^Trp recovers tRNA^Trp levels in trm10Δ strains, while another Trm10 substrate (tRNA^Gly) remains at a similar level in all strains. The specific role of the m¹G₉ tRNA modification is being investigated by analyzing known mechanisms of tRNA^Trp quality control, using S. cerevisiae trm10Δ strains with 5FU. tRNA structures that correlate with active substrates for Trm10 activity are also being determined with nuclease and chemical footprinting, as well as 2’ hydroxyl acylation analyzed by primer extension (SHAPE). Together these studies provide further understanding of tRNA structural elements that promote methylation by Trm10 and the biological impact of loss of this highly conserved modification.

Keywords: tRNA modification, tRNA levels, SHAPE

4. The c terminal domain of histone H1 is integral for altering transcription factor binding to nucleosomes

Nathaniel L. Burge (OSBP), Michael G. Poirier (Physics)

Abstract:
Redacted abstract

Keywords: Histone H1, Chromatin, Transcription

5. Elucidating the role of the mesoderm in let-60/Ras-mediated over-proliferation of epithelial cells in C. elegans

Marcos Corchado (Department of Molecular Genetics, The Ohio State University), Komal Rambani (Biomedical Sciences Graduate Program, The Ohio State University), Helen Chamberlin (Department of Molecular Genetics, The Ohio State University), Gustavo Leone (Department of Biochemistry, Medical College of Wisconsin)

Abstract:
Mesoderm-to-epithelium signaling plays an important role in the maintenance and progression of tumors, as mesodermal-derived cells comprise much of the microenvironment during cancer. The influence of the tumor microenvironment has been well documented, however, identification of the genes important in mediating environment-tumor interactions has been limited due to the complexity of stromal tissue in mammals. To overcome this challenge, we developed a simple model for the mesoderm-to-epithelium signaling using the nematode C. elegans. In this system, we genetically engineered let-60/Ras-sensitized animals to have tissue-specific activity of RNAi in the mesoderm. Using this strain, a genome-wide RNAi screen was performed to identify mesoderm-specific suppressors of let-60/Ras. To validate RNAi experiments, we generated genetically chimeric null animals and confirmed the cell non-autonomous suppression activity of two genes, hpo-18 and szy-5. hpo-18 encodes a mitochondria-specific ATPase while szy-5 encodes a zinc-finger domain-containing protein. To better understand how these two genes impact mesoderm-epithelium communication, we have focused on characterizing their role in the Anchor Cell (AC), muscle, and gonad, the three tissues that comprises the mesoderm. Previous research has shown the influence of AC mitochondria localization and ATP enrichment on basement membrane (BM) degradation and subsequent effects on vulva development. Therefore, we performed fluorescent imaging of the AC mitochondria and the BM structural protein lam-1 upon knockdown of hpo-18 and szy-5 which showed no difference in mitochondrial localization or BM integrity compared to wildtype. Similarly, RNAi knockdown show no effect on gonad morphology and muscle function. These results suggest that these cellular processes still occur under loss of either gene. The value of the tissue-specific RNAi screens presented in this research is the ability to identify let-60/Ras suppressors that otherwise may have been masked by lethal or developmentally arrested phenotypes in whole animal knockdown. To this extent, we present novel regulators of mesoderm-to-epithelium tissue communication and epithelial cell proliferation.

Keywords: Cancer, Microenvironment, C elegans

6. Pl1-Rhoades paramutation is associated with molecular changes at downstream tandem repeats

Natalie C. Deans (Department of Molecular Genetics and Centers for Applied Plant Sciences and RNA Biology, The Ohio State University, Columbus, OH, 43210), Joy-El R.B. Talbot (Department of Molecular Genetics, The Ohio State University, Columbus, OH, 43210; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200), Cristian Sez-Gonzlez, Mowei Li (Department of Molecular Genetics and Centers for Applied Plant Sciences and RNA Biology, The Ohio State University, Columbus, OH, 43210), Chris Watkins, Darren Heavens (The Earlham Institute, Norwich Research Park, Norwich, NR4 7UH, UK), Mario Caccamo (The Earlham Institute, Norwich Research Park, Norwich, NR4 7UH, UK; NIAB, Cambridge, CB3 0LE, UK), Jay B. Hollick (Department of Molecular Genetics and Centers for Applied Plant Sciences and RNA Biology, The Ohio State University; Department of Molecular and Cell Biology, University of California, Berkeley)

Abstract:
In maize, paramutations result in meiotically-heritable regulatory changes of certain alleles including Pl1-Rhoades which encodes a transcription factor required for anthocyanin production[1,2]. A strongly-expressed Pl1-Rhoades allele (denoted Pl-Rh ) is suppressed in trans when combined with a transcriptionally and post-transcriptionally repressed Pl1-Rhoades allele (denoted Pl´ ), and both alleles are subsequently sexually transmitted in a Pl´-like state. Mutant screens have identified at least sixteen loci whose functions are required to maintain repression (rmr) of Pl´ [ 3,4]. Known RMR proteins include subunits of a plant specific DNA-dependent RNA polymerase (Pol IV)[5,6] and others that influence 24nt RNAs[4,7,8] which, in Arabidopsis, facilitate repressive chromatin modifications. Although structural analyses of other maize alleles subject to paramutation identify distinct repeats as a common feature[9,10,11], the mechanisms and specific sequences that facilitate paramutation remain largely unknown. Here we report that a region downstream of the Pl1-Rhoades coding sequence conferring strong expression and paramutation behavior[12] contains five ~2kb direct repeats while the weakly paramutagenic CML52 and nonparamutagenic B73 haplotypes have three and one, respectively. Repeat RNA levels correlate with Pl1-Rhoades expression. Pol IV-dependent 24nt RNAs at these repeats indicate RMR proteins operate at this feature. Additional fractionation-based experiments revealed alternate nucleosome profiles within these repeats distinguishing allele states. How these repeats to facilitate paramutations remains an open question. We are currently identifying additional molecular features distinguishing alternate Pl1-Rhoades states and characterizing these regulatory transitions to determine how maize generates, maintains, and transmits meiotically-heritable regulatory variation.

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 Hale et al. 2007 PLoS Biol. 5, 2156 | 5 Erhard et al. 2009 Science 323, 1201 | 6 Stonaker et al. 2009 PLoS Genet. 5, e1000706 | 7 Nobuta et al. 2008 PNAS 105, 14958 | 8 Barbour et al. 2012 Plant Cell 24, 1761 | 9 Stam et al. 2002 Genes and Dev. 16, 1906 | 10 Kermicle et al. 1995 Genetics 141, 361 | 11 Goettel and Messing 2013 Theor. Appl. Genet. 126, 159 | 12 Erhard et al. 2013 Plant Cell 25, 808

Keywords: Enhancer, Gene regulation, Paramutation

7. Design and synthesis of potent DNA/RNA binding shortest di, tri and cyclic bifacial peptide nucleic acids (bPNAs)

Dr. Shekaraiah Devari (The Ohio State University), Prof. Dennis Bong (The Ohio State University)

Abstract:
Design and synthesis of a small library of alpha/beta isopeptide-nucleic acid has been untangle to explore the influence of various backbone carbon chain length and spacers in between the two monomers on the hybridization properties of the DNA or RNA. The screening of melamine (M) conjugated various amino acid derivatives, Diaminopropionic acid (Dp), Diaminobutyric acid (Db), ornithine (O) and lysine (K), have been performed. The hydrophobic amino acid D/L-alanine, D/L-proline and glycine has been introduced as a spacer. Melting temperature (Tm) analysis suggests that dipeptide having alpha-K2M bPNA exhibited the significant binding compared to various bPNAs having the spacer amino acids, i.e D or L-alanine, D or L-proline and glycine with both T/U rich DNA/RNAs. Furthermore, the mixed monomers containing bPNAs fail to exhibit the suitable binding T/U rich DNAs/RNAs. Interestingly, the cyclic bPNA having 4C linker K2M diketopiperazine (DKP) determines the greater binding against T/U rich DNAs/RNAs with respect to the linear di and tri-peptides as well as other DKPs (diketopiperazine's) with 1-3C linkers according to the thermal melting analysis (Tm).

References:
1) S. Miao, Y. Liang, I. Marathe, J. Mao, C. DeSantis, and D. Bong, J. Am. Chem. Soc. 2019, 141, 9365−9372.
2) Y. Liang, S. Miao, J. Mao, C. DeSantis, and D. Bong, Biochemistry 2020, 59, 2410−2418.

Keywords: RNA and DNA binding , bPNAs, Thermal melting

8. Investigating the inhibitory role of initiation factor eIF2A on translation in vitro

Daisy J. DiVita (The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Michael G. Kearse (The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
Canonical translation uses the heterotrimeric eukaryotic initiation factor 2 (eIF2) as the initiator tRNA (tRNA_i^Met) carrier and as a central node of regulation. However, eIF2 was not the first tRNA_i^Met carrier identified in eukaryotes. eIF2A (a monomer that is non-homologous to eIF2) was the initial tRNA_i^Met binding protein discovered, but its role in translation was overshadowed after eIF2 was identified. Recent reports have shown that eIF2A is required for cancer progression, not part of the canonical set of initiator factors, displays uncharacteristic GTP-independence for tRNA binding (unlike other tRNA-binding translation factors), and is able to stimulate initiation at CUG codons using Leu-tRNA^CUG; however, the exact function of eIF2A remains unknown. To determine how eIF2A functions in translation initiation, we programed in vitro translation extracts with recombinant human eIF2A. Our preliminary data show a 30-fold decrease in reporter activity when recombinant eIF2A is present, suggesting eIF2A is an inhibitor of translation. Here we will pursue testing whether eIF2A is competing for tRNA_i^Met or inhibiting 80S formation. Together, these mechanistic insights will shed light on how non-canonical initiation factors are used and regulate eukaryotic translation.

Keywords: Translational control, Ribosome, Protein synthesis

9. Investigating the role of EFTUD2 in pre-mRNA splicing and EJC deposition

Caleb M. Embree (Department of Molecular Genetics, Center for RNA Biology), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology)

Abstract:
During the gene expression pathway, pre-mRNA splicing is one of the earliest and most important RNA processing steps, which occurs co-transcriptionally. The end result of pre-mRNA splicing is an mRNP particle comprised of mRNA and RNA binding proteins deposited during splicing. One complex of RNA binding proteins, the exon junction complex (EJC), plays important roles in steps ranging from pre-mRNA splicing in the nucleus to mRNA degradation in the cytoplasm. Though the EJC is deposited by the spliceosome, the responsible spliceosomal proteins and their precise function in EJC deposition is not fully known. EFTUD2, a splicing regulator and member of the U5 snRNP that sits adjacent to the EJC within the activated spliceosome, may have a role in EJC deposition. Mutations in EFTUD2 and the EJC core factor EIF4A3 cause similar cranio-facial disorders, hinting that they function in a similar pathway. Using clinically relevant C-terminally truncated EFTUD2 proteins we are investigating the relationship between EFTUD2 and the EJC. Our work shows that the C-terminal truncations of EFTUD2 destabilize the protein, which suggests that these mutations likely cause EFTUD2 haploinsufficiency in patients. Our hypothesis is that such a haploinsufficiency leads to defects in spliceosome and/or EJC assembly resulting in splicing errors. Using EFTUD2 haploinsufficient (or knockdown) HEK293 cells, we plan to identify the splicing errors via RNA-Seq and assess transcriptome-wide spliceosome assembly via spliceosome profiling. Together these data will allow us to determine how reduced levels of EFTUD2 impairs spliceosome and/or EJC assembly leading to splicing defects.

Keywords: Exon Junction Complex, Splicing, developmental disorders

10. Role of the sarcin-ricin loop (SRL) of 23S rRNA in assembly of the 50S ribosomal subunit

Sepideh Fakhretaha Aval (Ohio state Biochemistry program, The Ohio State University. Department of Microbiology, The Ohio State University. Center for RNA biology, The Ohio State University), Kurt Fredrick (Ohio state Biochemistry program, The Ohio State University. Department of Microbiology, The Ohio State University. Center for RNA biology, The Ohio State University)

Abstract:
The sarcin-ricin loop (SRL) is one of the most conserved segments of the large subunit ribosomal RNA. Translational GTPases, such as EF-G and EF-Tu, and IF2, interact with the SRL, and this interaction is essential for GTP hydrolysis. Cleavage and modification of the SRL by cytotoxins α-sarcin and ricin disrupt GTPase-ribosome interaction, leading to reduced protein synthesis and cell death. However, the full role of the SRL remains unclear. A study from the Noller group showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in E. coli. These ΔSRL ribosomes were purified from cells and found to be completely inactive in protein synthesis. Surprisingly, further analysis showed that ΔSRL ribosomes exhibit a major assembly defect. In particular, block 4 of the 50S subunit, which includes the components of the peptidyl transferase center, fails to fold. We hypothesize that this assembly defect results from disruption of interactions with one or more translational GTPases rather than an intrinsic rRNA folding problem. During the assembly of large subunit, SRL folds early on as part of block 1. We envision that GTPases could then transiently bind via the SRL and facilitate subsequent folding events. Here, we describe in vitro 50S subunit reconstitution experiments to investigate this hypothesis.

References:
Moazed D, Robertson JM, Noller HF. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature. 1988;334(6180):362-364. doi:10.1038/334362a0
García-Ortega L, Alvarez-García E, Gavilanes JG, Martínez-del-Pozo A, Joseph S. Cleavage of the sarcin-ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding. Nucleic Acids Res. 2010;38(12):4108-4119. doi:10.1093/nar/gkq151
Lancaster L, Lambert NJ, Maklan EJ, Horan LH, Noller HF. The sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit. RNA. 2008;14(10):1999-2012. doi:10.1261/rna.1202108
Davis JH, Williamson JR. Structure and dynamics of bacterial ribosome biogenesis. Philos Trans R Soc Lond B Biol Sci. 2017;372(1716):20160181. doi:10.1098/rstb.2016.0181

Keywords: Sarcin-ricin loop, Ribosome assembly, Translational GTPases

11. The effect of global Sertad4 KO on post-MI cardiac remodeling

Ashley Francois (Molecular, Cellular and Developmental Biology MCDB ), Lynn Marcho (Department of Physiology & Cell Biology, Ohio State University ), Erin McGrail (Department of Physiology & Cell Biology, Ohio State University ), Alessandro Canella (Department of Physiology & Cell Biology, Ohio State University ), Paul Janssen (Department of Physiology & Cell Biology, Ohio State University ), Richard Gumina (Department of Internal Medicine, Ohio State University )

Abstract:
Heart failure (HF) affects millions of adults in the US and is prevalence is projected to increase 46% by 2030. HF is characterized by the heart's inability to sufficiently perfuse the body, resulting in edema, pulmonary congestion and multiorgan dysfunction. One common cause of HF is myocardial infarction (MI). At the ischemic site of damage, activated fibroblasts help establish a stabilizing scar. However, fibroblast activation can extend beyond the damaged area and cause excess deposition of extracellular matrix in previouslt healthy areas, leading to HF and possible death. The nuclear protein, Sertad4 (SERTA domain-containing protein 4) is a member of the SERTAD family of proteins, characterized by a conserved SERTA domain. Other members of this family have been identified as cell cycle regulators and transcriptional co-factors, but little is known about the role of Sertad4. In 2019, cell culture experiments identified Sertad4 as a potential regulator of cardiac fibroblast activation and we have observed increased protein expression of Sertad4 in human ischemic heart failure samples. We sought to determine the effect of a global Sertad4 knockout on post -MI cardiac remodeling in mice. After 4 weeks of permanent LAD ligation, echocardiography was performed to measure systolic function. Relative to wild-type controls, the Sertad4 KO mice showed preserved systolic function as evident by improved ejection fraction and fractional shortening. B-Gal staining in the Sertad4/LacZ reporter also showed robust Sertad4/LacZ expression in the infarct scar which extended into non ischemic tissue. This data supports the notion that Sertad4 has a key role in cardiac remodeling in response to ischemic injury. Future directions include investigating Sertad4 in cardiac inflammation, investigation Sertad4 molecular mechanisms, and cell-type specific disruption of Sertad4.

Keywords: cardiac

12. Insights into P. aeruginosa phage infection using RB-TnSeq

Marissa R. Gittrich (Department of Microbiology, The Ohio State University, Columbus, OH, USA.), Courtney Sanderson (Department of Microbiology, The Ohio State University, Columbus, OH, USA.), Jonathan Leopold (Department of Microbiology, The Ohio State University, Columbus, OH, USA.), Cara Noel (Department of Microbiology, The Ohio State University, Columbus, OH, USA.), Vivek K. Mutalik (Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America)

Abstract:
Pseudomonas aeruginosa causes an estimated 32,600 multi-drug resistant infections in hospitalized patients yearly and is categorized as a serious threat by the CDC due to intrinsic and acquired resistance to many current antibiotics. Due to this resistance, there have been studies into other antimicrobials, including phages viruses that infect bacteria, to treat bacterial infections. For a phage to successfully infect a host, the phage must enter the host and modify the host metabolism. Due to the phage reliance on the host, the bacteria can rapidly develop phage resistance by mutation of host genes necessary for the phage. There have been many studies testing the therapeutic capabilities of various Pseudomonas phages, but little is known about the host genes required by many Pseudomonas phages beyond receptor genes. To rapidly and comprehensively identify host factors involved in phage infection, we used random barcode transposon site sequencing (RB-TnSeq) to generate a 3000 mutant genome-wide loss-of-function transposon library in Pseudomonas aeruginosa PAO1. We challenged this library with two well-characterized Pseudomonas phages, phiKZ and LUZ19, at MOIs ranging from 10 to 0.01. Using RB-TnSeq, we confirmed receptors and a gene that increased mucoid production previously shown to confer resistance to the phages as well as additional host factors formerly not known to be involved in phage infection. This library will allow us to expand our knowledge on the dynamics of phage-host interactions that can then be applied to guide phage therapeutics.

Keywords: Phages, P aeruginosa, RB-TnSeq

13. Title not available online - please see the booklet.

Volha Golubeva (Medical Scientist Training Program and Biomedical Sciences Graduate Program, Center for RNA Biology, OSU, Columbus OH), Lisa Dorn (Medical Scientist Training Program, Center for RNA Biology, OSU, Columbus OH), Federica Accornero (Center for RNA Biology, Davis Heart and Lung Research Institute and Department of Physiology and Cell Biology, OSU, Columbus OH)

Abstract not available online - please check the booklet.

14. Self-Assembly of GalNAc-Cyanuric Acid and Melamine Derivatives as a Nucleic Acid Delivery System

Maricarmen Gonzalez (Department of Chemistry and Biochemistry), Dr. Shekaraiah Devari (Department of Chemistry and Biochemistry), Dr. Dennis Bong (Department of Chemistry and Biochemistry)

Abstract:
We have designed and synthesized GalNAc-coated nanoparticles that carry DNA plasmid cargo for hepatocyte-targeted delivery and directed protein expression. Nanoparticle assembly is driven by cyanuric acid (CA) and melamine (M) recognition. Hydrocarbon chains terminated with tertiary amino groups at one end and melamine at the other were synthesized to yield carbon chains of 4-8 in length. Similarly, hydrocarbon chains of C4-C8 in length were synthesized with N-Acetylgalactosamine (GalNAc) at one end and cyanuric acid at the other. GalNAc is bound by the asialoglycoprotein receptor found on the surface of liver cells and is well-established as a ligand for liver-targeted delivery. A bromide-terminated GalNAc chain was used to alkylate a dibenzylated cyanuric acid derivative that enables monoalkylation. The cationic melamine lipids electrostatically coat the DNA backbone to form a loose particle assembly (~800 nm), which is condensed by treatment with the CA-GalNAc lipid, resulting in ~200 nm particles with low polydispersity. Consequently, the self-assembly results in a nucleic acid enclosed GalNAc complex in the form of DNA-M-CA-GalNAc, which may then be able to enter cells through receptor mediated endocytosis. Current studies are focused on the evaluation of plasmid delivery as judged by expression of RFP, as well as optimization of delivery as a function of hydrocarbon chain length.

Keywords:

15. Remodeling of the m6A landscape in the heart reveals few conserved post-transcriptional events underlying cardiomyocyte hypertrophy

Scott Hinger (Dept. of Physiology and Cell Biology, OSU), Jiangbo Wei (Dept. of Chemistry, Dept. of Biochemistry & Molecular Biolgy, Institute for Biophysical Dynamics, HHMI, The University of Chicago, IL), Lisa Dorn, Bryan Whitson, Paul M.L. Janssen, Chuan He, Federica Accornero

Abstract:
Regulation of gene expression plays a fundamental role in cardiac stress-responses.
Modification of coding transcripts by adenosine methylation (m6A) has recently emerged as a critical post-transcriptional mechanism underlying heart disease. Thousands of mammalian mRNAs are known to be m6A-modified, suggesting that remodeling of the m6A landscape may play an important role in cardiac pathophysiology. Here we found an increase in m6A content in human heart failure samples. We then adopted genome-wide analysis to define all m6A-regulated sites in human failing compared to non-failing hearts and identified targeted transcripts involved in histone modification as enriched in heart failure. Further, we compared all m6A sites regulated in human hearts with the ones occurring in isolated rat hypertrophic cardiomyocytes to define cardiomyocyte-specific m6A events conserved across species. Our results identified 38 shared transcripts targeted by m6A during stress conditions, and 11 events that are unique to unstressed cardiomyocytes. Of these, further evaluation of select mRNA and protein abundances demonstrates the potential impact of m6A on post-transcriptional regulation of gene expression in the heart.

Keywords:

16. Reverse (3'-5') RNA polymerases: Applications beyond tRNAHis maturation

Malithi I. Jayasinghe (The Ohio State University), Krisna J. Patel (The Ohio State University)

Abstract:
Redacted abstract

References:
[1] Gu, W., Jackman, J. E., Lohan, A. J., Gray, M. W., and Phizicky, E. M. (2003) tRNA[His] maturation: An essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNA[His], Genes & Development 17, 2889-2901.
[2] Jackman, J. E., and Phizicky, E. M. (2006) tRNA^His guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G_-1 addition, Proceedings of the National Academy of Sciences of the United States of America 103, 8640-8645.
[3] Rao, B. S., Maris, E. L., and Jackman, J. E. (2011) tRNA 5'-end repair activities of tRNA(His) guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea, Nucleic Acids Research 39, 1833-1842.
[4] Long, Y. C., Abad, M. G., Olson, E. D., Carrillo, E. Y., and Jackman, J. E. (2016) Identification of distinct biological functions for four 3'-5' RNA polymerases, Nucleic Acids Research 44, 8395-8406

Keywords: Reverse (3-5) RNA polymerase, 5-end labeling, Applications

17. Understanding the biological significance of multiple zebrafish Trm10 homologs

Ben Jepson (MCDB)

Abstract:
The tRNA m1R9 methyltransferase (Trm10) family enzymes methylate the N-1 atom of purine residues at the ninth position of a subset of tRNAs. Trm10 enzymes are ubiquitous throughout Eukarya and Archaea, with eukaryotes encoding up to three homologs of Trm10. Humans, for example, encode the three homologs TRMT10A, TRMT10B and TRMT10C. Previously, we showed that human TRMT10A and TRMT10B have distinct, non-redundant biochemical activities. Human TRMT10A, like yeast Trm10, catalyzes m1G9 formation on multiple tRNA species whereas TRMT10B forms m1A9 specifically on tRNAAsp. To further probe the significance of these distinct enzyme activities and test their generality across multiple vertebrate models, we are using Danio rerio (zebrafish), which also encodes two cytosolic homologs, Trmt10a and Trmt10b. Like human TRMT10A, zebrafish Trmt10a rescues the trm10∆ phenotype in yeast and methylates yeast tRNAs in vivo. Conversely, neither zebrafish Trmt10b nor human TRMT10B are capable of rescuing the phenotype or methylating yeast substrates in vivo. Intriguingly, however, we discovered that human and zebrafish TRMT10B homologs differ significantly in their in vitro activities. In contrast to the human enzymes, zebrafish Trmt10a and Trmt10b do not show the same pattern of unique in vitro substrate specificities, although they do modify some tRNAs with different catalytic rates. These studies reveal that determinants of substrate specificity of individual Trm10 homologs are complex, and often can not be fully recapitulated by in vitro analysis. In order to fully elucidate the biological functions of these enzymes, we are using mutant zebrafish lines to analyze the specific roles of each enzyme in tRNA modification in vivo.

Keywords: tRNA modification, Trm10, m1R9

18. Disease-associated point mutations in a bifunctional aminoacyl-tRNA synthetase elicit the integrated stress response

Danni Jin (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210), Nathan Kudlapur (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210), Ronald Wek (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, 46202), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210)

Abstract:
Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that catalyze the charging of specific amino acids onto cognate tRNAs. Human glutamyl-prolyl-tRNA synthetase (EPRS) is a bifunctional AARS with two catalytic domains (ERS and PRS), an N-terminal GST domain and a linker region connecting the catalytic cores. Previous work revealed a role for EPRS in the cellular integrated stress response (ISR), wherein the translation of eprs and a few other genes are selectively upregulated under stress conditions¹. In this work, we investigate the effects of two EPRS point mutations recently linked to human diseases: P14R in the GST domain and E205G in the ERS catalytic domain. Patients possessing P14R/E205G compound heterozygous mutations suffer from diabetes and bone disease. WT ERS constructs were first purified and shown to display robust tRNA binding and catalytic activity in vitro. When tested individually, both point mutants displayed similar tRNA binding affinity as WT but were defective in amino acid activation. The E205G mutation also resulted in a significant loss of aminoacylation activity. The P14R mutant displayed near-WT levels of overall aminoacylation efficiency, but a 10-fold reduced k_cat and similarly reduced K_M, suggesting an altered catalytic mechanism. When expressed in HEK293T cells, EPRS P14R displayed significant proteolysis in the linker region, suggesting a long-range conformational effect of the point mutation. This conformational effect was confirmed by limited-protease digestion analyses in vitro. Preliminary studies also showed increased expression of ISR factors ATF4 and Chop in patient fibroblasts. We propose that the aminoacylation defects and conformational changes in EPRS mutants sensitize patient cells to stress, triggering an increased ISR that diminishes cell viability. This work has important implications for understanding AARS-associated human disease mechanisms and development of new therapeutics.

References:
1. Young, S. K. & Wek, R. C. Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response. J. Biol. Chem. 291, 16927–16935 (2016).

Keywords: Aminoacyl-tRNA synthetase, EPRS , Integrated stress response

19. Oxidative Stress Regulates E. coli Alanyl-tRNA Synthetase Activity

Arundhati Kavoor (MCDB), Dr. Paul Kelly (MCDB)

Abstract:
Accuracy during translation ensures faithful conversion of the genetic code. Aminoacyl tRNA-synthetases (aaRS) are essential enzymes that attach an amino acid to a cognate tRNA. The aaRS first activates an amino acid in an ATP-dependent manner to form an aminoacyl adenylate. Next, the aaRS attaches the aminoacyl adenylate to the terminal adenosine residue of the cognate tRNA. The aminoacylated tRNA is transported to the ribosome by the elongation factor Tu and participates in translation. AaRSs play a major role in maintaining translational accuracy by attaching the correct amino acids to their cognate tRNAs. Although the active site pockets of many aaRSs are able to discriminate against the wrong amino acid, the shared chemicophysical structure of some amino acids can lead to mis-activated amino acids. To prevent mistranslation, some aaRS have an additional editing domain capable of hydrolyzing mis-activated amino acids. Recent observations have shown that during oxidative stress, reactive oxygen species (ROS) can oxidize aaRSs resulting in either a positive or negative effect on aminoacylation fidelity. As defects in alanyl-tRNA synthetase (AlaRS) fidelity have been shown to cause defects from bacteria to mice, we wanted to determine the effect of oxidation on AlaRS activity. Here, we show that oxidation of AlaRS does not affect cognate activation of alanine or the editing activity against its non-cognate substrates, serine and glycine. However, oxidative stress increased the rate of tRNA^Ala aminoacylation threefold, accompanied by the oxidation of four residues in AlaRS. Current work is focused on better understanding the regulation of AlaRS and other aaRSs that are critical in maintaining translational accuracy.

References:
1. Ibba M, Söll D (2000) Aminoacyl-tRNA synthesis. Annu Rev Biochem. 69:617–650.
2. Ling J, Reynolds N, Ibba M (2009) Aminoacyl-tRNA synthesis and translational quality control. Annu Rev Microbiol. 63:61–78.
3. Ling J, Söll D (2010) Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site. Proc Natl Acad Sci USA. 107:4028–4033.
4. Steiner, R. E., Kyle, A. M., Ibba, M. (2019). Oxidation of phenylalanyl-tRNA synthetase positively regulates translational quality control. Proceedings of the National Academy of Sciences. 116(20), 10058-10063.
5. Kelly P, et al. (2019) Alanyl-tRNA synthetase quality control prevents global dysregulation of the Escherichia coli proteome. mBio. 10:e02921-19.

Keywords: tRNA, Aminoacyl-tRNA synthetase

20. Determining the Role of Gasdermin C in Intestinal Health

Andrea R. Keller (Department of Biological Chemistry and Pharmacology, Ohio State University), Maria M. Mihaylova (Department of Biological Chemistry and Pharmacology, Ohio State University)

Abstract:
Redacted abstract

Keywords: intestinal health, aging

21. Role of HIV-1 5′UTR RNA structure and conformational dynamics in genome packaging

Jonathan P. Kitzrow (Department of Chemistry and Biochemistry, Center for RNA Biology and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Shuohui Lui (Department of Chemistry and Biochemistry, Center for RNA Biology and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Shiqin Miao (Department of Chemistry and Biochemistry, Center for RNA Biology and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Kevin Jamison (Department of Physics, The Ohio State University, Columbus, OH 43210), Dennis Bong (Department of Chemistry and Biochemistry, Center for RNA Biology and Center for Retroviral Research, The Ohio State University), Michael Poirier (Department of Physics, The Ohio State University, Columbus, OH 43210)

Abstract:
The highly conserved 5′UTR of HIV-1 genomic RNA (gRNA) is central to the regulation of virus replication. Biochemical and NMR experiments support a model in which the 5′UTR can adopt at least two mutually exclusive conformational states. In one state, the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed and the genome is competent for dimerization and packaging into assembling virions. According to this model the conformation of the 5′UTR determines the fate of the genome. The number of 5′ guanosines has also been implicated in the localization of HIV-1 gRNA; transcripts with three 5′ guanosines (3G) are abundant in the cytoplasm, whereas transcripts with a single 5′ guanosine (1G) are preferentially packaged into budding virions.[1, 2] We previously characterized a 238-nt region of the 5′UTR lacking the 5′-TAR/polyA domain using both ensemble and single molecule FRET assays.[3] This study confirmed that the 5′UTR was conformationally dynamic and revealed how the dynamics were modulated by host factor and viral protein binding. The impact of the number of 5′ guanosines on the ensemble of RNA conformations and their dynamics is unknown. Here, we investigate the full 5′UTR using a bifacial peptide nucleic acid strategy to position internal FRET dyes. Native in-gel FRET and RNA structure-probing studies revealed that 1G and 3G 5′UTRs adopt a different ensemble of RNA conformations. Single molecule studies to probe differences in RNA dynamics are underway. These results have implications for the development of RNA-targeted therapeutics that may interfere with selective packaging of HIV-1 gRNA.

References:

1. Masuda, T., et al., Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA. Sci Rep, 2015. 5: p. 17680.
2. Kharytonchyk, S., et al., Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome. Proc Natl Acad Sci U S A, 2016. 113(47): p. 13378-13383.
3. Brigham, B.S., et al., Intrinsic conformational dynamics of the HIV-1 genomic RNA 5'UTR. Proc Natl Acad Sci U S A, 2019. 116(21): p. 10372-10381.

Keywords: RNA Structure, FRET, HIV

22. Identifying inhibitors of the FraB deglycase as therapeutics for Salmonella-mediated gastroenteritis

Jamison Law (Department of Chemistry and Biochemistry, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Non-typhoidal Salmonella enterica (Salmonella) annually causes significant morbidity/mortality (154 million/121,000 worldwide), healthcare-related costs ($12 billion, USA), and meat spoilage (13 million pounds recalled in 2018, USA) ^1-3. No vaccines are available and extant antibiotics are disfavored because they exacerbate the infection ^4-6. Few drug targets exist for Salmonella due to its versatility in exploiting redundancy/overlap in metabolic pathways ^7-8. However, fructose-asparagine (F-Asn), an Amadori compound, was recently discovered as a nutrient utilized by Salmonella due to the activity of five proteins encoded in the fraRBDAE operon ⁹. Surprisingly, ΔfraB mutants could grow in glucose but not in glucose + F-Asn. This growth inhibition is due to the toxic buildup of 6-phosphofructose-aspartate, the substrate of FraB ¹⁰. The absence of FraB in mammals and most members of the human microbiota makes FraB an attractive anti-Salmonella target ¹¹. Therefore, to identify potential inhibitors of FraB, we sought to leverage the ~500,000-compound library from the ICCB-Longwood Screening Facility at Harvard. To this end, cell-based (Ahmer, OSU) and biochemical assays were developed to enable high-throughput screening of compounds capable of inhibiting Salmonella growth and FraB activity, respectively. Of 200,000 compounds that we tested, ~150 were confirmed as bona fide inhibitors of FraB with 18 hits overlapping between both assays. Ongoing biochemical studies with these compounds seek to characterize their K_i values and mode of inhibition. Further medicinal chemistry-based optimization of these hits, however, requires a high-resolution structure of FraB (± substrate/inhibitor). To overcome poor outcomes in our longstanding crystallization efforts, we recently used the Surface Entropy Reduction approach to gain insights into mutations that would promote crystal formation. One such mutant, FraB K275A-E276A, yielded robust crystals that diffracted to 2.0 Å and a structure! This key advance bolsters co-crystallization prospects and helps forge an exciting path towards Salmonella-specific therapeutics.

References:
1. Sabag-Daigle et al. Sci Rep. 2016;6:28117.
2. Kirk MD et al. PLoS Med. 2015;12(12):e1001921.
3. Scharff. J Food Prot. 2012;75(1):123-131.
4. Summary of Recall Cases in Calendar Year 2018. In: United States Department of Agriculture Food Safety and Inspection Services; 2019.
5. Gopinath et al. Proc Natl Acad Sci USA. 2014;111(44):15780-15785.
6. Diard et al. Curr Biol. 2014;24(17):2000-2005.
7. Wiström et al. Ann Intern Med. 1992;117(3):202-208.
8. Becker et al. Nature. 2006;440(7082):303-307.
9. Steeb B et al. PLoS Pathog. 2013;9(4):e1003301.
10. Ali et al. PLoS Pathog. 2014;10(6):e1004209.
11. Sabag-Daigle et al. Appl Environ Microbiol. 2018;84(5).

Keywords: Salmonella, crystallography, therapeutic

23. Modifying Duchenne muscular dystrophy severity in zebrafish by regulating Tgfβ

Geremy T. Lerma (Ohio State University), Natalie M. Aloi (Ohio State University), Joseph C. Beljan (Ohio State University), Jared C. Talbot (Ohio State University)

Abstract:
Duchenne Muscular Dystrophy (DMD) is a dystrophinopathy characterized by progressive muscle degeneration and weakness that affects approximately 1 in 3500 live male births. Despite development of promising therapeutics, there is currently no cure. Transforming Growth Factor β (TGFβ) is a multifunctional cytokine that is aberrantly activated in muscular dystrophies, including DMD. TGFβ exacerbates disease progression by promoting inflammation and fibrosis in muscle with chronic injury. Genome-wide association studies identified variant alleles of TGFβ regulatory proteins which corresponded with age to loss of ambulation in DMD patients. These proteins are Latent TGFβ Binding Protein 4 (LTBP4), a protein responsible for TGFβ secretion and deposition into the extracellular matrix (ECM), and Thrombospondin 1 (THBS1), a protein responsible for activation of TGFβ in the ECM. Protective LTBP4 and THBS1 alleles result in reduced TGFβ signaling while risk alleles cause the opposite. We are utilizing chemical and genetic approaches in the zebrafish DMD (dmd) disease model to investigate how TGFβ (Tgfβ) signaling influences the dmd phenotype. Transient inhibition of Tgfβ receptors before significant muscle degeneration begins results in significant and sustained rescue of dmd mutant muscle integrity and ultrastructure at later stages when untreated controls show severe damage. We have generated and are characterizing loss-of-function alleles of ltbp4, thbs1a, and thbs1b (zebrafish have two copies of THBS1) to mimic protective decreased expression alleles described in humans. Double ltbp4;dmd and thbs1b;dmd mutants show partial rescue of dmd mutant muscle integrity and increased survivorship compared to dmd single mutant siblings. A long-term goal of our work is to further define the cell-specific regulation mechanisms that control TGFβ signaling through LTBP4 and THBS1 to potentially identify novel therapeutic targets.

Keywords: Zebrafish, Dystrophy, Tgf-beta

24. Context-sensitive cleavage of folded DNAs by loop-targeting bPNAs

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

Abstract:
Herein, we demonstrate context-dependent molecular recognition of DNA by synthetic bPNA iron and copper complexes, using oxidative backbone cleavage as a chemical readout for binding. Oligoethylenimine bPNAs displaying iron·EDTA or copper·phenanthroline sites were found to be efficient chemical nucleases for designed and native structured DNAs with T-rich single-stranded domains. Cleavage reactivity depends strongly on structural context, as strikingly demonstrated with DNA substrates of the form (GGGTTA)_n. This repeat sequence from the human telomere is known to switch between parallel and antiparallel G-quadruplex (G4) topologies with a change from potassium to sodium buffer: notably, bPNA−copper complexes efficiently cleave long repeat sequences into ∼22-nucleotide portions in sodium, but not potassium, buffer. We hypothesize preferential cleavage of the antiparallel topology (Na⁺) over the parallel topology (K⁺) due to the greater accessibility of the TTA loop to bPNA in the antiparallel (Na⁺) form. Similar ion-sensitive telomere shortening upon treatment with bPNA nucleases can be observed in both isolated and intracellular DNA from PC3 cells by quantitative polymerase chain reaction. Live cell treatment was accompanied by accelerated cellular senescence, as expected for significant telomere shortening. Taken together, the loop-targeting approach of bPNA chemical nucleases complements prior intercalation strategies targeting duplex and quadruplex DNA. Structurally sensitive loop targeting enables discrimination between similar target sequences, thus expanding bPNA targeting beyond simple oligo-T sequences. In addition, bPNA nucleases are cell membrane permeable and therefore may be used to target native intracellular substrates.

Keywords: context-sensitive, chemical nuclease, telomere shortening

25. Occlusion of the Anti-Shine-Dalgarno in the Bacteroidetes ribosome

Zakkary A. McNutt (Department of Microbiology & Center for RNA Biology & The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA), Bappaditya Roy, Bethany L. Boleratz, Dean E. Watkins, Kurt Fredrick (Department of Microbiology & Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Vikash Jha, Dushyant Jahagirdar, Kaustuv Basu, Joaquin Ortega (Department of Anatomy and Cell Biology & Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada), Elan A. Shatoff, Ralf Bundschuh (Department of Physics & Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA)

Abstract:
Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the conserved anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein, bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. In those Flavobacteriia that have an alternative ASD, the fully complementary sequence lies upstream of the bS21 gene in all cases, indicative of natural covariation. In the other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.

Keywords: Translation , Shine Dalgarno, Bacteroidetes

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

Tyler Mendes (Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, USA, Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA ), Norman R Groves (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA, Center for Applied Plant Sciences, The Ohio State University, Columbus, OH, USA), Iris Meier (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA, Center for Applied Plant Sciences, The Ohio State University, Columbus, OH, USA, Center for RNA Biology, The Ohio State )

Abstract:
Fertilization is a key component of plant reproduction that is necessary for agriculture. While the basic process of fertilization has been understood for centuries, the mechanism underlying how plant sperm is trafficked to the egg remains poorly understood. To achieve fertilization, the growing pollen tube transports the sperm cell (SCs) to ovules. The SCs are physically connected via a cytoplasmic projection from the lead sperm cell to the vegetative nucleus (VN); this combined complex is referred to as the Male Germ Unit (MGU). Previous research has shown that Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes at the VN nuclear envelope facilitate MGU movement. The LINC complex spans the inner and outer nuclear membranes and connects the nucleoplasm to the cytoskeleton. Null mutants in genes for two plant LINC complex subunits, WIP and WIT, result in a VN movement defect, wherein the SCs lead while the VN trails behind. This defect correlates with a defect in pollen tube burst and, in turn, a loss of seed set. We hypothesize that WIT and WIP act as adapter proteins between the VN envelope and unknown cytoskeletal motor proteins. In plants, there are two kinds of cytoskeletal motor proteins: Kinesins and Myosins. While Myosins have been well-studied in pollen-tube tip growth, the role of Kinesins in pollen tubes has not been established. To determine which motors are involved in VN movement, we have screened insertional mutants in 17 pollen-expressed Kinesins (PEK1-17) for fertility defects. Kinesin-14s represent a significant portion of the PEKs, with 8 of the 17 PEKs being Kinesin-14s. Two of these Kinesin-14s, PEK3 and PEK9, present mild fertility defects in insertional mutants. PEK3 and PEK9 are likely the result of a gene duplication event, leading to the possibility that a more severe fertility defect would be observed in a double mutant. Insertional mutants in a Kinesin-4, PEK14, displayed significant reductions in seed set, on the order of the reduction observed in wit mutants, indicating it may play a role in MGU movement. Future experiments will establish if these genes are associated with the VN envelope, physically interact with WIP and/or WIT, and are required for pollen tube burst.

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.
4. Cai G, Cresti M. (2009). “Organelle motility in the pollen tube: a tale of 20 years.” Journal of Experimental Botany. 60(2):495-508.
5. Heslop-Harrison J, et al. (1988) “Cytoskeletal elements, cell shaping and movement in the angiosperm pollen-tube.” Journal of Cell Science. 91:49-60.

Keywords: Male Germ Unit, Pollen Tube, Kinesin

27. Bifacial PNAs destabilize MALAT1 by 3′ poly(A) tail displacement from the U-rich Internal Loop

Shiqin Miao (Department of Chemistry and Biochemistry), Debmalya Bhunia (Department of Chemistry and Biochemistry), Shekar Devari (Department of Chemistry and Biochemistry), Yufeng Liang (Department of Chemistry and Biochemistry), Oliver Munyaradzi (Department of Chemistry and Biochemistry), Sarah Rundell (Department of Chemistry and Biochemistry)

Abstract:
We report herein a new class of synthetic reagents for targeting the element for nuclear expression (ENE) in MALAT1, a long noncoding RNA upregulated in many cancers. The cis-acting ENE contains a U-rich internal loop (URIL) that forms an 11 base UAU-rich triplex stem with the truncated 3′ poly-A terminus of MALAT1, protecting the terminus from exonuclease digestion and greatly extending transcript lifetime. Bifacial peptide nucleic acids (bPNAs) similarly bind URILs via base triple formation between two uracil bases and a synthetic base, melamine. We synthesized a set of low molecular weight bPNAs comprised of α-linked peptide, isodipeptide and diketopiperazine backbones and evaluated their ENE binding efficacy in vitro via oligo-A strand displacement and consequent exonuclease sensitivity. Degradation was greatly enhanced by bPNA treatment in the presence of exonucleases, with ENE halflife plunging to 6 min from >24h. RNA digestion kinetics could clearly distinguish between bPNAs with similar URIL affinity, highlighting the utility of functional assays for evaluating synthetic RNA binders. In vitro activity was mirrored by a 50% knockdown of MALAT1 expression in pancreatic cancer (PANC-1) cells upon treatment with bPNAs, consistent with intracellular digestion triggered by a similar ENE A-tail displacement mechanism. Pulldown from PANC-1 total RNA with biotinylated bPNA enriched MALAT1 >4500X, supportive of bPNA-URIL selectivity. Together, these experiments establish the feasibility of native transcript targeting by bPNA in both in vitro and intracellular contexts. Reagents such as bPNAs may be useful tools for investigation of transcripts stabilized by cis-acting poly(A) binding RNA elements.

Keywords: MALAT1, PNA, RNA targeting

28. Helical bPNA targeting U-rich RNA/T-rich DNA

Oliver Munyaradzi (Chemistry and Biochemistry, Ohio State University), Dennis Bong (Chemistry and Biochemistry, Ohio State University)

Abstract:
We present helical bifacial peptide nucleic acids (bPNA) that recognize and bind U-rich RNA or T-rich DNA with high affinity and specificity. bPNA is an α-PNA containing lysine residues (K^2M) functionalized with two melamines (M) per lysine. Despite previous work from our group demonstrating the ability of bPNA to bind to 6x6 symmetric internal loops containing U/T through formation of U-M-U or T-M-T base triples, enhanced binding affinity still remains to be explored. We hypothesized that lowering the significant entropic cost of triplex formation by pre-organizing the peptide backbone may improve binding affinity. Initial structured bPNAs included α-helical I: (A₃K^2M)₃ and the PP-II helix II: (PPK^2M)₃. Triplexes containing pre-organized helix I or II and T₆C₄T₆ ssDNA had greater thermal stability (T_m = 60 ^oC, 61 ^oC) than those containing unstructured bPNA – (βAK^2M)₃ (T_m = 57 ^oC). Although triplexes containing RNA were less stable, those containing helical bPNA were still more stable (T_m = 30 ^oC for I and II with U₆C₄U₆ vs. 25 ^oC for unstructured (βAK^2M)₃.
To reduce the binding footprint to 4x4 symmetric loops, we sought improved binding properties by optimizing display of melamine along the helix face. We synthesized a series of single-turn stapled helical bPNAs containing two K^2M residues with varied spacing between them for optimal placement of melamine along the helix face. Triplexes containing stapled K3Z3 (K^2MAZAK^2MAZ; Z=azido-Ala) and T₄C₄T₄ ssDNA were more stable (T_m = 36.5 ^oC) than those containing unstapled K3Z3 (T_m = 35.3 ^oC). The effect of melamine placement along the helix face on the thermodynamics of binding were evaluated by van’t Hoff analysis of melting curves. Binding studies with RNA are underway, while future work will explore inclusion of other nucleobases to extend this work beyond U-rich RNA or T-rich DNA.

Keywords: RNA-binding peptide, stapled, U-rich

29. Negative cooperativity in nucleotide binding to a AAA+ ATPase hexamer

Rodrigo Muzquiz (Presenting Author), Kelly Karch (Department of Chemistry and Biochemistry, OSU), Vycki Wysocki (Department of Chemistry and Biochemistry, OSU), Mark Foster (Department of Chemistry and Biochemistry, OSU )

Abstract:
Hexameric helicases are motor proteins that play important physiological roles in cellular replication, chromosome packaging, and transcription termination3. The Escherichia coli (E.coli) Rho factor is an essential motor protein involved in regulating protein expression1. This ring-shaped protein binds nascent mRNA in its central pore and fuels its translocation by ATP hydrolysis. This allows it to move down the substrate to contact the RNA polymerase, thereby terminating transcription1,3. The process of ATP and RNA binding lead to a conformational change of an open ring (lock-washer) to a closed ring. Currently, it is not understood how ATP and RNA binding are coupled on a single subunit and whether the ATP or RNA bound to one subunit affects the affinity for these ligands on neighboring subunits. We have implemented native mass spectrometry (nMS) in tandem with surface induced dissociation (SID) to probe the allosteric and structural properties of Rho. nMS allows us to see the populations of each ATP bound state of Rho rather than just the solution averages. ATP titration experiments of apo-Rho have shown that there is negative cooperativity in binding. These experiments have also shown that Rho exists in variable oligomeric states (dimer, trimer, etc.) during electrospray process that are not normally observed in solution under these same conditions. nMS experiments of other AAA+ ATPases have observed similar features with variability in spraying under the same conditions. From this we can fit the data to a statistical thermodynamic model to quantify the cooperative coupling free energies between subunits. Our next steps we plan to optimize spraying conditions and nucleotide analogs to reduce the variability in oligomeric conformations observed so that the data fitting can yield more accurate parameters. We also want to look at how the presence of RNA shifts the populations of ATP bound states of Rho. By using SID we can also compare the binding energies of the subunits for the apo and holo state of Rho, where different oligomeric conformations will be present at higher dissociation energies.

References:
1. Mitra P.; Ghosh G.; Hafeezunnisa M.; Sen R. Rho protein: roles and mechanisms. Annu Rev Microbiol. 2017, 8 (71) 687. DOI: 10.1146/annurev-micro-030117-020432
2. Yu Y.; Liu H.; Yu Z.; Witkowska H.E.; Cheng Y. Stoichiometry of nucleotide binding to proteasome AAA+ ATPase hexamer established by native mass spectrometry. Mol Cell Proteomics. 2020, 19 (12): 1997. DOI: 10.1074/mcp.RA120.002067
3. Patel, S. S., & Picha, K. M. (2000). Structure and Function of Hexameric Helicases. Annual Review of Biochemistry, 69(1), 651–697. https://doi.org/10.1146/annurev.biochem.69.1.651

Keywords: native mass spectrometry, cooperativity, transcription termination

30. Modeling Ultrasound Stimuli in Simulations of Ion Channels for Sonogenetics

Brandon Neel (Department of Chemistry & Biochemistry, OSBP), Elakkiya Tamilselvan (Biophysics), Harsha Mandayam Bharathi (Department of Chemistry & Biochemistry), Marcos Sotomayor (Department of Chemistry & Biochemistry)

Abstract:
Optical activation of neurons using light-sensitive ion channels (optogenetics) has provided invaluable and unprecedented insight into neuronal circuits and brain function. However, optogenetics is limited by the depth that light can reach through brain tissue. An alternative methodology, termed sonogenetics, uses low-intensity ultrasound that can propagate through brain tissue to activate worm TRPN or human TRPA1 ion channel proteins transfected into selected neurons. Intriguingly, both proteins have a large number of ankyrin repeats that assemble in long solenoid like structures that might provide an elastic attachment to the cytoskeleton. While the molecular mechanisms by which ultrasound activates these ion channels are unknown, cholesterol and ankyrin repeats have been shown to be required for ultrasound-mediated activity of TRPA1. Here we present modeling efforts to simulate TRPN and TRPA1 structures in a hydrated membrane bilayer in the presence and absence of ultrasound stimuli. We have created all-atom models of TRPN and TRPA1 and equilibrated these systems, with and without cholesterol. In addition, we have developed an approach that allows us to incorporate ultrasound in the simulations as a global oscillatory change in system pressure. Simulations of TRPA1 using this approach, with and without constraints on ankyrin repeats to mimic cytoskeletal attachment, are ongoing. We expect that these simulations will provide insight into ultrasound effects on ion channels and may assist in engineering protein sensors for sonogenetics.

Keywords: Channel protein, Simulations, Sonogenetics

31. The role of RNA polymerase IV in effecting heritable regulatory changes

Benjamin P. Oakes (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, Ohio 43210), Jay B. Hollick (Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210; Centers for RNA Biology and Applied Plant Sciences, The Ohio State University, Columbus, Ohio 43210)

Abstract:
Paramutation is a behavior in which one parental allele at a given locus facilitates a meiotically heritable change at the other¹. This behavior occurs at specific alleles of multiple maize loci encoding transcriptional activators of flavonoid biosynthesis including red1 (r1), booster1 (b1), purple plant1 (pl1), and pericarp color1 (p1)². The Pl1-Rhoades (Pl1-Rh) allele can exist in a highly expressed reference state (Pl-Rh) or an epigenetically repressed paramutant state (denoted Pl′ )³. Pl1-Rh alleles in the Pl′ state often revert to Pl-Rh in plants homozygous for a null allele (rmr6-1) of a gene encoding the RNA polymerase (RNAP) IV largest subunit⁴ indicating that RNAP IV maintains the heritable information specifying Pl1-Rh paramutation. Because RNAP IV both sources 24 nucleotide RNAs⁵ and controls the heritable regulatory status of Pl1-Rh⁴, we hypothesize that RNAP IV, and potentially small RNAs (sRNAs) in general, condition the inheritance of genome-wide regulatory information. To test this idea, seedling RNA-seq and sRNA-seq profiles of heterozygous BC₅ progeny from sibling rmr6-1 mutant and heterozygous fathers were compared to identify heritable RNAP IV-dependent effects. RNA abundances of 140 genes were either significantly enriched or depleted in the progeny of mutant rmr6-1 fathers as compared to progeny of heterozygous fathers. Additionally, 962 sRNA clusters were significantly enriched or depleted in an identical comparison. These differences point to other alleles, like Pl1-Rh, whose dysregulation in the absence of RNAP IV might persist through meiosis. Future studies aim to determine the role of environmental factors in effecting heritable changes at Pl1-Rh and other alleles as Bernard Mikula showed that the extent of heritable changes brought about by paramutations occurring at r1 is influenced by the environment during early development⁶.

References:
1. Brink Genetics (1956) | 2. Hollick Nat Rev Gen (2017) | 3. Hollick et al. Genetics (1995) | 4. Hollick et al. Genetics (2005) | 5. Erhard et al. Science (2009) | 6. Mikula Genetics (1995)

Keywords: Epigenetics, Paramutation, Inheritance

32. Epigenetic regulation of nuclear lamina-associated heterochromatin domains by HAT1 and the acetylation of newly synthesized histones

Liudmila Popova (MCBD), Prabakaran Nagarajan, Callie M. Lovejoy (Department of Biological Chemistry and Pharmacology, The Ohio State University), Benjamin Sunkel (Nationwide Childrens Research Institute), Michael A. Freitas (Department of Cancer Biology and Genetics), Benjamin Stanton (Nationwide Childrens Research Institute), Mark R. Parthun (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
When eukaryotic cells divide, not all of the information necessary for proper functioning of daughter cells is encoded in the primary DNA sequence of their genomes. Regulatory information, such is chemical modifications to histone proteins, is inherited epigenetically during cell division. In mammals, faithful transmission of epigenetic information is essential for maintenance of cell identity, genome integrity, and control of cell proliferation.

During DNA replication, parental histones are recycled and deposited behind the replication fork and newly synthesized histones are delivered to sites of replication via the pathway of replication-coupled chromatin assembly. Histone Acetyltransferase 1 (Hat1) plays an important role in replication-coupled chromatin assembly by acetylating lysines 5 and 12 in the tail of the newly synthesized histone H4.

Here, we report that HAT1 is a key regulator of the epigenetic inheritance of chromatin states and genome architecture. HAT1 is a global negative regulator of H3K9me2 and K9me3. ATAC-Seq analysis demonstrates that HAT1 regulates accessibility of specific chromosomal domains, termed HAT1-dependent accessibility domains (HADs). HADs range in size from 0.1 to 10 Mb, are AT-rich, gene poor and heterochromatic. HADs correspond to regions of the genome with the highest density of H3 K9 methylation. HADs also display a high degree of overlap with Lamina-Associated Domains suggesting that HAT1 and acetylation of newly synthesized histones regulate the association between heterochromatin and the nuclear lamina following DNA replication. We also propose a model for the epigenetic regulation of histone H3 methylation by HAT1 and newly synthesized histone acetylation and for the role of histone modification dynamics in the association of nascent chromatin with the nuclear lamina.

Keywords: Hat1, Epigenetics, Nuclear Lamina-Heterochromatin Interactions

33. Changes in Translation Drive Heart Growth During Development and Regeneration in Zebrafish.

Anupama Rao (Department of Biological Chemistry and Pharmacology, The Ohio State University), Baken Lyu (Department of Biological Chemistry and Pharmacology, The Ohio State University), Ariel Bazzini (Stowers Institute), Antonio Giraldez (Yale University), Kenneth Poss (Duke University School of Medicine), Joseph Aaron Goldman (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
Heart disease is the leading cause of death in today’s world. Heart disease patients are prone to heart damage and subsequent loss of cardiac tissue. The mammalian heart lacks appreciable capacity to regenerate this lost tissue leaving the patients vulnerable to heart failure. However, zebrafish can regenerate large portions of cardiac tissues lost following injury. Many studies have surveyed epigenetic, genomic and transcriptional changes to yield important insights into cardiac regeneration, identifying many required factors in the process. Here, we show that changes in translation also play an important role in driving cardiac regeneration. Using ribosome profiling we documented the changes in translation efficiency that accompany heart regeneration and identified thousands of transcripts that change in their association with ribosomes. Specific components of the translational machinery may be critical for regulation of translation of these transcripts. There is transcriptional upregulation of translation initiation factors during regeneration, one of which is eif4e1c which is known to bind 5’ methylated caps. eIF4E acts as the limiting factor in the translation initiation, and its expression levels help determine the occurrence of mRNA translation. Here, we identified the translation initiation factor, eif4e1c and its role during zebrafish development and regeneration. Deletion of eif4e1c using CRISPR resulted in lower survival rates to adulthood after normal development although with overall growth deficits. Furthermore, eif4e1c is required for efficient proliferation of cardiomyocytes (CMs) in zebrafish hearts during normal growth and during regeneration. In sum, the findings of this study support a model of cardiac regeneration in which regulation of gene expression through translation is a critical component driving CM proliferation.

Keywords: Cardiac regeneration, Translation, Zebrafish

34. Structure-activity relationship of minimal bPNAs

Sarah Rundell (Chemistry and Biochemistry, Ohio State University), Shekariah Devari (Chemistry and Biochemistry, Ohio State University), Shiqin Miao (Chemistry and Biochemistry, Ohio State University)

Abstract:
Our lab utilizes bPNA+ which are α-PNAs with modified lysines bearing a melamine (M) base mimic (K^2M) that can bind to 4 T/U bases. Our recent publications show the ability of our (SK^2M)₃ bPNA+ to bind to RNA modified to include two genetically-encoded U6 domains. We hypothesized that insights into the drivers of DNA/RNA binding could be obtained by structural variation and thermodynamic characterization of the DNA and RNA binding properties of a library of 4M bPNAs. A 4M bPNA library was prepared by SPPS that sampled different backbone topologies while maintaining overall charge, and subject to Van’t Hoff analysis. All of the derivatives had better binding to DNA (avg T_m = 40^oC) than to RNA (avg T_m = 24°C). Tripeptide, dipeptide and diketopiperazine bPNA scaffolds were studied, with the DKP exhibiting the strongest DNA and RNA binding. Overall, we find that a wide range of variation may be tolerated within the tripeptide scaffold, while significant gains in binding are observed upon cyclic constraint of the bPNA backbone. .

Keywords: bPNA, DNARNA, melamine

35. Probing the structure and function of the Bacillus subtilis thrS T-box riboswitch

Alexander T. Runyon (Microbiology Department, The Ohio State University), Tina M. Henkin (Microbiology Department, The Ohio State University)

Abstract:
Many amino acid-related genes in Gram-positive bacteria are regulated at the level of transcription attenuation by cis-acting RNA structures known as T-box riboswitches. A specific uncharged tRNA that corresponds to the amino acid specificity of the downstream gene is used as the regulatory ligan. ~95% of T-box riboswitches are predicted to fold into the canonical pattern, which includes a set of conserved sequence and structural elements. Current biochemical understanding of T-box riboswitch function derives from noncanonical models that lack one or more of these conserved elements. The Bacillus subtilis thrS riboswitch is located upstream of the threonyl-tRNA synthetase coding sequence and contains all of the major conserved elements, making it a canonical riboswitch. Unlike other canonical T-box RNAs, tRNA-dependent antitermination in vitro has been demonstrated with thrS, which allows comparisons with biochemical data obtained from non-canonical riboswitches. Point mutations have been generated in conserved elements in thrS and in vitro transcription and tRNA binding assays indicate that the conserved elements in thrS are critical for tRNA-dependent antitermination activity, a subset of which are critical for tRNA binding. Structural studies performed with SHAPE confirm the overall structural pattern and mutational disruption of conserved elements. In addition, some elements of thrS have been modified to resemble the corresponding elements of the non-canonical ileS US riboswitch found in Actinobacteria. These mutations reduce thrS function, although not as severely as point mutations that disrupt key elements. Analysis of the thrS system provide a better understanding of the function of canonical T-box RNAs, which represent the major class of these elements found in nature.

References:
Grundy FJ, Henkin TM. 1993. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74:475–482.
Sherwood AV, Frandsen JK, Grundy FJ, Henkin TM. 2018. New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch. Proc Natl Acad Sci USA 115:3894–3899.
Putzer H, Condon C, Brechemier-Baey B, Brito R, Grunberf-Manago M. 2002. Transfer RNA‐mediated antitermination in vitro. Nucleic Acids Res. 30:3026–3033.
Suddala KC, Zhang J. 2019. High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nat Struct Mol Biol 26:1114–1122.
Rollins SM, Grundy FJ, Henkin TM. 1997. Analysis of cis-acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene. Mol Microbiol 25:411–421.

Keywords: tRNA, Riboswitch, T-box

36. Determining how open reading frame length regulates translation re-initiation and mRNA stability

Paul Russell (Cellular, Molecular, and Biochemical Sciences Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Michael G. Kearse (Cellular, Molecular, and Biochemical Sciences Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
Approximately 50% of all mammalian transcripts contain major regulatory elements known as upstream open reading frames (uORFs). Since uORFs exist in the 5’ untranslated region (UTR) and are very commonly encoded by an AUG start codon, they directly compete against the downstream primary open reading frame (ORF) for the scanning ribosome. Previous studies have shown that uORFs do not completely inhibit translation of the functional gene product as ribosomes, after translating uORFs, can re-initiate. Additionally, uORFs have been implicated in regulating the stability of the mRNA. Despite these observations in vitro and in vivo, the factors that drive these regulatory mechanisms remain poorly defined. Here we use re-initiation specific luciferase reporters with different length uORFs in rabbit reticulocyte lysate and in HeLa cells to assess uORF length dependency on re-initiation and mRNA stability. Our data shows re-initiation is more favorable after translating short uORFs and that mRNA stability is decreased with increasing uORF length. These findings suggest that uORF length influences alterations in the composition of ribosomal-bound proteins and that retention of specific initiation factors is essential for mediating re-initiation and mRNA stability. Future studies will need to be performed to determine the identification of the critical factors.

Keywords: translational control, uORF, ribosome

37. Translational control by the fragile X mental retardation protein, FMRP

MaKenzie R. Scarpitti 1,2 (1Department of Biological Chemistry and Pharmacology, 2Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Michael Kearse 1,2 (1Department of Biological Chemistry and Pharmacology, 2Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
Fragile X syndrome is caused by the loss of expression of the fragile X mental retardation protein (FMRP). Loss of functional FMRP is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is an RNA-binding protein that is thought to inhibit translation elongation of its target transcripts, supporting the hypothesis that FXS is caused by an imbalance in protein synthesis at neuronal synapses. In neurons, FMRP must achieve a certain degree of specificity to facilitate healthy neuronal development and maintenance. Biochemical binding assays indicate that specific RNA-binding domains of FMRP have high affinity to structured RNAs, such as pseudoknots and G-quadruplexes. Global analysis of FMRP mRNA binding shows an extreme preference to the coding sequence. These data support the leading model in the field—FMRP is targeted to mRNAs by affinity to structures within mRNA coding regions and stalls elongating ribosomes to inhibit translation elongation. However, work over the years from multiple groups shows that FMRP inhibits in vitro translation of every mRNA tested. Here, we directly test if FMRP uses RNA secondary and tertiary structure to target specific transcripts for translation elongation inhibition. Our preliminary data indicate that FMRP in fact does not require pseudoknots nor G-quadruplexes in coding regions to inhibit translation. Biochemical dissection of FMRP suggests that the last two domains, which are an annotated RGG box and an unstructured and positively charged C-terminal domain, contain the translational inhibition activity. However, each domain alone cannot inhibit translation in vitro. These data point toward a mechanism where the RGG box and C-terminal domains work synergistically to inhibit translation, directly challenging the leading model of FMRP-mediated translation inhibition.

Keywords: RNA binding protein, Fragile X mental retardation protein, Translation elongation

38. Characterization and application of Methanocaldococcus jannaschii RNase P, an ancient tRNA processing enzyme.

Vaishnavi Sidharthan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Bela Haifa Khairunisa (Genetics, Bioinformatics, and Computational Biology Graduate Program, Virginia Tech, Blacksburg, VA), Hong Duc Phan (The Ohio State Biochemistry Program, and Center for RNA Biology, The Ohio State University), Biswarup Mukhopadhayay (Department of Biochemistry, Virginia Tech, Blacksburg, VA), Venkat Gopalan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University)

Abstract:
RNase P catalyzes removal of the 5'-leader of precursor-tRNAs (pre-tRNAs). The ribonucleoprotein form of this enzyme is comprised of a catalytic RNase P RNA (RPR) and a variable number of RNase P protein (RPP) cofactors: one in bacteria, up to five in archaea, and up to ten in eukaryotes. Since the five archaeal RPPs have eukaryotic homologs, the simpler composition and biochemical tractability of archaeal RNase P has inspired its use as an experimental surrogate for the larger eukaryotic relative. The recent cryo-EM structure¹ of in vitro reconstituted RNase P from Methanocaldococcus jannaschii (Mja, a hyperthermophilic archaeon) has yielded insights into the arrangement of all six subunits and the active site. However, the stoichiometry for one of the RPPs is inconsistent with findings from native mass spectrometry (MS) of in vitro assembled Mja RNase P². Also, the higher protein content in archaeal/eukaryotic RNase P mirrors the separation of the archaeal/eukaryotic transcriptional machineries from the simpler bacterial version and motivates our investigation of potential crosstalk between RNase P and other cellular machineries³. Towards the use of MS/MS-based proteomics to characterize the subunit make-up, post-synthetic modifications, and the interactome of Mja RNase P, we are leveraging a new method⁴ for transforming Mja with foreign DNA and constructing in-frame gene deletions or affinity tagging of a target gene. Specifically, we will replace the native Mja RPR with an affinity-tagged variant; we verified that this variant, post-reconstitution with the 5 RPPs, is functionally comparable to the untagged RPR. Also, we are exploring the use of RNase P as a gene knockdown tool⁵, an important goal given the lack of functional annotation for ~50% of Mja genes and paucity of functional genomics methods (e.g., CRISPR) for use in thermophiles. To this end, we have used in vitro assays to direct our design of external guide sequences, which upon binding to a target mRNA forms a pre-tRNA-like bipartite complex and renders the mRNA a substrate for RNase P cleavage. Results from ongoing pilot studies that target the Mja sulfite reductase mRNA⁶ will be shared.

References:
1. Wan, F.; Wang, Q.; Tan, J.; Tan, M.; Chen, J.; Shi, S.; Lan, P.; Wu, J.; Lei, M., Nat Commun 2019, 10, 2617.
2. Lai, L. B.; Tanimoto, A.; Lai, S. M.; Chen, W. Y.; Marathe, I. A.; Westhof, E.; Wysocki, V. H.; Gopalan, V., Nucleic Acids Res 2017, 45, 7432-7440.
3. Serruya, R., Orlovetskie, N., Reiner, R., Dehtiar-Zilber, Y., Wesolowski, D., Altman, S., Jarrous, N., Nucleic Acids Res 2015, 43, 5442-5450.
4. Susanti, D.; Frazier, M. C.; Mukhopadhyay, B., Front Microbiol 2019, 10, 1256.
5. Cho, I. M.; Kazakov, S. A.; Gopalan, V., J Mol Biol 2011, 405, 1121-1127.
6. Johnson, E. F.; Mukhopadhyay, B., J Biol Chem 2005, 280, 38776-33786.

Keywords: RNase P, Mja, knockdown tool

39. Stepwise DNA bending accommodates assembly of Cre recombination complexes

Kye Stachowski (Department of Chemistry and Biochemistry), Andrew Norris (Department of Chemistry and Biochemistry), Devante Potter (Department of Microbiology), Vicky Wysocki (Department of Chemistry and Biochemistry), Mark Foster (Department of Chemistry and Biochemistry)

Abstract:
Gene editing technologies are a hot topic due to the rise of the CRISPR-Cas system, but due to off-target effects and unwanted DNA double strand breaks (DSBs), less error prone systems should be explored. Cre recombinase is a site-specific, tyrosine recombinase that catalytically recombines DNA without creating DSBs, utilizes no cofactors, and functions with single-nucleotide precision.1 An activated, tetrameric complex forms when two Cre protomers bind to one, 34-base pair, loxP DNA sequence that contains two Cre binding sites, followed by antiparallel assembly of two such Cre2-loxP complexes.2 Crystallographic studies of tetrameric structures of Cre show that the loxP substrates are bent at ~ 108°, and the complexes feature many interprotomer protein-protein contacts, hallmarks of an activated complex.3,4 Structural details regarding assembly and activation of Cre pre-tetrameric intermediate structures are unknown.

Here we used protein engineering to isolate Cre-loxP and Cre2-loxP complexes. Then we determined their structures along with a tetrameric complex using cryo-EM to resolutions of 5.1Å, 4.5Å and 3.2Å, respectively. We found that as Cre assembles into an activated complex, the bend of the loxP site becomes more pronounced as each intermediate is reached. The progressive DNA bending is accompanied by increased protein-protein interactions. Our work shows how tetramerization is required for Cre to become activated to recombine DNA. We also used 3D variability analysis to uncover motion in the tetramer that shows how the protein-protein interface plasticity is important for activation of Cre. These new insights could prove useful in design of new Cre variants with engineered site-specificity and improved recombination efficiency.

References:
1. Abremski, K. & Hoess, R. Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. J. Biol. Chem. 259, 1509–14 (1984).
2. Ringrose, L. et al. Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination. J. Mol. Biol. 284, 363–384 (1998).
3. Guo, F., Gopaul, D. N. & Van Duyne, G. D. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389, 40–46 (1997).
4. Ghosh, K., Guo, F. & Van Duyne, G. D. Synapsis of loxP sites by cre recombinase. J. Biol. Chem. 282, 24004–24016 (2007).

Keywords: Cryo-EM, DNA Bending, Cre Recombinase

40. Role of the Leader/Trailer structure of the 17S precursor in 30S subunit biogenesis

Benjamin R. Warner (Department of Microbiology, The Ohio State University)

Abstract:
Ribosome assembly is a complex process, involving rRNA transcription, processing, and modification as well as rRNA folding and r protein binding. rRNAs are transcribed from long operons containing the three rRNA genes as a long primary transcript. The flanking sequences for the 16S and 23S are complementary and form long helices known as Leader/Trailer (L/T) helices. These helices serve as the sight of processing by several different RNases. The L/T structure of the 17S (precursor to the 16S) consists of a L/T helix as well as several leader helices. Previous data has shown this sequence is important for forming functional 30S subunits. To determine what role the L/T structure is playing in assembly, we set out to systematically disrupt the L/T structure. We were able to support one of the predicted structures based on our covariation analysis. We also showed that removing either the leader, trailer or both results in complete loss of actively translating 30S subunits. We are further targeting elements of the L/T structure to determine the critical core structure needed for 30S subunit assembly. Lastly, we are developing an in vitro FRET assay to measure 30S assembly in real time.

References:
Abdi, N. M. & Fredrick, K. Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli. Rna 11, 1624–1632 (2005).
Bunner, A. E. & Williamson, J. R. Stable isotope pulse-chase monitored by quantitative mass spectrometry applied to E. coli 30S ribosome assembly kinetics. Methods 49, 136–141 (2009).
King, T. C., Sirdeskmukh, R. & Schlessinger, D. Nucleolytic processing of ribonucleic acid transcripts in procaryotes. Microbiol. Rev. 50, 428–451 (1986).
Mizushima, S. & Nomura, M. Assembly mapping of 30S ribosomal proteins from E. coli. Nature 226, 1214–1218 (1970).
Young, R. A. & Steitz, J. A. Complementary sequences 1700 nucleotides apart from a ribonuclease III cleavage site in Escherichia coli ribosomal precursor RNA. PNAS. U. S. A. 75, 3593–3597 (1978).

Keywords: ribosome biogenesis, RNA structure, precursor RNA

41. Uncovering the role of an unusual transmembrane helix in the LptB2FGC ATP-binding cassette transporter

Andrew Wilson (Microbiology, The Ohio State University), Natividad Ruiz (Microbiology, The Ohio State University)

Abstract:
Gram-negative bacteria possess an inner membrane (IM) and an outer membrane (OM), which delineate the cytoplasm and periplasmic compartment. The outer leaflet of the OM is primarily formed by an essential layer of tightly packed molecules of lipopolysaccharide (LPS). LPS is synthesized in the IM, after which it must be transported to the cell surface. The cellular machinery responsible for this transport process is comprised of seven essential lipopolysaccharide transport proteins, LptA-G. The molecular “motor” of this machinery, LptB2FGC, is an ATP-binding cassette (ABC) transporter embedded in the IM. Generally, ABC transporters contain two cytoplasmic nucleotide-binding domains (LptB2) physically coupled to two transmembrane domains (LptF and LptG). However, the structure of the LptB2FGC transporter deviates from all other identified ABC transporters in that the single transmembrane α-helix from the protein LptC (TMC) is inserted into one of the contact interfaces between LptF and LptG. Interestingly, TMC is highly conserved yet it was previously reported that removing this membrane anchor from LptC yields no observable phenotypes in Escherichia coli, calling into question whether TMC has a role in LPS transport. Here, we used a genetic approach to elucidate several phenotypes for a ∆TMC mutant strain of E. coli, demonstrating a bona fide role for TMC in LPS transport. Suppressor analysis revealed that removal of TMC improves LPS transport in mutants with ATP-binding defects in LptB2. Furthermore, we show that removal of TMC is not a general suppressor of LPS transport defects, as the effect is localized to a specific step in the LPS transport cycle. Finally, we observed that removing TMC affects LptC levels, suggesting a role of TMC in the assembly of LptC into the Lpt ABC transporter.

Keywords: ABC transporter, lipopolysaccharide transport, suppressor analysis

43. NMD activation by UPF3 paralogs is dictated by their mid-domain but not their EJC binding.

Zhongxia Yi (Center for RNA Biology, the Ohio State University), Rene M. Arvola, Sean Myers, Corinne N. Dilsavor, Rabab Abu Alhasan, Bayley N. Carter, Robert D. Patton, Ralf Bundschuh, Guramrit Singh

Abstract:
Nonsense-mediated mRNA decay (NMD) degrades aberrant transcripts with premature termination codons (PTC) and also regulates ~10% of mammalian transcriptome. While three core NMD factors, Upf1p, Upf2p and Upf3p, are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, with its paralog UPF3A suggested to only weakly activate or even suppress NMD. The mechanism of UPF3B-independent NMD and the mRNA substrates of this distinct NMD branch remains unknown. NMD is enhanced by the presence of an exon junction complex (EJC) downstream of a stop codon. EJC is a multi-protein complex of heterogenous composition that marks exon-exon junctions. In current models of NMD, UPF3B activates NMD by bridging the UPF complex (UPF1-UPF2-UPF3B) at the terminating ribosome to the downstream EJC. This model, however, fails to explain how UPF3 can activate NMD without EJC in various organisms from yeast to human. To characterize the UPF3B-dependent and -independent human NMD pathway, we have used CRISPR-Cas9 to knockout UPF3B in the human colorectal carcinoma HCT116 cell line. RNA-seq from the knockout and wildtype (WT) cells show that, in HCT116 cells, a vast majority of NMD targets can undergo UPF3B-independent NMD. UPF3B loss preferentially affects some, but not all, EJC-mediated NMD substrates. We find that while UPF3A is almost completely dispensable for NMD in WT HCT116 cells, it strongly activates NMD after UPF3B loss. Together, UPF3A and UPF3B regulate a significant portion of EJC-mediated NMD. Further, UPF3-dependent NMD can be regulated by a specific composition of EJC that contains the CACS3 protein. Surprisingly, complementation of human UPF3 double knockout cells with human and mouse UPF3A paralogs shows that the EJC-binding domain is not essential for UPF3 function in NMD but the conserved mid domain, previously shown to engage with translation release factors, is required for a full UPF3 NMD activity. Taken together, we show that while UPF3-dependent NMD can be regulated by EJC, UPF3 plays a more active role in triggering NMD than simply bridging the EJC to the UPF complex.

Keywords: Nonsense-mediated mRNA decay, UPF3, Exon junction complex

44. tRNA recognition by the T. brucei methyltransferase Trm140

Aubree A. Zimmer (Ohio State Biochemistry Program, Department of Microbiology, OSU Center for RNA Biology), Katherine M. McKenney (Ohio State Biochemistry Program, Department of Microbiology, OSU Center for RNA Biology), Mary Anne T. Rubio (Department of Microbiology, OSU Center for RNA Biology), Juan D. Alfonzo (Ohio State Biochemistry Program, Department of Microbiology, OSU Center for RNA Biology)

Abstract:
Transfer RNAs (tRNAs) are central to protein synthesis by converting the information found in protein-coding regions of the genome into a precise polypeptide chain as dictated by the codons in mRNA. However, before becoming functional in translation, tRNAs are subject of several processing steps such as intron removal, CCA addition, cleavage of 5′ leader and 3′ trailer sequences, along with extensive chemical modifications necessary for proper function. For instance, the conversion of A to I at the wobble base of the anticodon increases decoding capabilities, while pseudouridine at position 55 is evolutionarily conserved and plays a key role in tRNA stability. While many tRNA modification enzymes across a wide variety of species have been described, how these enzymes target a specific tRNA substrate among a pool of similar non-substrate molecules is still unclear. In Trypanosoma brucei, we have described a methylation at position 32 of all three tRNAThr, which requires a deaminase (ADAT 2/3) and a methyltransferase (Trm140) working together while showing intertwining functionality to convert an encoded C₃₂ to m3U₃₂—in what we describe as enzyme co-activation. Our laboratory aims to understand enzyme co-dependence by first understanding the biochemical interactions between the two enzymes and tRNA. In vitro binding studies using both enzymes and tRNA mini substrates shows Trm140 and ADAT 2/3 can only bind synergistically to a complete tRNA cloverleaf. We also show both enzymes can bind non-substrate tRNA’s with equal affinity to substrate, but only modify tRNAThr. The results presented here aim at elucidating how the Trm140 methylase recognizes and binds its cognate tRNA substrate, highlighting nuances in substrate specificity by tRNA processing enzymes.

References:
1. Rubio MAT et al. 2017. Editing and methylation at a single site by functionally interdependent activities. Nature 542: 494–497
2. McKenney KM, Rubio MAT, Alfonzo JD. 2018. Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei. RNA 24: 56-66.
5. McKenney KM, 2018. Investigating the basis of tRNA editing and modification enzyme coactivation in Trypanosoma brucei. PhD Dissertation. The Ohio State University, OH.

Keywords: tRNA editing, Methyltransferase, Trypanosoma

45. Development of bifacial Peptide Nucleic Acids for Probing Structural Motifs Containing Non-canonical Pairs in RNA and DNA

Xue Tang (Department of Chemistry and Biochemistry), Debmalya Bhunia (Department of Chemistry and Biochemistry)

Abstract:
RNA is an important target for chemical probes and therapeutics; however, RNA non-canonical pair (NCP) targets are underexploited due to lack of understanding and identifying of the noncovalent interactions between NCP motifs and small molecules. Our group has previously reported bPNA(+) triplex hybridization for targeting and fluorescence labeling UxU and TxT NCPs in structured RNA and DNA. To extend the binding repertoire of bPNAs beyond oligo-U/T bulges, we designed and synthesized bPNA variants that recognize other NCPs in addition to UxU and TxT. We then carried out binding screens for these bPNA variants using DNA and RNA duplexes with internal bulge sequences that have NCPs. We have observed clear band shifts on native gels for bPNA that have good binding behavior with certain NCPs. Thus, new binding strategies for structural motifs containing NCPs have been identified. With the broadened recognition of bPNA, we generally provided a solution for specifically labeling various native RNA sequences that contain NCP motifs, which can be further exploited to develop fluorescent probes that function in vitro and in vivo.

Keywords:

46. Investigation of the dynamics of the SMK box riboswitch

Haoyun Yang (Department of Chemistry and Biochemistry, OSU), Mark Foster (Department of Chemistry and Biochemistry, OSU), Tina Henkin (Department of Microbiology, OSU)

Abstract:
The conformational dynamics of non-coding RNAs are coupled to vital cellular processes such as metabolite sensing, site specific RNA catalysis and the hierarchy of ordered ribonucleoprotein assemblies. However, the detailed mechanistic description of the structures and RNA functional conformational dynamics remain poorly understood. To fill this knowledge gap, we propose to study a model system, the SMK box, S-adenosylmethionine (SAM) metabolite-binding/SAM-III riboswitch RNA, a compact RNA that undergoes ligand-mediated conformational changes to alter ribosome binding and thereby regulate translation1,2. Previously, X-Ray crystallography analysis of the SMK aptamer bound to SAM established the structure of the ligand-bound, repressed state3. In the absence of SAM, SMK is proposed to be in equilibrium between an alternative fold (ISO) that has no obvious ligand binding site and a ligand binding competent state (PRIMED) evidenced by mutation and chemical probing experiments4,5. Thus, a conformation selection model is speculated. To understand how the SMK box riboswitch is able to undergo conformational fluctuations between alternative structures in order to enable its function. We propose to first use stopped-flow and 2-aminopurine labeled RNA to reveal the ligand binding model that best describes this riboswitch and extract thermodynamics parameters that can provide structural insight of the binding steps. From this, we can use NMR relaxation dispersion (RD) experiments to investigate the conformational fluctuations of the RNA with atomic resolution.

References:
1. Fuchs RT, Grundy FJ, Henkin TM. S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA. Proc Natl Acad Sci U S A. 2007;104(12):4876-4880. doi:10.1073/pnas.0609956104

2. Fuchs RT, Grundy FJ, Henkin TM. The S(MK) box is a new SAM-binding RNA for translational regulation of SAM synthetase. Nat Struct Mol Biol. 2006;13(3):226-233. doi:10.1038/nsmb1059

3. Lu C, Smith AM, Fuchs RT, et al. Crystal structures of the SAM-III/S(MK) riboswitch reveal the SAM-dependent translation inhibition mechanism. Nat Struct Mol Biol. 2008;15(10):1076-1083. doi:10.1038/nsmb.1494

4. Lu C, Smith AM, Ding F, Chowdhury A, Henkin TM, Ke A. Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch. J Mol Biol. 2011;409(5):786-799. doi:10.1016/j.jmb.2011.04.039

5. Wilson RC, Smith AM, Fuchs RT, Kleckner IR, Henkin TM, Foster MP. Tuning riboswitch regulation through conformational selection. J Mol Biol. 2011;405(4):926-938. doi:10.1016/j.jmb.2010.10.056

Keywords:

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