Poster abstracts

Poster number 138 submitted by Mary Anne Johnson

The Role of Arc1p’s OB-Fold-Mediated Editing Activity In Vivo

Mary Anne Johnson (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, United States ), Hannah Lathrop (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, United States ), Rylan Watkins (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, United States ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH, United States )

Abstract:
Faithful translation of genetic information is essential for cell viability. Aminoacyl-tRNA synthetases (ARSs) charge tRNAs with their cognate amino acids but occasionally mischarge tRNAs with similarly sized non-cognate amino acids, making editing essential for faithful protein synthesis. In eukaryotes, subsets of ARSs assemble into multi-synthetase complexes (MSCs), the smallest of which is found in Saccharomyces cerevisiae. All known MSCs include at least one scaffolding protein containing an oligonucleotide/oligosaccharide binding (OB)-fold. While the OB-fold was previously thought to channel tRNA to adjacent synthetases, we discovered that the OB-fold deacylates aminoacyl-tRNA (aa-tRNA). In yeast, the OB-fold protein Arc1p edits several aa-tRNAs in vitro, including Ala-tRNAPro. Whether this editing activity contributes to translational fidelity in cells remains unknown. We hypothesize that Arc1p prevents misincorporation events in yeast. Using kinetic growth assays, we found that arc1Δ cells have a growth defect under standard conditions, which cold stress exacerbates, potentially reflecting increased translational errors. To test for misincorporation directly, we designed a GFP reporter that fluoresces upon Ala-tRNAPro incorporation. We observed no change in fluorescence in the arc1Δ mutants compared to wild-type, indicating that other mischarging events may better explain the growth phenotype. We are screening additional candidates now. This work will establish whether Arc1p-mediated editing prevents mischarging events in vivo, expanding our understanding of a novel family of tRNA editing domains.

References:
1. Watkins RR, Bockelman S, Vradi A, Grabarkewitz K, Pyun A, Stark J, Wysocki VH, Alfonzo JD, Musier-Forsyth K. Unexpected enzymatic function of an ancient nucleic acid-binding fold. Nucleic Acids Res. 2025 Apr 22;53(8):gkaf328. doi: 10.1093/nar/gkaf328. PMID: 40274265; PMCID: PMC12021450.

2. Frechin M, Kern D, Martin RP, Becker HD, Senger B. Arc1p: anchoring, routing, coordinating. FEBS Lett. 2010 Jan 21;584(2):427-33. doi: 10.1016/j.febslet.2009.11.037. PMID: 19914242.

3. Kuzmishin Nagy AB, Bakhtina M, Musier-Forsyth K. Trans-editing by aminoacyl-tRNA synthetase-like editing domains. Enzymes. 2020;48:69-115. doi: 10.1016/bs.enz.2020.07.002. Epub 2020 Sep 8. PMID: 33837712.

Keywords: tRNA, aminoacyl-tRNA synthetases, oligo-binding fold