Poster abstracts

Poster number 91 submitted by Nisha Makkar

Investigating biological significance of tRNA acetyltransferase Tan1, methyltransferase Trm1, and pseudouridine synthase Pus1 tRNA substrates in Saccharomyces cerevisiae

Nisha Makkar (The Ohio State University Chemistry and Biochemistry), Isobel Bowles (The Ohio State University Biochemistry Program), Jane Jackman (The Ohio State University Chemistry and Biochemistry)

Abstract:
Three highly conserved tRNA-modifying enzymes that lack phenotypes when deleted in Saccharomyces cerevisiae under normal growth conditions are tRNA acetyltransferase 1 (Tan1), methyltransferase 1 (Trm1), and pseudouridine synthase 1 (Pus1). However, upon their individual deletion, S. cerevisiae strains experience heightened sensitivity to stress induced by chemotherapeutic 5-fluorouracil (5FU), resulting in a growth defect. We hypothesized that destabilization of tRNA in these deletion strains due to hypomodification may impair cellular coping with 5FU cytotoxicity and that certain tRNA species may be more affected by these lost modifications. We showed that in tan1Δ strains lacking the 12th position N-4-acetylcytidine (ac4C12) modification, only individual overexpression of two of the six Tan1 substrates, tRNASer(CGA) and tRNASer(UGA), suppresses hypersensitivity to 5FU. Correspondingly, individually overexpressing tRNASer(CGA) and tRNASer(UGA) also rescued growth hypersensitivity of the trm1Δ strain to 5FU, while that of the 16 other substrate tRNAs containing m22G26 dimethylation performed by Trm1 in S. cerevisiae did not. Moreover, overexpressing individual tRNA substrates did not ameliorate the elevated toxicity of 5FU in pus1Δ strains lacking pseudouridylation by Pus1. To identify the quality control pathway encountered by hypomodified tRNASer(CGA) and tRNASer(UGA), an enzyme related to sensing several such pathways was deleted in combination with Tan1 and arrested the 5FU-induced growth defect. The effects of modification by Tan1 on tRNA stability are also being examined with northern blotting, yet, the specific genes related to decay of these hypomodified tRNAs remain undetermined, as well as how 5FU toxicity specifically impacts small non-coding RNA. Such studies have important implications for elucidating the biological impact of these highly conserved tRNA modifications.

References:
Gustavsson, M., & Ronne, H. (2008). Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast. RNA, 14(4), 666–674. doi:10.1261/rna.966208

Hopper, A.K. & Huang H.Y. (2015). Quality control pathways for nucleus-encoded eukaryotic tRNA biosynthesis and subcellular trafficking. American Society for Microbiology, 35(12). doi:10.1128/MCB.00131-15

Howell, N.W. and Jackman, J.E. (2023). Impact of chemical modification on tRNA function. eLS, John Wiley & Sons, Ltd (Ed.). doi:10.1002/9780470015902.a0028527

Phizicky, E.M. & Hopper, A.K. (2010). tRNA biology charges to the front. Genes & development, 24(17), 1832–1860. doi:10.1101/gad.1956510

Keywords: tRNA modification, ac4C12, m22G26