Poster abstracts
Poster number 12 submitted by Ben Jepson
Analysis of tRNA methyltransferase homologs from zebrafish reveals similarities and differences to human Trm10 enzymes
Ben Jepson (Department of Chemistry and Biochemistry, OSU), Jane Jackman (Department of Chemistry and Biochemistry, OSU)
Abstract:
The tRNA m1R9 methyltransferase (Trm10) family enzymes methylate the N-1 atom of purine residues at the ninth position of tRNAs. Higher eukaryotes encoding up to three homologs of Trm10. Human TRMT10A catalyzes m1G9 formation on multiple tRNAs, whereas TRMT10B forms m1A9 specifically on human tRNAAsp. Familial TRMT10A mutations are associated with human disease, further supporting the non-redundant function of the two homologs in vivo. We sought to develop a vertebrate model system to further probe the significance of these distinct activities using Danio rerio and have created mutant lines for the cytosolic homologs. Like human TRMT10A, expression of zebrafish Trmt10a rescues the trm10∆ growth phenotype in S. cerevisiae 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. However, zebrafish Trmt10a and Trmt10b do not show the same pattern of in vitro substrate specificities as the human homologs. Since the modification status of zebrafish tRNAs had not been previously determined, we carried out a genome-wide study to map tRNA modification in zebrafish using mim-tRNAseq on wild-type embryos. We have now identified and validated multiple tRNA modifications in zebrafish, providing the first global map of tRNA modification in this organism, including target tRNAs for m1R9 modification. Analysis of R9 modification in the trmt10a and trmt10b mutant fish show that although each homolog acts on unique tRNA substrates similar to modification in humans, other substrates are shared between homologs, consistent with our in vitro experiments with purified enzymes. Overall, our data reveals that while the zebrafish enzymes share important similarities with their human counterparts there are distinct differences in some patterns of activity, deeper investigation of which could help uncover important insights into complex substrate specificities of Trm10 homologs.
Keywords: tRNA modification, Trm10, m1R9