Poster abstracts
Poster number 63 submitted by Ben Jepson
Assessing the biological function of cytoplasmic tRNA methyltransferase (Trm10) enzymes in Danio rerio
Ben Jepson (Dept. of Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Dept. of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Transfer RNAs (tRNAs) are highly modified across all domains of life. The m1R9 tRNA methyltransferase (Trm10) catalyzes the methylation of the N-1 atom of the purine nucleotides Adenine and Guanine at position nine of substrate tRNAs. Trm10 family enzymes are conserved across all archaeal and eukaryotic organisms. In Saccharomyces cerevisiae, there is only one Trm10 enzyme. However, in higher eukaryotes, there are multiple Trm10 homologs in individual organisms. Humans, for example, have one mitochondrial (TRMT10C) and two cytoplasmic (TRMT10A and TRMT10B) Trm10 enzymes. Mutations in one of the cytoplasmic paralogs, TRMT10A, have been implicated in diseases that include glucose metabolic and neurodegenerative disorders. This observation suggests that these two cytoplasmic enzymes have non-redundant roles in the cell, and previous work in the lab has shown that TRMT10A and TRMT10B have different enzymatic activities and tRNA substrate specificities. Here we use Danio rerio (zebrafish), which also encodes two cytoplasmic Trm10 enzymes as a model organism in order to investigate their biological function. We use in vitro biochemical assays to show that zebrafish trmt10a and trmt10b show similar enzymatic activities and substrate specificities. However, only zebrafish trmt10a is capable of complementing the 5-FU hypersensitivity phenotype of a trm10∆ S. cerevisiae strain, consistent with the proposed non-redundant roles for these enzymes in vivo. Future work will attempt to fill the gap in knowledge of substrate specificity from two directions. The first approach will utilize an RNA-seq based method to analyze the modification status of zebrafish tRNAs in an attempt to identify potential substrates for zebrafish trmt10a/b; these findings will be used in further experiments using recombinant zebrafish Trm10 proteins. The second approach will take advantage of existing zebrafish trmt10a/b mutant strains, where total RNA will be isolated and analyzed using primer extension. Completion of this work will leave us with a greater understanding of the differing substrate specificities of the cytoplasmic Trm10 homologs in zebrafish and will help to shed some light on the distinct functionalities of the Trm10 family as a whole.
Keywords: tRNA modification, tRNA methylation, Trm10