Poster abstracts
Poster number 67 submitted by Olivia Roumaya
Investigation of a unique vertebrate tRNA modification by Trmt10b in zebrafish
Olivia Roumaya (Ohio State Biochemistry Program), Ben Jepson (Ohio State University), Thomas Gallagher (Department of Molecular Genetics, Ohio State University), Sharon Amacher (Department of Molecular Genetics, Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry, Ohio State University)
Abstract:
The tRNA methyltransferase 10 (Trm10) family methylates the N-1 atom of select purines at position 9 (m1R9) in the tRNA core using S-adenosyl methionine (SAM) and is ubiquitously expressed in Eukarya and Archaea. While most archaeal and single-celled eukaryotic species contain only one homolog of Trm10, vertebrates contain two cytoplasmic (Trmt10a and Trmt10b) and one mitochondrial (Trmt10c) enzyme. We and others have established that human and zebrafish (Danio rerio) Trmt10a enzymes perform m1G9-modification on a number of tRNAs, while Trmt10b has a non-redundant role in catalyzing m1A9-modification solely on tRNAAsp in human cells and in zebrafish embryos. Intriguingly, while the biological modification pattern catalyzed by human Trmt10b catalyzes m1A9 on tRNAAsp can be recapitulated in vitro with purified enzyme, recombinantly expressed and purified zebrafish trmt10b cannot catalyze the correct modification when tested in vitro. To investigate whether Trmt10b activity can be reconstituted in the context of another eukaryote, human and zebrafish Trmt10b were expressed in Saccharomyces cerevisiae trm10Δ strains. We demonstrated that human Trmt10b activity does not modify S. cerevisiae tRNAAsp, but exhibits the correct m1A9 modification activity on both human and zebrafish tRNAAsp species when they are also introduced into the yeast strain. However, zebrafish trmt10b activity is not able to catalyze any detectable modification on either yeast, human or zebrafish tRNAAsp in this heterologous system, indicating that something unique to the zebrafish cellular environment may be required for m1A9 catalysis. To study the significance of these paralogs in a vertebrate organism, our lab generated homozygous mutant trmt10a and trmt10b zebrafish. To further probe the mechanism of zebrafish trmt10b, we will use a proximity labeling assay to determine potential interactions necessary for catalysis in zebrafish embryos. To identify features of tRNAAsp important for catalysis, a mutant tRNAAsp library will be created and specific residues required for trmt10b recognition will be identified via ordered two-template relay sequencing. Altogether, this work will provide a further understanding of the mechanism and biological impact of the highly conserved vertebrate Trmt10a and Trmt10b enzymes.
Keywords: tRNA modification, Trm10, m1R9