Poster abstracts
Poster number 96 submitted by Olivia Roumaya
Investigation of vertebrate tRNA methyltransferases Trmt10a and Trmt10b in zebrafish
Olivia Roumaya (Ohio State Biochemistry Program, Ohio State University), Ben Jepson (Molecular, Cellular and Developmental Biology, Ohio State University), Aidan Manning (Department of Biomolecular Engineering, University of California Santa Cruz), 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 purines at position 9 (m1R9) in tRNA using S-adenosyl methionine and is ubiquitously expressed in Eukarya. Vertebrates contain 3 Trm10 paralogs, Trmt10a and Trmt10b, that act on nuclear tRNA, and Trmt10c, that acts on mitochondrial tRNA. To investigate these paralogs in vertebrates, we created homozygous loss-of-function mutants for trmt10a and trmt10b in zebrafish (Danio rerio). Using ordered two-template relay sequencing (OTTR-Seq), we established that zebrafish Trmt10a catalyzes m1G9-modification on several tRNAs, while Trmt10b catalyzes m1A9-modification solely on tRNAAsp, identical to the known substrate specificities of the human orthologs. OTTR-Seq also predicted a modification at the G6 position in the acceptor stem on several zebrafish tRNAs. While this modification had not yet been characterized in fish, it is highly conserved across all domains as m2G6 or m2,2G6. These data also showed that loss of trmt10a and trmt10b modification in the zebrafish mutants impacted the predicted abundance of G6 modification in some, but not all tRNAs. We used primer extension to examine these predictions and found that m1G9 is crucial for G6 modification on some Trmt10a substrates, tRNAPro-AGG, tRNAPro-CGG, and tRNAPro-TGG, which all have a decreased G6 modification abundance when lacking m1G9. However, when Trmt10a substrate, tRNAGly-CCC lacks m1G9, the amount of G6 modification does not change, highlighting the importance of m1G9 for G6 modification on some tRNAs but not others. Interestingly, the sole substrate of Trmt10b, tRNAAsp-GTC, also had decreased G6 modification when lacking m1A9, demonstrating the importance of the m1A9 modification on tRNAAsp-GTC for the first time. While tRNA modification circuits are well established in the anticodon loop and T-loop, this is, to our knowledge, the first example of interplay between different modifications in and near the acceptor stem. As the mechanism of this modification circuit is not yet known, we predict that the m1R9 modification could act as a structural component on specific tRNAs, enabling G6 modification to occur. Together, these studies demonstrate a novel circuit between two tRNA modifications and opens the door on future work to determine their precise order and mechanism.
Keywords: tRNA, Trm10, Zebrafish