Poster abstracts

Poster number 45 submitted by Olivia Roumaya

A new Trm10-dependent tRNA modification circuit identified in the acceptor stem of zebrafish tRNA

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, Sharon Amacher (Department of Molecular Genetics, Ohio State University), Jane Jackman (Department of Chemistry and Biochemistry, Ohio State University)

Abstract:
One of the most ubiquitous modifications in eukaryotic tRNA is N-1 methylation of position 9 purines (m1R9) by the tRNA methyltransferase 10 (Trm10) family. In fungi, a single Trm10 enzyme installs m1G9 on many tRNAs, whereas vertebrates encode three paralogs: Trmt10a and Trmt10b, which modify nuclear tRNAs, and Trmt10c, which modifies mitochondrial tRNAs. We characterized homozygous loss-of-function mutants for trmt10a and trmt10b in zebrafish (Danio rerio) using ordered two-template relay sequencing (OTTR-Seq) and demonstrated that Trmt10a catalyzes m1G9-modification on several tRNAs, while Trmt10b catalyzes m1A9-modification solely on tRNA-Asp, identical to the known substrate specificities of the human orthologs we established previously. We recently showed that m1G9 contributes to tRNA quality control and stability in Saccharomyces cerevisiae, and loss of human TRMT10A is linked to disease, suggesting conserved functions across eukaryotes. However, the biochemical role of the vertebrate‑specific m1A9 remains unknown.

Here, we analyzed the OTTR-Seq data from trmt10a-/- and trmt10b-/- embryos to identify if other modifications were impacted by loss of m1R9 and saw that the abundance of modification-dependent mismatches at the G6 position was significantly impacted by the loss of m1R9. Although the G6 modification in zebrafish has not been characterized, m2G6 has been described in tRNA across life domains. By primer extension, we validated the interdependence of m1R9 modifications on G6 modification, demonstrating that loss of m1G9 results in decreased abundance of G6 modification on Trmt10a substrates, tRNA-Pro-AGG, CGG, and TGG. Interestingly, the sole tRNA substrate of Trmt10b, tRNA-Asp-GTC, also exhibited decreased G6 modification when lacking m1A9, demonstrating a biological role for the m1A9 modification on tRNA-Asp-GTC for the first time. While tRNA‑modification circuits are well characterized in the anticodon loop and T‑loop, this is the first example of coordinated modifications within or near the acceptor stem. Together, these findings reveal a new modification circuit near the acceptor stem and highlight the need to define biochemical interplay between these conserved enzymes.

Keywords: tRNA, Trm10, tRNA modification circuit