Poster abstracts
Poster number 24 submitted by Nathan Howell
A family expands: Characterization of human TRMT10B, a multifunctional tRNA methyltransferase
Nathan Howell (Ohio State University), Jane Jackman (Ohio State University)
Abstract:
By linking genetic information with protein synthesis, tRNA performs a critical role in biology. To satisfy the cell's need for a high quality pool of tRNA, substantial resources are invested into producing functional tRNA molecules, including an extensive system of post-transcriptional nucleotide modifications. Through this universal process, chemical functional groups are changed, rearranged, and added to individual nucleotide residues in a pattern that is specific for each target tRNA. One such modification is the addition of a methyl group to the N-1 atom of ninth-position purines (m1N9), which occurs in archaea and eukarya and is catalyzed by the Trm10 family of enzymes. Initially discovered in S. cerevisiae, this SPOUT methyltransferase family was believed to comprise members that only exhibit m1G9 activity. Recently however, several archaeal and mitochondrial Trm10 homologs were unexpectedly demonstrated to form m1A9, either in addition to or instead of m1G9; this activity is not well understood, particularly in light of different pKa values exhibited by the methylated atoms. Intriguingly, higher eukaryotes encode multiple Trm10 paralogs, including two cytoplasmic enzymes in humans: TRMT10A and TRMT10B. TRMT10A is suggested to be responsible for formation of all m1G9 in human cytosolic tRNA, whereas TRMT10B has been resistant to biochemical characterization. However, yeast complementation experiments and human disease models suggest that TRMT10B is not functionally redundant with TRMT10A. Using in vitro biochemical assays, primer extension, and HPLC analysis, we report that TRMT10B is indeed a tRNA methyltransferase, and is responsible for the newly reported m1A9 modification in human tRNA-Asp. Furthermore, our data support the role of TRMT10B as a multifunctional methyltransferase capable of both A and G methylation, forming distinct patterns of previously unknown methylation products in a substrate-dependent manner on human tRNA. Human TRMT10B is the first eukaryotic cytosolic enzyme to display such unique functionality in terms of its target nucleotide identity and substrate discrimination. Our results not only illuminate the enzyme responsible for a newly identified m1A9 in human tRNA, but shed further light on the unusually complex biochemical repertoire of the Trm10 enzyme family.
Keywords: tRNA modifications, enzymology, Trm10