Mechanisms and biological functions of SPOUT methyltransferases

PROJECT SUMMARY/ ABSTRACT Transfer RNAs (tRNAs) are the universal adaptor molecules necessary to convert the nucleic acid-based genetic code into protein sequence during protein synthesis (translation) by the ribosome. This process is universally conserved and fundamental to all life, and, as such, defects in the molecular players of translation, including tRNAs, result in diverse human diseases. Specific chemical modifications such as methylation are common in tRNA, but a detailed understanding of the enzymes that incorporate them and their contributions to tRNA function (and disfunction in disease) have only recently emerged for a few select examples. Since the discovery of the tRNA methyltransferase (Trm10) in Saccharomyces cerevisiae, an accumulating body of evidence, including phenotypes in yeast and a multisymptomatic disease associated with human mutations, has established a significant role for Trm10 in tRNA biology. To better understand the implications of Trm10 modification, the mechanisms by which Trm10 family enzymes specifically recognize and act on their substrate tRNA, and the impact of tRNA modifications on important cellular processes need to be addressed. This project will determine the molecular basis for Trm10 mechanism and function using a multi-disciplinary approach. Genetic, biochemical and molecular enzymology approaches will be combined with structural analyses of enzyme-tRNA complexes using synthetic analogs of the native methyl donor, S-adenosyl-L-methionine, to uniquely identify the role of Trm10 in the maintenance of a high-quality pool of tRNA. A newly developed vertebrate model for Trm10 function will enable investigation of previously challenging questions on Trm10's role in the biological function of multicellular eukaryotes. The studies will be performed in three complementary but independent aims that will: 1) Determine how specific tRNA substrates are selected for modification by yeast and vertebrate Trm10 enzymes using structural, biochemical and genetic approaches; 2) Assess the molecular basis for and biological significance of the uniquely conserved vertebrate m1A9 modification exploiting a new vertebrate model for Trm10 function, and 3) Identify tRNA-specific functions for G9 modification in yeast and zebrafish using complementary genetic approaches in both model species. Collectively, the proposed studies will advance the fields of enzymology, RNA biochemistry, and tRNA biology by providing mechanistic and biological insight into a tRNA modification enzyme that is universally conserved among eukaryotes and is critically important for human health, yet whose molecular mechanism and biological functions are not at all understood. These results will also provide new insight into the dynamic landscape of tRNA modifications in multicellular eukaryotes.