Recognition and catalytic mechanism of tRNA m3C methyltransferase METTL6

Post-transcriptional RNA modifications regulate the RNA structural and functional landscape, and thus play a central role in many cellular processes. Accordingly, aberrant RNA modification pathways contribute to a variety of human diseases, including cancer and neurological disorders. However, recognition and catalytic mechanisms of most RNA-modifying proteins remain obscure, due to sparse structural data. Recently, the cryogenic electron microscopy structure of N3-methyl-cytosine (m3C) methyltransferase METTL6 revealed seryl-tRNA synthetase (SerRS) as a cofactor for tRNASer selection, but left the cooperative role of SerRS and the reaction process undefined. Here, combining computer simulations and in vitro experiments, we define the mechanism of concerted substrate identification by METTL6 and SerRS, demonstrating that SerRS pre-organizes the tRNASer variable arm for METTL6 recruitment. We show that once METTL6 is stably docked onto the SerRS-tRNASer complex, thereby ensuring the precise alignment of the reactants, the methylation reaction proceeds spontaneously. Our results establish the catalytic mechanism of m3C methyltransferases and provide the foundation for rational design of targeted therapeutics.