Throll, P.; Dolce, L. G.; Lastres, P. R.; Arnold, K.; Tengo, L.; Basu, S.; Kaiser, S.; Schneider, R.; Kowalinski, E.

Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. However, our understanding of the substrate selection and catalysis mode of the m3C RNA methyltransferases is currently still lacking. Here, we report the cryo-electron microscopy structure of the m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We reveal how METTL6 and SerRS jointly coordinate the long variable arm of tRNASer in their interface. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, based on the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA binding mode for m3C RNA methyltransferases including METTL2 and METTL8, suggesting that these mammalian paralogues use similar ways to engage their respective tRNA substrates and co-factors.