Structural basis of tRNA recognition by the m3C-RNA-methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase

Summary Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m 3 C) is crucial for cellular translation fidelity 1 . Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances 2,3 . However, our understanding of the substrate selection and catalysis mode of the m 3 C RNA methyltransferases is currently still lacking. Here, we report the cryo-electron microscopy structure of the m 3 C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNA Ser . Through the complex structure, we identify the tRNA binding domain of METTL6. We show that SerRS acts as the tRNA Ser substrate selection factor for METTL6. We reveal how METTL6 and SerRS jointly coordinate the long variable arm of tRNA Ser 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 tRNA Ser methylation. Finally, based on the structure of METTL6 in complex with SerRS and tRNA Ser , we postulate a universal tRNA binding mode for m 3 C RNA methyltransferases including METTL2 and METTL8, suggesting that these mammalian paralogues use similar ways to engage their respective tRNA substrates and co-factors.