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

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. Here, we report the cryo-electron microscopy structure of the human m³C 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 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, on the basis of the structure of METTL6 in complex with SerRS and tRNA^Ser, we postulate a universal tRNA-binding mode for m³C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors.