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.

tRNA 反密码子环中 32 位胞嘧啶甲基化为 3-甲基胞嘧啶 (m ³ C) 对于细胞翻译保真度至关重要。 RNA 甲基转移酶的错误调节导致这种修饰可能导致侵袭性癌症和代谢紊乱。在这里，我们报道了与丝氨酰-tRNA合成酶（SerRS）及其共同底物tRNA ^Ser复合的人m ³ C tRNA甲基转移酶METTL6的冷冻电镜结构。通过复杂的结构，我们鉴定了 METTL6 的 tRNA 结合域。我们证明 SerRS 充当 METTL6 的 tRNA ^Ser底物选择因子。我们证明 SerRS 增强了 METTL6 的甲基化活性，并且 METTL6 和 SerRS 之间的直接接触对于有效的 tRNA ^Ser甲基化是必要的。最后，根据 METTL6 与 SerRS 和 tRNA ^Ser复合物的结构，我们假设 m ³ C RNA 甲基转移酶（包括 METTL2 和 METTL8）具有通用的 tRNA 结合模式，表明这些哺乳动物旁系同源物使用相似的方式来接合各自的tRNA 底物和辅因子。