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 反密码子环中的胞嘧啶 32 甲基化为 3-甲基胞嘧啶 （m³C） 对于细胞翻译保真度至关重要。设置这种修饰的 RNA 甲基转移酶的失调会导致侵袭性癌症和代谢紊乱。在这里，我们报道了人 m³C tRNA 甲基转移酶 METTL6 与 seryl-tRNA 合成酶 （SerRS） 及其共同底物 tRNA^Ser 复合物中的冷冻电子显微镜结构。通过复杂的结构，我们确定了 METTL6 的 tRNA 结合结构域。我们表明 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 之间的直接接触是高效 tRNA^Ser 甲基化所必需的。最后，基于 METTL6 与 SerRS 和 tRNA^Ser 复合物的结构，我们假设 m³C RNA 甲基转移酶（包括 METTL2 和 METTL8）的通用 tRNA 结合模式，表明这些哺乳动物旁系同源物使用类似的方式结合它们各自的 tRNA 底物和辅因子。