Mutually exclusive substrate selection strategy by human m(3)C RNA transferases METTL2A and METTL6

tRNAs harbor the most diverse posttranscriptional modifications. The 3-methylcytidine (m(3)C) is widely distributed at position C32 (m(3)C32) of eukaryotic tRNA(Thr) and tRNA(Ser) species. m(3)C32 is decorated by the single methyltransferase Trm140 in budding yeasts; however, two (Trm140 and Trm141 in fission yeasts) or three enzymes (METTL2A, METTL2B and METTL6 in mammals) are involved in its biogenesis. The rationale for the existence of multiple m(3)C32 methyltransferases and their substrate discrimination mechanism is hitherto unknown. Here, we revealed that both METTL2A and METTL2B are expressed in vivo. We purified human METTL2A, METTL2B, and METTL6 to high homogeneity. We successfully reconstituted m(3)C32 modification activity for tRNA(Thr) by METT2A and for tRNA(Ser)(GCU) by METTL6, assisted by seryl-tRNA synthetase (SerRS) in vitro. Compared with METTL2A, METTL2B exhibited dramatically lower activity in vitro. Both G35 and t(6)A at position 37 (t(6)A37) are necessary but insufficient prerequisites for tRNA(Thr) m(3)C32 formation, while the anticodon loop and the long variable arm, but not t(6)A37, are key determinants for tRNA(Ser)(GCU) m(3)C32 biogenesis, likely being recognized synergistically by METTL6 and SerRS, respectively. Finally, we proposed a mutually exclusive substrate selection model to ensure correct discrimination among multiple tRNAs by multiple m(3)C32 methyltransferases.