tRNAs harbor the most diverse posttranscriptional modifications. The 3-methylcytidine (m3C) is widely distributed at position C32 (m3C32) of eukaryotic tRNAThr and tRNASer species. m3C32 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 m3C32 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 m3C32 modification activity for tRNAThr by METT2A and for tRNASer(GCU) by METTL6, assisted by seryl-tRNA synthetase (SerRS) in vitro. Compared with METTL2A, METTL2B exhibited dramatically lower activity in vitro. Both G35 and t6A at position 37 (t6A37) are necessary but insufficient prerequisites for tRNAThr m3C32 formation, while the anticodon loop and the long variable arm, but not t6A37, are key determinants for tRNASer(GCU) m3C32 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 m3C32 methyltransferases.

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m3C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m3C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m3C32 modification of human mitochondrial tRNAThr (hmtRNAThr) was not reliant on t6A modification at A37 (t6A37), while that of hmtRNASer(UCN) critically depended on i6A modification at A37 (i6A37). We clarified the hmtRNAThr substrate recognition mechanism, which was obviously different from that of hmtRNASer(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m3C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m3C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.

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