Mitochondrial RNA m3C methyltransferase METTL8 relies on an isoform-specific N-terminal extension and modifies multiple heterogenous tRNAs

• Published in: Science bulletin (Beijing) 2023-09, Vol.68 (18), p.2094-2105
• Main Authors: Huang, Meng-Han, Wang, Jin-Tao, Zhang, Jian-Hui, Mao, Xue-Ling, Peng, Gui-Xin, Lin, Xiuying, Lv, Daizhu, Yuan, Chen, Lin, Huan, Wang, En-Duo, Zhou, Xiao-Long
• Format: Article
• Language: English
• Subjects: 3-methylcytidine; Aminoacylation; Methyltransferase; tRNA
• ISSN: 2095-9273
• DOI: 10.1016/j.scib.2023.08.002

[Display omitted] Methyltransferase-like 8 (METTL8) encodes a mitochondria-localized METTL8-Iso1 and a nucleolus-distributed METTL8-Iso4 isoform, which differ only in their N-terminal extension (N-extension), by mRNA alternative splicing. METTL8-Iso1 generates 3-methylcytidine at position 32 (m3C32) of mitochondrial tRNAThr and tRNASer(UCN). Whether METTL8-Iso4 is an active m3C32 methyltransferase and the role of the N-extension in mitochondrial tRNA m3C32 formation remain unclear. Here, we revealed that METTL8-Iso4 was inactive in m3C32 generation due to the lack of N-extension, which contains several absolutely conserved modification-critical residues; the counterparts were likewise essential in cytoplasmic m3C32 biogenesis by methyltransferase-like 2A (METTL2A) or budding yeasts tRNA N3-methylcytidine methyltransferase (Trm140), in vitro and in vivo. Cross-compartment/species tRNA modification assays unexpectedly found that METTL8-Iso1 efficiently introduced m3C32 to several cytoplasmic or even bacterial tRNAs in vitro. m3C32 did not influence tRNAThrN6-threonylcarbamoyladenosine (t6A) modification or aminoacylation. In addition to its interaction with mitochondrial seryl-tRNA synthetase (SARS2), we further discovered an interaction between mitochondrial threonyl-tRNA synthetase (TARS2) and METTL8-Iso1. METTL8-Iso1 substantially stimulated the aminoacylation activities of SARS2 and TARS2 in vitro, suggesting a functional connection between mitochondrial tRNA modification and charging. Altogether, our results deepen the mechanistic insights into mitochondrial m3C32 biogenesis and provide a valuable route to prepare cytoplasmic/bacterial tRNAs with only a m3C32 moiety, aiding in future efforts to investigate its effects on tRNA structure and function.