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

Meng-Han Huang; Jin-Tao Wang; Jian-Hui Zhang; Xue-Ling Mao; Gui-Xin Peng; Xiuying Lin; Daizhu Lv; Chen Yuan; Huan Lin; En-Duo Wang; Xiao-Long Zhou

doi:10.1016/j.scib.2023.08.002

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

Sci Bull (Beijing). 2023 Sep 30;68(18):2094-2105. doi: 10.1016/j.scib.2023.08.002. Epub 2023 Aug 2.

Authors

Meng-Han Huang¹, Jin-Tao Wang¹, Jian-Hui Zhang², Xue-Ling Mao¹, Gui-Xin Peng³, Xiuying Lin⁴, Daizhu Lv⁵, Chen Yuan⁶, Huan Lin⁷, En-Duo Wang⁸, Xiao-Long Zhou⁹

Affiliations

¹ Key Laboratory of RNA Science and Engineering, State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
² Key Laboratory of RNA Science and Engineering, State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China.
³ Key Laboratory of RNA Science and Engineering, State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
⁴ State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China; School of Life Sciences, Hainan University, Haikou 570228, China.
⁵ Analysis and Testing Center, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China.
⁶ School of Pharmacy, China Pharmaceutical University, Nanjing 211198, China.
⁷ State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China.
⁸ Key Laboratory of RNA Science and Engineering, State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: edwang@sibcb.ac.cn.
⁹ Key Laboratory of RNA Science and Engineering, State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China. Electronic address: xlzhou@sibcb.ac.cn.

PMID: 37573249
DOI: 10.1016/j.scib.2023.08.002

Abstract

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 tRNA^Thr and tRNA^Ser(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 tRNA^ThrN⁶-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.

Keywords: 3-methylcytidine; Aminoacylation; Methyltransferase; tRNA.

MeSH terms

COVID-19*
Humans
Methyltransferases / genetics
Protein Isoforms
RNA, Mitochondrial / genetics
RNA, Transfer / genetics

Substances

RNA, Mitochondrial
RNA, Transfer
Protein Isoforms
Methyltransferases