Molecular basis for human mitochondrial tRNA m3C modification by alternatively spliced METTL8

Abstract

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.

Selective degradation of tRNASer(AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease

“…We have successfully purified mature SARS2 from E. coli ( 32 , 33 ). We obtained mature forms of wild-type, Ins12, and dupC, and then performed thermal shift assays (TSA) to determine the stability of purified proteins in vitro ( 42 ).…”

“…A construct expressing the full-length mature form of SARS2 with a C-terminal His 6 -tag was developed according to a previous report ( 33 ). Constructs expressing Ins12 and dupC were generated using mutagenesis kits (TOYOBO, Osaka, Japan).…”

Ovarian cancer subtypes based on the regulatory genes of RNA modifications: Novel prediction model of prognosis

“…For ac4C, writers included NAT10 and THUMPD1; and erasers and readers remain unknown ( 18 , 19 ). For m3C, only one writer METTL8 was discovered, and erasers and readers were still undetected ( 20 ). For m6Am, writers included PCIF1, METTL3, and METTL4; eraser involved FTO; and no readers were discovered ( 21 – 24 ).…”

Integrative analysis of m3C associated genes reveals METTL2A as a potential oncogene in breast Cancer

“…METTL6 interacts with seryl-tRNA synthetase (SerRS, encoded by SARS1) to mediate the biogenesis of m3C32 modification in human tRNA Ser [ 9 ]. In contrast to the preceding three cytoplasmic m3C32-modifying enzymes, it had been established that METTL8 functioned in mitochondria [ 10 , 11 ].…”

“…The m3C32 modification of mitochondria tRNA Ser and tRNA Thr were catalyzed by the mitochondrial protein METTL8 and the high expression of METTL8 contributed an enhanced respiratory chain activity in pancreatic cancer [ 10 ]. The alternative mRNA splicing research reported that one of the isoforms of METTL8, METLL8-iso1, had been investigated to be transported to mitochondrial with the assistance of its N-terminal mitochondrial targeting sequence [ 11 ]. Aside from the classical m3C32 modification of mitochondrial tRNAs, upregulated of METTL8 by Yin Yang 1 (YY1) transcription factor mediated the m3C modification on the mRNA of AT-rich interactive domain-containing protein 1A and attenuated its translation, which induced the migration of breast cancer cell lines [ 17 ].…”