This study is led by Profs. Xiao-Long Zhou and En-Duo Wang (CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences).
tRNA is a key adaptor molecule in mRNA translation. There are a large number of post-transcriptional modifications on tRNA, which regulate the speed and fidelity of protein synthesis. 3-Methylcytosine (m 3 C) modification is widely found at position 32 (m 3 C32) of the anticodon loops of several cytoplasmic and mitochondrial tRNAs in eukaryotes.
A previous study by the same lab has found that the m 3 C32 modification of human cytoplasmic tRNAs was mediated by METTL2A/2B and METTL6, while that of human mitochondrial tRNA Thr (hmtRNA Thr ) and tRNA Ser (UCN) (hmtRNA Ser (UCN)) is catalyzed by METTL8; Human METTL8 generates two protein isoforms of different lengths by alternative splicing of mRNA. The long-length form, METTL8-Iso1, was targeted into mitochondria to catalyze the m 3 C32 modification of hmtRNA Thr and hmtRNA Ser (UCN); while the short-length form, METTL8-Iso4, was located in the nucleolus with unknown function. The only difference between the two isoforms is a 28-amino acid N-terminal extension peptide in METTL8-Iso1. Whether METTL8-Iso4 has m 3 C32 methyltransferase activity and the role of the N-terminal extension of METTL8-Iso1 in mitochondrial tRNA m 3 C32 modification is unknown. It is also unclear whether cytoplasmic or mitochondrial m 3 C32 modification enzymes can cross-recognize tRNAs from different cellular compartments. In addition, since most tRNA m 3 C32 modifications require N 6 -threonylcarbamoyl adenosine modification at position 37 (t 6 A37) in the anticodon loop as a prerequisite, the preparation of tRNA molecules containing only m 3 C32 modification has not been fully achieved.
To address these questions, the researchers confirmed the conservation of the N-terminal extension (N-extension) of METTL8-Iso1 through multiple sequence alignment. In vitro enzyme activity determination revealed that METTL8-Iso4 had no m 3 C32 modification activity. They further proved that the N-extension of METTL8-Iso1 acted as a key tRNA-binding element in the catalytic process. Two completely conserved amino acid residues in all METTL2A/2B/8 proteins were identified. METTL8-Iso1 was able to mediate m 3 C32 modification for both cytoplasmic and E. coli tRNAs, which was not reliant on t 6 A37. However, cytoplasmic m 3 C32 modification enzymes METTL2A and METTL6 were unable to catalyze m 3 C32 modification of mitochondrial tRNA, indicating that METTL8-Iso1 has a more relaxed substrate specificity. The m 3 C32 modification did not affect the t 6 A37 modification and aminoacylation levels of hmtRNA Thr . Finally, they also revealed that METTL8-Iso1 interacted with mitochondrial seryl-tRNA synthetase (SARS2) and mitochondrial threonyl-tRNA synthetase (TARS2), respectively, and significantly promoted aminoacylation activity of SARS2 and TARS2.
In summary, this work reveals the molecular mechanism of mitochondrial tRNA m 3 C32 biogenesis mediated by METTL8, which relies on a specific N-extension as a key RNA-binding element. METTL8 had a broad spectrum of heterogenous tRNA substrates, which provided a basis for preparation of tRNAs containing only a m 3 C moiety. This work provides a comprehensive understanding of the conservation and difference between cytoplasmic and mitochondrial tRNA m 3 C modification.
https://doi.org/10.1016/j.scib.2023.08.002
Science Bulletin