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NSUN3 methylase initiates 5-formylcytidine biogenesis in human mitochondrial tRNAMet

Nature Chemical Biology volume 12, pages 546–551 (2016)Cite this article

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Abstract

In human mitochondria, the AUA codon encodes methionine via a mitochondrial transfer RNA for methionine (mt-tRNAMet) that contains 5-formylcytidine (f5C) at the first position of the anticodon (position 34). f5C34 is required for deciphering the AUA codon during protein synthesis. Until now, the biogenesis and physiological role of f5C34 were unknown. We demonstrate that biogenesis of f5C34 is initiated by S-adenosylmethionine (AdoMet)-dependent methylation catalyzed by NSUN3, a putative methyltransferase in mitochondria. NSUN3-knockout cells showed strong reduction in mitochondrial protein synthesis and reduced oxygen consumption, leading to deficient mitochondrial activity. We reconstituted formation of 5-methylcytidine (m5C) at position 34 (m5C34) on mt-tRNAMet with recombinant NSUN3 in the presence of AdoMet, demonstrating that NSUN3-mediated m5C34 formation initiates f5C34 biogenesis. We also found two disease-associated point mutations in mt-tRNAMet that impaired m5C34 formation by NSUN3, indicating that a lack of f5C34 has pathological consequences.

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Figure 1: Determination of the carbon source for f5C34 in human mt-tRNAMet.
Figure 2: Lack of f5C34 in NSUN3-KO cells.
Figure 3: NSUN3 is required for mitochondrial activity.
Figure 4: In vitro reconstitution of m5C34 and impact of pathogenic mutations on m5C34 formation by NSUN3.

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Acknowledgements

We are grateful to Y. Sakaguchi, K. Miyauchi, T. Ohira, S. Kimura, H. Hojo, H. Lin, T. Chujo, Y. Ikeuchi and other members of Tsutomu Suzuki's laboratory for technical support and insightful discussions. Special thanks to O. Nureki, I. Koyama-Honda, N. Mizushima (University of Tokyo), C. Ushida (Hirosaki University) and Primetech Inc. (Japan) for materials and technical support. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and the Japan Society for the Promotion of Science (JSPS) (to Tsutomu Suzuki and Takeo Suzuki).

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  1. Saori Nakano and Takeo Suzuki: These authors contributed equally to this work.

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  1. Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan

    Saori Nakano, Takeo Suzuki, Layla Kawarada, Hiroyoshi Iwata, Kana Asano & Tsutomu Suzuki

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  1. Saori Nakano
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Tsutomu Suzuki and Takeo Suzuki designed this study. S.N., L.K. and H.I. performed experiments. K.A. assisted with biochemical work. All authors discussed the results. Tsutomu Suzuki, Takeo Suzuki. and S.N. wrote the paper. Tsutomu Suzuki supervised the work.

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Correspondence to Tsutomu Suzuki.

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Nakano, S., Suzuki, T., Kawarada, L. et al. NSUN3 methylase initiates 5-formylcytidine biogenesis in human mitochondrial tRNAMet. Nat Chem Biol 12, 546–551 (2016). https://doi.org/10.1038/nchembio.2099

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