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RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis

Nature Structural & Molecular Biology volume 19, pages 900–905 (2012)Cite this article

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Abstract

The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation. tRNA methylation analysis of the double-knockout mice demonstrated complementary target-site specificities for Dnmt2 and NSun2 and a complete loss of cytosine-C5 tRNA methylation. Steady-state levels of unmethylated tRNAs were substantially reduced, and loss of Dnmt2 and NSun2 was further associated with reduced rates of overall protein synthesis. These results establish a biologically important function for cytosine-C5 tRNA methylation in mammals and suggest that this modification promotes mouse development by supporting protein synthesis.

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Figure 1: Characterization of Dnmt2−/−;NSun2−/− mice.
Figure 2: Cellular and molecular pathology of the Dnmt2−/−;NSun2−/− phenotype.
Figure 3: tRNA methylation analysis.
Figure 4: Analysis of tRNA levels.
Figure 5: Analysis of protein-synthesis rates.

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Acknowledgements

We thank M. Bewerunge-Hudler and O. Heil for mRNA-expression profiling, C. Falckenhayn and G. Raddatz for analyzing 454 sequencing results, J. Hummel-Eisenbeiß and K. Hanna for technical assistance, and J. Burhenne for providing LC-MS. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP1463 to F.L., FOR1082 to M.S., M.H. and F.L. and STO 859/2-1 to G.S.). S.H. is supported by a bridging project grant from the DKFZ-ZMBH Alliance, and F.T. is supported by the Institute of Genetics and Biophysics A. Buzzati-Traverso, C.N.R., Italy.

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Authors and Affiliations

  1. Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany

    Francesca Tuorto, Reinhard Liebers, Tanja Musch, Matthias Schaefer & Frank Lyko

  2. Helmholtz Junior Research Group Posttranscriptional Control of Gene Expression, Deutsches Krebsforschungszentrum, Heidelberg, Germany

    Sarah Hofmann & Georg Stoecklin

  3. Institute of Pharmacy, Johannes Gutenberg University of Mainz, Mainz, Germany

    Stefanie Kellner & Mark Helm

  4. Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK

    Michaela Frye

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  1. Francesca Tuorto
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  2. Reinhard Liebers
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Contributions

F.L. conceived the study. F.T. performed the mouse husbandry and the mouse phenotyping. M.F. contributed the NSun2−/− mice. F.T., R.L. and T.M. performed the RNA bisulfite sequencing. S.K. performed the LC-MS analysis. F.T. established the MEF cell lines. F.T., R.L., T.M. and S.H. characterized the MEF cell lines. F.T., M.S., S.H., S.K., M.H., G.S. and F.L. designed the experiments and interpreted the results. F.L. wrote the manuscript with contributions from F.T. and G.S. as well as input from the other coauthors.

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Correspondence to Frank Lyko.

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Tuorto, F., Liebers, R., Musch, T. et al. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat Struct Mol Biol 19, 900–905 (2012). https://doi.org/10.1038/nsmb.2357

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