Article | Published:

N6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics

Nature Structural & Molecular Biology volume 23, pages 110115 (2016) | Download Citation

Abstract

N6-methylation of adenosine (forming m6A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m6A in mRNA decoding. Although m6A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m6A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m6A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m6A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants GM51266 and GM099687 to J.D.P.; by grants from the Knut and Alice Wallenberg Foundation (RiboCORE) and the Swedish Research Council and the Human Frontier Science Program to M.E.; by NIH grants GM111858 to S.E.O'L.; by grants from the Israel Science Foundation (ISF) grant no. 1667/12), the Israeli Centers of Excellence (I-CORE) Program (ISF grants no. 41/11 and no. 1796/12) and the Ernest and Bonnie Beutler Research Program to G.R.; by a Human Frontier Science Program long-term fellowship to D.D.; and by a Stanford Bio-X fellowship to J. Choi. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, US Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research, NIH, US National Center for Research Resources, Biomedical Technology Program, and the US National Institute of General Medical Sciences. G.R. is supported as a member of the Sagol Neuroscience Network and by the Kahn Family Foundation. We thank P. Agris (University of Albany) for a human ASL reagent and members of Puglisi laboratory for discussion. J. Choi thanks J.B. Choi for support.

Author information

Author notes

    • Dan Dominissini

    Present address: Department of Chemistry, University of Chicago, Chicago, Illinois, USA.

Affiliations

  1. Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA.

    • Junhong Choi
    • , Jin Chen
    • , Alexey Petrov
    • , Arjun Prabhakar
    • , Seán E O'Leary
    •  & Joseph D Puglisi
  2. Department of Applied Physics, Stanford University, Stanford, California, USA.

    • Junhong Choi
    •  & Jin Chen
  3. Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Uppsala, Sweden.

    • Ka-Weng Ieong
    •  & Måns Ehrenberg
  4. Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, California, USA.

    • Hasan Demirci
  5. Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California, USA.

    • Hasan Demirci
    •  & S Michael Soltis
  6. Program in Biophysics, Stanford University, Stanford, California, USA.

    • Arjun Prabhakar
  7. Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel.

    • Dan Dominissini
    •  & Gideon Rechavi
  8. Israel & Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

    • Gideon Rechavi

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Contributions

J. Choi, K.-W.I. and H.D. performed all the experiments and the data analysis; J. Choi performed single-molecule experiments; K.-W.I. performed bulk kinetic experiments; H.D. performed X-ray crystallography, with the help of S.M.S. in material preparations. D.D. and G.R. provided reagents and conceived the project with J. Choi, K.-W.I., H.D., J. Chen, M.E. and J.D.P. J. Chen, A. Petrov and A. Prabhakar assisted in reagent preparation. J. Choi, K.-W.I., H.D., S.E.O'L., M.E. and J.D.P. wrote manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Joseph D Puglisi.

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DOI

https://doi.org/10.1038/nsmb.3148

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