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

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


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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Referenced accessions


  1. 1.

    From birth to death: the complex lives of eukaryotic mRNAs. Science 309, 1514–1518 (2005).

  2. 2.

    et al. Widespread occurrence of N6-methyladenosine in bacterial mRNA. Nucleic Acids Res. 43, 6557–6567 (2015).

  3. 3.

    et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

  4. 4.

    , & Reversible RNA adenosine methylation in biological regulation. Trends Genet. 29, 108–115 (2013).

  5. 5.

    et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).

  6. 6.

    et al. N6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

  7. 7.

    in Fine-Tuning of RNA Functions by Modification and Editing Vol. 12 (ed. Grosjean, H.) 141–177 (Springer, 2005).

  8. 8.

    , , & Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

  9. 9.

    et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18–29 (2013).

  10. 10.

    et al. m6A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15, 707–719 (2014).

  11. 11.

    et al. RNA-methylation-dependent RNA processing controls the speed of the circadian clock. Cell 155, 793–806 (2013).

  12. 12.

    et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).

  13. 13.

    et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

  14. 14.

    et al. N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat. Cell Biol. 16, 191–198 (2014).

  15. 15.

    et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 24, 1403–1419 (2014).

  16. 16.

    et al. N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function. Genomics Proteomics Bioinformatics 11, 8–17 (2013).

  17. 17.

    et al. High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Cell 155, 1409–1421 (2013).

  18. 18.

    et al. Structure and thermodynamics of N6-methyladenosine in RNA: a spring-loaded base modification. J. Am. Chem. Soc. 137, 2107–2115 (2015).

  19. 19.

    et al. N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518, 560–564 (2015).

  20. 20.

    et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519, 486–490 (2015).

  21. 21.

    et al. High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence. Proc. Natl. Acad. Sci. USA 111, 664–669 (2014).

  22. 22.

    , & Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection. Proc. Natl. Acad. Sci. USA 109, 131–136 (2012).

  23. 23.

    , , , & tRNA selection and kinetic proofreading in translation. Nat. Struct. Mol. Biol. 11, 1008–1014 (2004).

  24. 24.

    , , , & Coordinated conformational and compositional dynamics drive ribosome translocation. Nat. Struct. Mol. Biol. 20, 718–727 (2013).

  25. 25.

    et al. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010).

  26. 26.

    et al. Human tRNA(Lys3)(UUU) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism. J. Mol. Biol. 416, 467–485 (2012).

  27. 27.

    et al. Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function. RNA 16, 2319–2324 (2010).

  28. 28.

    et al. Structure of the 30S ribosomal subunit. Nature 407, 327–339 (2000).

  29. 29.

    et al. pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA. Proc. Natl. Acad. Sci. USA 108, 79–84 (2011).

  30. 30.

    , , , & The role of fluctuations in tRNA selection by the ribosome. Proc. Natl. Acad. Sci. USA 104, 13661–13665 (2007).

  31. 31.

    et al. Translational tuning optimizes nascent protein folding in cells. Science 348, 444–448 (2015).

  32. 32.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  33. 33.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  34. 34.

    et al. Modified uridines with C5-methylene substituents at the first position of the tRNA anticodon stabilize U.G wobble pairing during decoding. J. Biol. Chem. 283, 18801–18811 (2008).

  35. 35.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  36. 36.

    , & Irreversible chemical steps control intersubunit dynamics during translation. Proc. Natl. Acad. Sci. USA 105, 15364–15369 (2008).

  37. 37.

    et al. Site-specific labeling of the ribosome for single-molecule spectroscopy. Nucleic Acids Res. 33, 182–189 (2005).

  38. 38.

    & Following the intersubunit conformation of the ribosome during translation in real time. Nat. Struct. Mol. Biol. 17, 793–800 (2010).

  39. 39.

    , & An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).

  40. 40.

    & Optimization of translation accuracy. Prog. Nucleic Acid Res. Mol. Biol. 31, 191–219 (1984).

  41. 41.

    , , & The kinetics of ribosomal peptidyl transfer revisited. Mol. Cell 30, 589–598 (2008).

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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.


  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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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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