Abstract

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

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References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

    & Modified nucleosides and bizarre 5′-termini in mouse myeloma mRNA. Nature 255, 28–33 (1975)

  6. 6.

    , & Methylated nucleotides block 5′ terminus of HeLa cell messenger RNA. Cell 4, 379–386 (1975)

  7. 7.

    et al. 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468, 452–456 (2010)

  8. 8.

    , & N6, O2′-dimethyladenosine a novel methylated ribonucleoside next to the 5′ terminal of animal cell and virus mRNAs. Nature 257, 251–253 (1975)

  9. 9.

    , & HeLa cell RNA (2′-O-methyladenosine-N6-)-methyltransferase specific for the capped 5′-end of messenger RNA. J. Biol. Chem. 253, 5033–5039 (1978)

  10. 10.

    , & 5′-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA. Biochemistry 15, 397–401 (1976)

  11. 11.

    et al. The fat mass and obesity associated gene (Fto) regulates activity of the dopaminergic midbrain circuitry. Nat. Neurosci. 16, 1042–1048 (2013)

  12. 12.

    et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767–772 (2015)

  13. 13.

    et al. FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat. Commun. 4, 1798 (2013)

  14. 14.

    et al. A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA. Sci. Rep. 1, 126 (2011)

  15. 15.

    et al. Fasting induced cytoplasmic Fto expression in some neurons of rat hypothalamus. PLoS One 8, e63694 (2013)

  16. 16.

    et al. A promoter-level mammalian expression atlas. Nature 507, 462–470 (2014)

  17. 17.

    et al. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Reports 8, 284–296 (2014)

  18. 18.

    et al. The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc. Natl Acad. Sci. USA 99, 12663–12668 (2002)

  19. 19.

    & Let me count the ways: mechanisms of gene regulation by miRNAs and siRNAs. Mol. Cell 29, 1–7 (2008)

  20. 20.

    , , & A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11, 1640–1647 (2005)

  21. 21.

    et al. Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res . 34, 4801–4815 (2006)

  22. 22.

    , , & Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010)

  23. 23.

    et al. 5′ UTR m6A promotes cap-independent translation. Cell 163, 999–1010 (2015)

  24. 24.

    et al. Decomposition of RNA methylome reveals co-methylation patterns induced by latent enzymatic regulators of the epitranscriptome. Mol. Biosyst. 11, 262–274 (2015)

  25. 25.

    et al. Dynamic m6A mRNA methylation directs translational control of heat shock response. Nature 526, 591–594 (2015)

  26. 26.

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

  27. 27.

    et al. Inactivation of the Fto gene protects from obesity. Nature 458, 894–898 (2009)

  28. 28.

    et al. Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations. Am. J. Hum. Genet. 85, 106–111 (2009)

  29. 29.

    & Towards a molecular understanding of microRNA-mediated gene silencing. Nat. Rev. Genet. 16, 421–433 (2015)

  30. 30.

    , & The absolute frequency of labeled N-6-methyladenosine in HeLa cell messenger RNA decreases with label time. J. Mol. Biol. 124, 487–499 (1978)

  31. 31.

    , , , & in Current Protocols in Nucleic Acid Chemistry Vol. 43 (ed S. et al. Beaucage) 3.19.11–13.19.27 (John Wiley & Sons, Inc., 2010)

  32. 32.

    , , & A base-labile group for 2′-OH protection of ribonucleosides: a major challenge for RNA synthesis. Chemistry 14, 9135–9138 (2008)

  33. 33.

    et al. Chemical solid-phase synthesis of 5′-triphosphates of DNA, RNA, and their analogues. Org. Lett. 12, 2190–2193 (2010)

  34. 34.

    et al. Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase. RNA 18, 856–868 (2012)

  35. 35.

    et al. X-ray structure and activities of an essential Mononegavirales L-protein domain. Nat. Commun. 6, 8749 (2015)

  36. 36.

    et al. Development of specific dengue virus 2′-O- and N7-methyltransferase assays for antiviral drug screening. Antiviral Res. 99, 292–300 (2013)

  37. 37.

    , , & The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc. Natl Acad. Sci. USA 99, 12663–12668 (2002)

  38. 38.

    , , & Analysis of mRNA decapping. Methods Enzymol. 448, 3–21 (2008)

  39. 39.

    , , , & A short adaptive path from DNA to RNA polymerases. Proc. Natl Acad. Sci. USA 109, 8067–8072 (2012)

  40. 40.

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

  41. 41.

    et al. Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347, 1002–1006 (2015)

  42. 42.

    et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  43. 43.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)

  44. 44.

    et al. BRIC-seq: a genome-wide approach for determining RNA stability in mammalian cells. Methods 67, 55–63 (2014)

  45. 45.

    et al. Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat. Protocols 9, 263–293 (2014)

  46. 46.

    et al. Analysis of overrepresented motifs in human core promoters reveals dual regulatory roles of YY1. Genome Res. 17, 798–806 (2007)

  47. 47.

    et al. The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA. Nature 530, 441–446 (2016)

  48. 48.

    et al. The BioMart community portal: an innovative alternative to large, centralized data repositories. Nucleic Acids Res . 43 (W1), W589–W598 (2015)

  49. 49.

    , , , & An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments. Nucleic Acids Res . 43, e2 (2015)

  50. 50.

    & RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011)

  51. 51.

    et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007)

  52. 52.

    et al. starBase: a database for exploring microRNA-mRNA interaction maps from Argonaute CLIP-Seq and Degradome-Seq data. Nucleic Acids Res . 39, D202–D209 (2011)

  53. 53.

    , & Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor. Nature 534, 558–561 (2016)

  54. 54.

    , , , & The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat. Protocols 7, 1534–1550 (2012)

  55. 55.

    , , , & AREsite2: an enhanced database for the comprehensive investigation of AU/GU/U-rich elements. Nucleic Acids Res . 44 (D1), D90–D95 (2016)

  56. 56.

    , , & Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105 (2009)

  57. 57.

    et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011)

  58. 58.

    , , & G-quadruplexes: the beginning and end of UTRs. Nucleic Acids Res . 36, 6260–6268 (2008)

  59. 59.

    & 5′-UTR G-quadruplex structures acting as translational repressors. Nucleic Acids Res . 38, 7022–7036 (2010)

  60. 60.

    et al. Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation. J. Biol. Chem. 289, 17299–17311 (2014)

  61. 61.

    et al. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell 20, 1278–1288 (2008)

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Acknowledgements

We thank P. Holliger for assistance with TGK-polymerase, A. O. Olarerin-George for assistance with data analysis, K. Meyer for early contributions on FTO-target mapping, and members of the Jaffrey laboratory for helpful comments and suggestions. This work was supported by NIH grants R01DA037755 (S.R.J.), P01HD67244 and R37HL87062 (S.S.G.), T32HD060600 and a Clinical and Translational Science Center Fellowship (A.V.G.), T32CA062948 (B.F.P.), and R01GM067005 (M.K.), by the French Centre National de la Recherche Scientifique (F.D.) and by the DFG (J.M. and B.L.).

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Affiliations

  1. Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, New York 10065, USA

    • Jan Mauer
    • , Anya V. Grozhik
    • , Deepak P. Patil
    • , Bastian Linder
    • , Brian F. Pickering
    • , Qiuying Chen
    • , Steven S. Gross
    •  & Samie R. Jaffrey
  2. Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854, USA

    • Xiaobing Luo
    • , Xinfu Jiao
    •  & Megerditch Kiledjian
  3. Department of Chemistry, IBMM UMR 5247, CNRS, Université de Montpellier ENSCM, UM Campus Triolet, Place E. Bataillon, 34095 Montpellier Cedex 05, France

    • Alexandre Blanjoie
    • , Jean-Jacques Vasseur
    •  & Françoise Debart
  4. Department of Physiology and Biophysics, Weill Cornell Medicine, Cornell University, New York, New York 10065, USA

    • Olivier Elemento
  5. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College, Cornell University, New York, New York 10065, USA

    • Olivier Elemento

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Contributions

S.R.J., M.K., J.M. and X.J. designed the experiments. J.M., X.L., X.J. and A.V.G. carried out the experiments. D.P.P. carried out the analysis of covariance. B.L. and B.F.P. analysed the previously published ribosome profiling datasets. F.D., J.V. and A.B. synthesized modified oligonucleotides. S.S.G. and Q.C. carried out mass spectrometry analysis. O.E. performed analysis on Fto knockout MeRIP-seq datasets. S.R.J. and J.M. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Samie R. Jaffrey.

Reviewer Information

Nature thanks O. Namy and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

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  1. 1.

    Supplementary Figure 1

    Uncropped images of western blots presented in Extended Data Fig. 3d, Numbers indicate protein standard molecular weight in kDa.

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  1. 1.

    Supplementary Tables

    This file contains Supplementary Tables 1-6 comprising: (1)-miCLIP-identified m6Am clusters including overlap with cage-tags and the core initiator motif (YYANW); (2) Annotated transcription start sites; (3) ANCOVA analysis of m6Am effect on mRNA half-life; (4) Sequences of modified oligonucleotides; (5) Relative stoichiometry of m6Am reads was determined by dividing normalized miCLIP reads by normalized RNA-Seq reads; (6) List of newly generated and previously published datasets used in the current study.

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https://doi.org/10.1038/nature21022

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