Abstract

N6-Methyladenosine (m6A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified ('m6A levels') or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A-level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m6A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3′ untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Gene Expression Omnibus

References

  1. 1.

    & Nucleic acid modifications with epigenetic significance. Curr. Opin. Chem. Biol. 16, 516–524 (2012).

  2. 2.

    , & Mapping and significance of the mRNA methylome. Wiley Interdiscip. Rev. RNA 4, 397–422 (2013).

  3. 3.

    & The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat. Rev. Mol. Cell Biol. 15, 313–326 (2014).

  4. 4.

    & Dynamic RNA modifications in disease. Curr. Opin. Genet. Dev. 26, 47–52 (2014).

  5. 5.

    N6-methyl-Adenosine modification in messenger and long non-coding RNA. Trends Biochem. Sci. 38, 204–209 (2013).

  6. 6.

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

  7. 7.

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

  8. 8.

    et al. m6A-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

    , , , & Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3, 1233–1247 (1997).

  13. 13.

    et al. A METTL3–METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).

  14. 14.

    et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 24, 177–189 (2014).

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 20.

    , , & Transcriptome-wide mapping of N6-methyladenosine by m6A-seq. Methods Enzymol. 560, 131–147 (2015).

  21. 21.

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

  22. 22.

    et al. Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA. RNA 19, 1848–1856 (2013).

  23. 23.

    , , , & Mapping of N6-methyladenosine residues in bovine prolactin mRNA. Proc. Natl. Acad. Sci. USA 81, 5667–5671 (1984).

  24. 24.

    , , , & Global analysis reveals multiple pathways for unique regulation of mRNA decay in induced pluripotent stem cells. Genome Res. 22, 1457–1467 (2012).

  25. 25.

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

  26. 26.

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

  27. 27.

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

  28. 28.

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

  29. 29.

    , & Nucleic acid related compounds. 12. The facile and high-yield stannous chloride catalyzed monomethylation of the cis-glycol system of nucleosides by diazomethane. J. Org. Chem. 39, 1891–1899 (1974).

  30. 30.

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

  31. 31.

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

  32. 32.

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

  33. 33.

    et al. MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-seq data. Nucleic Acids Res. 40, e61 (2012).

  34. 34.

    et al. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-seq data. Proc. Natl. Acad. Sci. USA 111, E5593–E5601 (2014).

  35. 35.

    et al. CFIm25 links alternative polyadenylation to glioblastoma tumour suppression. Nature 510, 412–416 (2014).

  36. 36.

    et al. A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation. Genes Dev. 29, 2037–2053 (2015).

  37. 37.

    , & Mechanisms and consequences of alternative polyadenylation. Mol. Cell 43, 853–866 (2011).

  38. 38.

    & Alternative 3′ UTRs act as scaffolds to regulate membrane protein localization. Nature 522, 363–367 (2015).

  39. 39.

    , , & Global promotion of alternative internal exon usage by mRNA 3′ end formation factors. Mol. Cell 58, 819–831 (2015).

  40. 40.

    & The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site. Genes Dev. 29, 889–897 (2015).

  41. 41.

    et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).

  42. 42.

    , & Nucleic acid modifications in regulation of gene expression. Cell Chem. Biol. 23, 74–85 (2016).

  43. 43.

    , , , & Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression. Genes Dev. 27, 2380–2396 (2013).

  44. 44.

    et al. Identification of N6,N6-dimethyladenosine in transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin. Molecules 16, 5168–5181 (2011).

  45. 45.

    & Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔCT) method. Methods 25, 402–408 (2001).

  46. 46.

    et al. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proc. Natl. Acad. Sci. USA 110, 2876–2881 (2013).

  47. 47.

    , & TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).

  48. 48.

    et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

  49. 49.

    , & Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

  50. 50.

    et al. APADB: a database for alternative polyadenylation and microRNA regulation events. Database (Oxford) 2014, bau076 (2014).

  51. 51.

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

  52. 52.

    , & Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

  53. 53.

    et al. An RNA map predicting Nova-dependent splicing regulation. Nature 444, 580–586 (2006).

  54. 54.

    et al. Genome-wide determination of a broad ESRP-regulated posttranscriptional network by high-throughput sequencing. Mol. Cell. Biol. 32, 1468–1482 (2012).

  55. 55.

    et al. The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B. RNA 18, 1041–1049 (2012).

Download references

Acknowledgements

This study was supported by an MGH startup and ECOR grants to C.C.G. This study was also supported by National Institutes of Health (NIH) grant R01GM088342 and an Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA and Rose Hills Foundation Research Award to Y.X. Y.X. is supported by an Alfred Sloan Research Fellowship. P.D. was supported by the National Research Foundation of Singapore through the Singapore–MIT Alliance for Research and Technology, National Institute of Environmental Health Science grants ES002109 and ES024615, and National Science Foundation grant CHE-1308839. A.C.M. was supported by NIH grant DK090122. We thank D. Mirsky for copy editing the manuscript. We thank J. Wan for technical support on data analyses. We thank H. Chang, P. Batista, and K. Jeffrey for reading the manuscript and providing helpful comments.

Author information

Author notes

    • Benoit Molinie
    •  & Jinkai Wang

    These authors contributed equally to this work.

Affiliations

  1. Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Benoit Molinie
    • , Nicholas Van Wittenberghe
    • , Benjamin D Howard
    • , Kaveh Daneshvar
    • , Alan C Mullen
    •  & Cosmas C Giallourakis
  2. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.

    • Jinkai Wang
    • , Zhi-xiang Lu
    •  & Yi Xing
  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Kok Seong Lim
    • , Roman Hillebrand
    •  & Peter Dedon
  4. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Alan C Mullen
    •  & Cosmas C Giallourakis
  5. Center for Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Cosmas C Giallourakis

Authors

  1. Search for Benoit Molinie in:

  2. Search for Jinkai Wang in:

  3. Search for Kok Seong Lim in:

  4. Search for Roman Hillebrand in:

  5. Search for Zhi-xiang Lu in:

  6. Search for Nicholas Van Wittenberghe in:

  7. Search for Benjamin D Howard in:

  8. Search for Kaveh Daneshvar in:

  9. Search for Alan C Mullen in:

  10. Search for Peter Dedon in:

  11. Search for Yi Xing in:

  12. Search for Cosmas C Giallourakis in:

Contributions

C.C.G., Y.X., B.M., and J.W. conceived of the project, analyzed the data, and wrote the manuscript with input from all authors. B.M. did the experimental work related to m6A-LAIC-seq along with Z.L., N.V.W., B.D.H., K.D., and A.C.M. Y.X. and J.W. did the computational analyses of data with input from C.C.G. and B.M. P.D., K.S.L., and R.H. synthesized m6Am and performed RNA mass spectrometry.

Competing interests

C.C.G., Y.X., B.M., and J.W. are in the process of filing a patent application for m6A-LAIC-seq.

Corresponding authors

Correspondence to Yi Xing or Cosmas C Giallourakis.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1-7

Excel files

  1. 1.

    Supplementary Table 1

    m6A-LAIC-seq generated genome-wide m6A levels in H1-ESC and GM12878 cell lines. The m6A level and mapped read pairs from m6A-LAIC-seq are shown for each gene.

  2. 2.

    Supplementary Table 2

    Hypermethylated lincRNAs. The lincRNAs with m6A levels greater than 0.6 in H1-ESC and GM12878 are shown respectively.

  3. 3.

    Supplementary Table 3

    Splicing changes between supernatant and eluate in H1-ESC and GM12878 cell lines. The information of the significantly changed alternative splicing events between supernatant and eluate in either H1-ESC or GM12878 is shown.

  4. 4.

    Supplementary Table 4

    Alternative poly(A) changes between supernatant and eluate in H1-ESC and GM12878. The information of the significantly changed alternative poly(A) sites between supernatant and eluate in H1-ESC and GM12878 is shown respectively.

  5. 5.

    Supplementary Table 5

    Summary of APA changes caused by knockdown of m6A writers in different cell lines.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.3898

Further reading