Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

FTO controls reversible m6Am RNA methylation during snRNA biogenesis

Abstract

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2′-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2′-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite d-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: FTO selectively demethylates small nuclear RNAs.
Fig. 2: Reversible N6,2′-O-dimethyladenosine (m6Am) in small nuclear RNAs.
Fig. 3: m2-snRNAs are reversibly regulated in oncometabolite-dependent cancer models.
Fig. 4: m2-snRNAs are incorporated into snRNPs.

Similar content being viewed by others

Code availability

All custom code used in this study can be obtained upon request from the corresponding author.

Data availability

Sequencing data that support the findings of this study have been deposited in the NCBI GEO database under accession number GSE107872. Other data shown in the article are available from the corresponding authors upon reasonable request.

References

  1. Hadjiolov, A. A., Venkov, P. V. & Tsanev, R. G. Ribonucleic acids fractionation by density-gradient centrifugation and by agar gel electrophoresis: a comparison. Anal. Biochem. 17, 263–267 (1966).

    Article  CAS  Google Scholar 

  2. Dreyfuss, G., Philipson, L. & Mattaj, I. W. Ribonucleoprotein particles in cellular processes. J. Cell. Biol. 106, 1419–1425 (1988).

    Article  CAS  Google Scholar 

  3. Lührmann, R. Functions of U-snRNPs. Mol. Biol. Rep. 14, 183–192 (1990).

    Article  Google Scholar 

  4. Kunkel, G. R., Maser, R. L., Calvet, J. P. & Pederson, T. U6 small nuclear RNA is transcribed by RNA polymerase III. Proc. Natl Acad. Sci. USA 83, 8575–8579 (1986).

  5. Dahlberg, J. E. & Lund, E. Structure and expression of U-snRNA genes. Mol. Biol. Rep. 12, 139–143 (1987).

    Article  CAS  Google Scholar 

  6. Baillat, D. et al. Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell 123, 265–276 (2005).

    Article  CAS  Google Scholar 

  7. Cory, S. & Adams, J. M. Modified 5′-termini in small nuclear RNAs of mouse myeloma cells. Mol. Biol. Rep. 2, 287–294 (1975).

    Article  CAS  Google Scholar 

  8. Busch, H., Reddy, R., Rothblum, L. & Choi, Y. C. SnRNAs, SnRNPs, and RNA processing. Annu. Rev. Biochem. 51, 617–654 (1982).

    Article  CAS  Google Scholar 

  9. Mattaj, I. W. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell 46, 905–911 (1986).

    Article  CAS  Google Scholar 

  10. Matera, A. G. & Wang, Z. A day in the life of the spliceosome. Nat. Rev. Mol. Cell Biol. 15, 108–121 (2014).

    Article  CAS  Google Scholar 

  11. Pellizzoni, L. Chaperoning ribonucleoprotein biogenesis in health and disease. EMBO Rep. 8, 340–345 (2007).

    Article  CAS  Google Scholar 

  12. Patel, S. B. & Bellini, M. The assembly of a spliceosomal small nuclear ribonucleoprotein particle. Nucleic Acids Res. 36, 6482–6493 (2008).

    Article  CAS  Google Scholar 

  13. Shukla, S. & Parker, R. Quality control of assembly-defective U1 snRNAs by decapping and 5′-to-3′ exonucleolytic digestion. Proc. Natl Acad. Sci. USA 111, E3277–E3286 (2014).

  14. Ishikawa, H. et al. Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis. Nucleic Acids Res. 42, 2708–2724 (2014).

    Article  CAS  Google Scholar 

  15. Karijolich, J. & Yu, Y. T. Spliceosomal snRNA modifications and their function. RNA Biol. 7, 192–204 (2010).

    Article  CAS  Google Scholar 

  16. Pendleton, K. E. et al. The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retention. Cell 169, 824–835.e14 (2017).

  17. Jia, G. et al. N 6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).

    Article  CAS  Google Scholar 

  18. Jia, G. et al. Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO. FEBS Lett. 582, 3313–3319 (2008).

    Article  CAS  Google Scholar 

  19. Mauer, J. et al. Reversible methylation of m6Am in the 5′ cap controls mRNA stability. Nature 541, 371–375 (2017).

    Article  CAS  Google Scholar 

  20. Wei, C., Gershowitz, A. & Moss, B. N6,O2′-Dimethyladenosine a novel methylated ribonucleoside next to the 5′ terminal of animal cell and virus mRNAs. Nature 257, 251–253 (1975).

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

    Article  Google Scholar 

  22. Hess, M. E. et al. The fat mass and obesity associated gene (Fto) regulates activity of the dopaminergic midbrain circuitry. Nat. Neurosci. 16, 1042–10489 (2013).

  23. Reddy, R., Henning, D., Epstein, P. & Busch, H. Primary and secondary structure of U2 snRNA. Nucleic Acids Res. 9, 5645–5658 (1981).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

  26. Mowry, K. L. & Steitz, J. A. Identification of the human U7 snRNP as one of several factors involved in the 3′ end maturation of histone premessenger RNA’s. Science 238, 1682–1687 (1987).

    Article  CAS  Google Scholar 

  27. Jawdekar, G. W. & Henry, R. W. Transcriptional regulation of human small nuclear RNA genes. Biochim. Biophys. Acta 1779, 295–305 (2008).

    Article  CAS  Google Scholar 

  28. Bohnsack, M. T. & Sloan, K. E. Modifications in small nuclear RNAs and their roles in spliceosome assembly and function. Biol. Chem. 399, 1265–1276 (2018).

    Article  CAS  Google Scholar 

  29. Wei, J. et al. Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm. Mol. Cell 71, 973–985.e5 (2018).

    Article  CAS  Google Scholar 

  30. Hogeweg, P. & Konings, D. A. U1 snRNA: the evolution of its primary and secondary structure. J. Mol. Evol. 21, 323–333 (1984–1985).

  31. Bringmann, P. & Luhrmann, R. Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6. FEBS Lett. 213, 309–315 (1987).

    Article  CAS  Google Scholar 

  32. Yang, Y. et al. UOK 262 cell line, fumarate hydratase deficient (FH-/FH-) hereditary leiomyomatosis renal cell carcinoma: in vitro and in vivo model of an aberrant energy metabolic pathway in human cancer. Cancer Genet. Cytogenet. 196, 45–55 (2010).

    Article  CAS  Google Scholar 

  33. Gerken, T. et al. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase. Science 318, 1469–1472 (2007).

  34. Aik, W. et al. Structural basis for inhibition of the fat mass and obesity associated protein (FTO). J. Med. Chem. 56, 3680–3688 (2013).

    Article  CAS  Google Scholar 

  35. Ward, P. S. et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting α-ketoglutarate to 2-hydroxyglutarate. Cancer Cell. 17, 225–234 (2010).

  36. Wang, F. et al. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science 340, 622–626 (2013).

    Article  CAS  Google Scholar 

  37. Rohle, D. et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 340, 626–630 (2013).

    Article  CAS  Google Scholar 

  38. Bennett Saidu, N. E. et al. Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells. Oncotarget 9, 9088–9099 (2018).

    PubMed  PubMed Central  Google Scholar 

  39. Gabanella, F. et al. Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs. PLoS One 2, e921 (2007).

    Article  Google Scholar 

  40. Lotti, F. et al. An SMN-dependent U12 splicing event essential for motor circuit function. Cell 151, 440–454 (2012).

    Article  CAS  Google Scholar 

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

  42. Bartosovic, M. et al. N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3′-end processing. Nucleic Acids Res. 45, 11356–11370 (2017).

    Article  CAS  Google Scholar 

  43. Wei, J. et al. Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm. Mol. Cell 71, 973–985.e975 (2018).

    Article  CAS  Google Scholar 

  44. Su, R. et al. R-2HG exhibits anti-tumor activity by targeting FTO/m6A/MYC/CEBPA Signaling. Cell 172, 90–105.e123 (2018).

    Article  CAS  Google Scholar 

  45. Ke, S. et al. m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev. 31, 990–1006 (2017).

    Article  CAS  Google Scholar 

  46. Pomeranz Krummel, D. A., Oubridge, C., Leung, A. K., Li, J. & Nagai, K. Crystal structure of human spliceosomal U1 snRNP at 5.5 A resolution. Nature 458, 475–480 (2009).

    Article  CAS  Google Scholar 

  47. Fury, M. G. & Zieve, G. W. U6 snRNA maturation and stability. Exp. Cell Res. 228, 160–163 (1996).

    Article  CAS  Google Scholar 

  48. Dvinge, H. RNA components of the spliceosome regulate tissue- and cancer-specific alternative splicing. bioRxiv Preprint at https://doi.org/10.1101/326983 (2018).

  49. Zhang, Z. et al. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 133, 585–600 (2008).

    Article  CAS  Google Scholar 

  50. Kaida, D. et al. U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation. Nature 468, 664–668 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  Google Scholar 

  53. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome. Biol. 15, 550 (2014).

    Article  Google Scholar 

  54. Carissimi, C., Saieva, L., Gabanella, F. & Pellizzoni, L. Gemin8 is required for the architecture and function of the survival motor neuron complex. J. Biol. Chem. 281, 37009–37016 (2006).

    Article  CAS  Google Scholar 

  55. Monecke, T., Dickmanns, A. & Ficner, R. Structural basis for m7G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1. Nucleic Acids Res. 37, 3865–3877 (2009).

    Article  CAS  Google Scholar 

  56. Schulz, D. & Rentmeister, A. An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity in E. coli cell lysate. RNA Biol. 9, 577–586 (2012).

    Article  CAS  Google Scholar 

  57. Chen, Q. et al. Untargeted plasma metabolite profiling reveals the broad systemic consequences of xanthine oxidoreductase inactivation in mice. PLoS One 7, e37149 (2012).

    Article  CAS  Google Scholar 

  58. Katz, Y., Wang, E. T., Airoldi, E. M. & Burge, C. B. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat. Methods 7, 1009–1015 (2010).

    Article  CAS  Google Scholar 

  59. Elemento, O., Slonim, N. & Tavazoie, S. A universal framework for regulatory element discovery across all genomes and data types. Mol. Cell 28, 337–350 (2007).

    Article  CAS  Google Scholar 

  60. Ray, D. et al. A compendium of RNA-binding motifs for decoding gene regulation. Nature 499, 172–177 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Keshari (MSKCC) for UOK262 cells. This work was supported by NIH grants R01DA037755 and R01CA186702 (S.R.J.), R01GM123977 (H.G.), R01NS102451 (L.P.), P01HD67244, P01HD067244 (M.S.), and UO1HL121828 (S.S.G.), by the French Centre National de la Recherche Scientifique (T.G., J.-J.V., F.D.), DFG Priority Program grant RE2796/3-2 (A.R.) and by a DFG Research Fellowship (J.M.).

Author information

Authors and Affiliations

Authors

Contributions

S.R.J., L.P. and J.M. designed the experiments. J.M. carried out the experiments. F.D., J.-J.V. and T.G. synthesized modified oligonucleotides. A.R. produced hTGS1. S.S.G. and M.S. carried out mass spectrometry analysis. V.D. performed analysis of snRNA and snRNP stability, binding and assembly. B.R.H. helped with RNA extraction and sample preparation for mass spectrometry analysis. H.G. carried out the computational splicing analysis. S.R.J. and J.M. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Samie R. Jaffrey.

Ethics declarations

Competing interests

S.R.J. is scientific founder of, advisor to, and owns equity in Gotham Therapeutics.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12

Reporting Summary

Supplementary Table 1

List of 26,087 annotated transcription start sites.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mauer, J., Sindelar, M., Despic, V. et al. FTO controls reversible m6Am RNA methylation during snRNA biogenesis. Nat Chem Biol 15, 340–347 (2019). https://doi.org/10.1038/s41589-019-0231-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0231-8

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing