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.

  • Letter
  • Published:

Identification of direct targets and modified bases of RNA cytosine methyltransferases

Nature Biotechnology volume 31, pages 458–464 (2013)Cite this article

Subjects

Abstract

The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine–mediated RNA immunoprecipitation (Aza-IP), a technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes. In addition, it revealed many tRNA and noncoding RNA targets not previously associated with NSUN2. Notably, we observed a high frequency of C→G transversions at the cytosine residues targeted by both enzymes, allowing identification of the specific methylated cytosine(s) in target RNAs. Given the mechanistic similarity of RNA cytosine methyltransferases, Aza-IP may be generally applicable for target identification.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: RNA cytosine methylation mechanism and Aza-IP experimental design.
Figure 2: Aza-IP analysis of DNMT2 RNA targets.
Figure 3: Aza-IP analysis of NSUN2 RNA targets.
Figure 4: New ncRNA targets and sites for human NSUN2, and validation through siRNA knockdown and bisulfite sequencing.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Motorin, Y., Lyko, F. & Helm, M. 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res. 38, 1415–1430 (2010).

    Article  CAS  Google Scholar 

  2. Squires, J.E. et al. Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res. 40, 5023–5033 (2012).

    Article  CAS  Google Scholar 

  3. Jurkowski, T.P. et al. Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism. RNA 14, 1663–1670 (2008).

    Article  CAS  Google Scholar 

  4. King, M.Y. & Redman, K.L. RNA methyltransferases utilize two cysteine residues in the formation of 5-methylcytosine. Biochemistry 41, 11218–11225 (2002).

    Article  CAS  Google Scholar 

  5. Goll, M.G. et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science 311, 395–398 (2006).

    Article  CAS  Google Scholar 

  6. Rai, K. et al. Dnmt2 functions in the cytoplasm to promote liver, brain, and retina development in zebrafish. Genes Dev. 21, 261–266 (2007).

    Article  CAS  Google Scholar 

  7. Schaefer, M. et al. RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage. Genes Dev. 24, 1590–1595 (2010).

    Article  CAS  Google Scholar 

  8. Schaefer, M. & Lyko, F. Solving the Dnmt2 enigma. Chromosoma 119, 35–40 (2010).

    Article  CAS  Google Scholar 

  9. Durdevic, Z. et al. Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2. EMBO Rep. 14, 269–275 (2013).

    Article  CAS  Google Scholar 

  10. Tuorto, F. et al. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat. Struct. Mol. Biol. 19, 900–905 (2012).

    Article  CAS  Google Scholar 

  11. Brzezicha, B. et al. Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the pre-tRNA Leu (CAA). Nucleic Acids Res. 34, 6034–6043 (2006).

    Article  CAS  Google Scholar 

  12. Frye, M. & Watt, F.M. The RNA methyltransferase Misu (NSun2) mediates Myc-induced proliferation and is upregulated in tumors. Curr. Biol. 16, 971–981 (2006).

    Article  CAS  Google Scholar 

  13. Hussain, S. et al. The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability. J. Cell Biol. 186, 27–40 (2009).

    Article  CAS  Google Scholar 

  14. Blanco, S. et al. The RNA-methyltransferase Misu (NSun2) poises epidermal stem cells to differentiate. PLoS Genet. 7, e1002403 (2011).

    Article  CAS  Google Scholar 

  15. Abbasi-Moheb, L. et al. Mutations in NSUN2 cause autosomal- recessive intellectual disability. Am. J. Hum. Genet. 90, 847–855 (2012).

    Article  CAS  Google Scholar 

  16. Khan, M.A. et al. Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability. Am. J. Hum. Genet. 90, 856–863 (2012).

    Article  CAS  Google Scholar 

  17. Martinez, F.J. et al. Whole exome sequencing identifies a splicing mutation in NSUN2 as a cause of a Dubowitz-like syndrome. J. Med. Genet. 49, 380–385 (2012).

    Article  CAS  Google Scholar 

  18. Santi, D.V., Garrett, C.E. & Barr, P.J. On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell 33, 9–10 (1983).

    Article  CAS  Google Scholar 

  19. Lu, L.W., Chiang, G.H., Medina, D. & Randerath, K. Drug effects on nucleic acid modification. I. A specific effect of 5-azacytidine on mammalian transfer RNA methylation in vivo. Biochem. Biophys. Res. Commun. 68, 1094–1101 (1976).

    Article  CAS  Google Scholar 

  20. Lu, L.J. & Randerath, K. Effects of 5-azacytidine on transfer RNA methyltransferases. Cancer Res. 39, 940–949 (1979).

    CAS  PubMed  Google Scholar 

  21. Schaefer, M., Hagemann, S., Hanna, K. & Lyko, F. Azacytidine inhibits RNA methylation at DNMT2 target sites in human cancer cell lines. Cancer Res. 69, 8127–8132 (2009).

    Article  CAS  Google Scholar 

  22. Liu, K., Wang, Y.F., Cantemir, C. & Muller, M.T. Endogenous assays of DNA methyltransferases: evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo. Mol. Cell Biol. 23, 2709–2719 (2003).

    Article  CAS  Google Scholar 

  23. Schermelleh, L. et al. Trapped in action: direct visualization of DNA methyltransferase activity in living cells. Nat. Methods 2, 751–756 (2005).

    Article  CAS  Google Scholar 

  24. Yang, X., Lay, F., Han, H. & Jones, P.A. Targeting DNA methylation for epigenetic therapy. Trends Pharmacol. Sci. 31, 536–546 (2010).

    Article  Google Scholar 

  25. Jackson-Grusby, L., Laird, P.W., Magge, S.N., Moeller, B.J. & Jaenisch, R. Mutagenicity of 5-aza-2′-deoxycytidine is mediated by the mammalian DNA methyltransferase. Proc. Natl. Acad. Sci. USA 94, 4681–4685 (1997).

    Article  CAS  Google Scholar 

  26. Auxilien, S., Guerineau, V., Szweykowska-Kulinska, Z. & Golinelli-Pimpaneau, B. The human tRNA m5C methyltransferase Misu is multisite-specific. RNA Biol. 9, 1331–1338 (2012).

    Article  CAS  Google Scholar 

  27. Becker, M. et al. Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling. Nucleic Acids Res. 40, 11648–11658 (2012).

    Article  CAS  Google Scholar 

  28. Motorin, Y. & Grosjean, H. Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme. RNA 5, 1105–1118 (1999).

    Article  CAS  Google Scholar 

  29. Hu, S., Wu, J., Chen, L. & Shan, G. Signals from noncoding RNAs: unconventional roles for conventional pol III transcripts. Int. J. Biochem. Cell Biol. 44, 1847–1851 (2012).

    Article  CAS  Google Scholar 

  30. Gerard, M.A. et al. The scaRNA2 is produced by an independent transcription unit and its processing is directed by the encoding region. Nucleic Acids Res. 38, 370–381 (2010).

    Article  CAS  Google Scholar 

  31. Sakita-Suto, S. et al. Aurora-B regulates RNA methyltransferase NSUN2. Mol. Biol. Cell 18, 1107–1117 (2007).

    Article  CAS  Google Scholar 

  32. Guelorget, A. & Golinelli-Pimpaneau, B. Mechanism-based strategies for trapping and crystallizing complexes of RNA-modifying enzymes. Structure 19, 282–291 (2011).

    Article  CAS  Google Scholar 

  33. Nix, D.A., Courdy, S.J. & Boucher, K.M. Empirical methods for controlling false positives and estimating confidence in ChIP-Seq peaks. BMC Bioinformatics 9, 523 (2008).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Todaro, G.J. & Green, H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17, 299–313 (1963).

    Article  CAS  Google Scholar 

  36. Dong, A. et al. Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA. Nucleic Acids Res. 29, 439–448 (2001).

    Article  CAS  Google Scholar 

  37. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  Google Scholar 

  38. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  39. Koboldt, D.C. et al. VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics 25, 2283–2285 (2009).

    Article  CAS  Google Scholar 

  40. Laslett, D. & Canback, B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 32, 11–16 (2004).

    Article  CAS  Google Scholar 

  41. Chan, P.P. & Lowe, T.M. GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Clapier (hDNMT2 protein expression and purification), K. Rai (MTase assay set-up), V. Planelles, University of Utah (gift of the lentiviral expression construct), C. Maximiliano Rêgo Monteiro Filho and S. Dehghanizadeh (lentiviral protein expression, assays and IP experiments), J. Xu and A. Yerra (help on data not shown), and X. Cheng, Emory University (gift of pQE9 plasmid). We thank B. Dalley and N. Moss (library preparation and sequencing), T. Parnell, D. Nix, B. Milash, Y. Sun and K. Boucher (help and advice on analysis) and the Center for High Performance Computing, especially W.R. Cardoen. We thank C.J. Burrows for advice on reaction mechanisms, and D.A. Jones, C.J. Burrows and D.R. Davis for many helpful comments. This work was supported by the Howard Hughes Medical Institute, the Samuel Waxman Foundation and the National Cancer Institute CA24014 (for core facilities).

Author information

Authors and Affiliations

  1. Department of Oncological Sciences, Howard Hughes Medical Institute, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA

    Vahid Khoddami & Bradley R Cairns

Authors
  1. Vahid Khoddami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bradley R Cairns
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.K. contributed to experimental design and approaches, performed all experiments and analyses, and helped write the paper; B.R.C. contributed to experimental design and approaches, data interpretation and wrote (with V.K.) the manuscript.

Corresponding author

Correspondence to Bradley R Cairns.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Result 1, Supplementary Methods 1–4, Supplementary Figures 1–10 and Supplementary Tables 1–6 (PDF 935 kb)

Supplementary Data set 1

DNMT2 Aza-IP (tRNAs and signature analysis reports) (XLS 2182 kb)

Supplementary Data set 2

DNMT2 Aza-IP (All Genes) (XLS 13974 kb)

Supplementary Data set 3

Differentially methylated sites in wt vs Dnmt2-null MEFs RNAs (XLS 38 kb)

Supplementary Data set 4

NSUN2 Aza-IP (All Genes) (XLS 7609 kb)

Supplementary Data set 5

NSUN2 Aza-IP (VarScan signature analysis reports) (XLS 8770 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Khoddami, V., Cairns, B. Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat Biotechnol 31, 458–464 (2013). https://doi.org/10.1038/nbt.2566

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2566

This article is cited by

Search

Advanced 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