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:

N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation

Nature Chemical Biology volume 15, pages 88–94 (2019)Cite this article

Subjects

A Publisher Correction to this article was published on 08 February 2019

This article has been updated

Abstract

N6-Methyladenosine (m6A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs (snRNA) in humans. Although mRNA m6A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m6A methyltransferases for rRNAs and the function of rRNA m6A modifications are unknown. Here we report a new m6A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m6A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m6A modification in translation and in tumor biology.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: ZCCHC4 methylates 28S rRNA in vitro.
Fig. 2: ZCCHC4 catalyzes 28S rRNA methylation in cells.
Fig. 3: ZCCHC4 activity affects translation.
Fig. 4: ZCCHC4 expression in tumor tissues and its effects on xenograft tumor progression.

Similar content being viewed by others

Change history

  • 08 February 2019

    In the version of this article originally published, the references were incorrectly re-ordered during production. The hyphen in “N6-methyladenosine” in the title was also superscript. The errors have been corrected in the HTML and PDF versions of the paper.

References

  1. Roundtree, I. A., Evans, M. E., Pan, T. & He, C. Dynamic RNA modifications in gene expression regulation. Cell 169, 1187–1200 (2017).

    Article  CAS  Google Scholar 

  2. Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293–306 (2014).

    Article  CAS  Google Scholar 

  3. Gilbert, W. V., Bell, T. A. & Schaening, C. Messenger RNA modifications: form, distribution, and function. Science 352, 1408–1412 (2016).

    Article  CAS  Google Scholar 

  4. Zhao, B. S., Roundtree, I. A. & He, C. Post-transcriptional gene regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Cui, Q. et al. m6A RNA methylation regulates the self-renewal and tumorigenesis of glioblastoma stem cells. Cell Rep. 18, 2622–2634 (2017).

    Article  CAS  Google Scholar 

  7. Alarcón, C. R., Lee, H., Goodarzi, H., Halberg, N. & Tavazoie, S. F. N 6-methyladenosine marks primary microRNAs for processing. Nature 519, 482–485 (2015).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Xiang, Y. et al. RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature 543, 573–576 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Liu, J. et al. m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer. Nat. Cell Biol. 20, 1074–1083 (2018).

    Article  CAS  Google Scholar 

  12. Huang, H. et al. Recognition of RNA N 6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat. Cell Biol. 20, 285–295 (2018).

    Article  CAS  Google Scholar 

  13. Piekna-Przybylska, D., Decatur, W. A. & Fournier, M. J. The 3D rRNA modification maps database: with interactive tools for ribosome analysis. Nucleic Acids Res. 36, D178–D183 (2008).

    Article  CAS  Google Scholar 

  14. van Nues, R. W. & Watkins, N. J. Unusual C´/D´ motifs enable box C/D snoRNPs to modify multiple sites in the same rRNA target region. Nucleic Acids Res. 45, 2016–2028 (2017).

    PubMed  Google Scholar 

  15. Liu, N. et al. Probing N 6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA. RNA 19, 1848–1856 (2013).

    Article  CAS  Google Scholar 

  16. Sergiev, P. V., Aleksashin, N. A., Chugunova, A. A., Polikanov, Y. S. & Dontsova, O. A. Structural and evolutionary insights into ribosomal RNA methylation. Nat. Chem. Biol. 14, 226–235 (2018).

    Article  CAS  Google Scholar 

  17. Natchiar, S. K., Myasnikov, A. G., Kratzat, H., Hazemann, I. & Klaholz, B. P. Visualization of chemical modifications in the human 80 S ribosome structure. Nature 551, 472–477 (2017).

    CAS  PubMed  Google Scholar 

  18. Frye, M., Harada, B. T., Behm, M. & He, C. RNA modifications modulate gene expression during development. Science 361, 1346–1349 (2018).

    Article  CAS  Google Scholar 

  19. Liu, J. et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N 6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).

    Article  CAS  Google Scholar 

  20. Ping, X. L. et al. Mammalian WTAP is a regulatory subunit of the RNA N 6-methyladenosine methyltransferase. Cell Res. 24, 177–189 (2014).

    Article  CAS  Google Scholar 

  21. Wang, X. et al. Structural basis of N 6-adenosine methylation by the METTL3-METTL14 complex. Nature 534, 575–578 (2016).

    Article  CAS  Google Scholar 

  22. Bokar, J. A., Shambaugh, M. E., Polayes, D., Matera, A. G. & Rottman, F. M. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N 6-adenosine)-methyltransferase. RNA 3, 1233–1247 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Pendleton, K. E. et al. The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retention. Cell 169, 824–835.e814 (2017).

    Article  CAS  Google Scholar 

  24. Warda, A. S. et al. Human METTL16 is a N 6-methyladenosine (m6A) methyltransferase that targets pre-mRNAs and various non-coding RNAs. EMBO Rep 18, 2004–2014 (2017).

    Article  CAS  Google Scholar 

  25. Mendel, M. et al. Methylation of structured RNA by the m6A writer METTL16 is essential for mouse embryonic development. Mol. Cell 71, 986–1000.e1011 (2018).

    Article  CAS  Google Scholar 

  26. Doxtader, K. A. et al. Structural basis for regulation of METTL16, an S-adenosylmethionine homeostasis factor. Mol. Cell 71, 1001–1011.e1004 (2018).

    Article  CAS  Google Scholar 

  27. Albrecht, M. & Lengauer, T. Novel Sm-like proteins with long C-terminal tails and associated methyltransferases. FEBS Lett. 569, 18–26 (2004).

    Article  CAS  Google Scholar 

  28. Benhalevy, D. et al. The human CCHC-type zinc finger nucleic acid-binding protein binds G-rich elements in target mRNA coding sequences and promotes translation. Cell Rep. 18, 2979–2990 (2017).

    Article  CAS  Google Scholar 

  29. Jones, M. R. et al. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat. Cell Biol. 11, 1157–1163 (2009).

    Article  CAS  Google Scholar 

  30. Wen, J. et al. Zc3h13 regulates nuclear RNA m6A methylation and mouse embryonic stem cell self-renewal. Mol. Cell 69, 1028–1038.e1026 (2018).

    Article  CAS  Google Scholar 

  31. Thomson, E., Ferreira-Cerca, S. & Hurt, E. Eukaryotic ribosome biogenesis at a glance. J. Cell Sci. 126, 4815–4821 (2013).

    Article  CAS  Google Scholar 

  32. Wang, X. et al. N 6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

    Article  Google Scholar 

  33. Wang, X. et al. N 6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

    Article  CAS  Google Scholar 

  34. Sloan, K. E. et al. Tuning the ribosome: the influence of rRNA modification on eukaryotic ribosome biogenesis and function. RNA Biol. 14, 1138–1152 (2017).

    Article  Google Scholar 

  35. Arai, T. et al. Single methylation of 23 S rRNA triggers late steps of 50 S ribosomal subunit assembly. Proc. Natl Acad. Sci. USA 112, E4707–E4716 (2015).

    Article  CAS  Google Scholar 

  36. Khatter, H., Myasnikov, A. G., Natchiar, S. K. & Klaholz, B. P. Structure of the human 80 S ribosome. Nature 520, 640–645 (2015).

    Article  CAS  Google Scholar 

  37. Punekar, A. S., Liljeruhm, J., Shepherd, T. R., Forster, A. C. & Selmer, M. Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase Rlm. J. Nucleic Acids Res. 41, 9537–9548 (2013).

    Article  CAS  Google Scholar 

  38. Golovina, A. Y. et al. The last rRNA methyltransferase of E. coli revealed: the yhiR gene encodes adenine-N 6 methyltransferase specific for modification of A2030 of 23 S ribosomal RNA. RNA 18, 1725–1734 (2012).

    Article  CAS  Google Scholar 

  39. 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 

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

    Article  CAS  Google Scholar 

  41. Tang, C. et al. ALKBH5-dependent m6A demethylation controls splicing and stability of long 3'-UTR mRNAs in male germ cells. Proc. Natl Acad. Sci. USA 115, E325–E333 (2018).

    Article  CAS  Google Scholar 

  42. Zhang, S. et al. m6A demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation program. Cancer Cell. 31, 591–606.e596 (2017).

    Article  CAS  Google Scholar 

  43. Li, Z. et al. FTO plays an oncogenic role in acute myeloid leukemia as a N 6-methyladenosine RNA demethylase. Cancer Cell 31, 127–141 (2017).

    Article  Google Scholar 

  44. Liu, F. et al. ALKBH1-mediated tRNA demethylation regulates translation. Cell 167, 816–828.e816 (2016).

    Article  CAS  Google Scholar 

  45. Kawarada, L. et al. ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications. Nucleic Acids Res. 45, 7401–7415 (2017).

    Article  CAS  Google Scholar 

  46. Stojković, V. & Fujimori, D. G. Radical SAM-mediated methylation of ribosomal RNA. Methods Enzymol. 560, 355–376 (2015).

    Article  Google Scholar 

  47. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  48. Hafner, M. et al. PAR-CliP–a method to identify transcriptome-wide the binding sites of RNA binding proteins. J. Vis. Exp. 41, 2034 (2010).

    Google Scholar 

  49. Esposito, A. M. et al. Eukaryotic polyribosome profile analysis. J. Vis. Exp. 40, 1948 (2010).

    Google Scholar 

Download references

Acknowledgements

We thank X. Wang, L. Hu, H. Shi, F. Liu, and J. Wei from C.H.’s laboratory for discussions and for sharing experiments materials. We thank J. Tauler for editing the manuscript. This work was supported by the National Institutes of Health grants HG008935 (to C.H.) and GM113194 (to T.P. and C.H.). C.H. is an investigator of the Howard Hughes Medical Institute (HHMI). This work also supported by National Natural Science Foundation of China (81602513; to J.C.) and funds from Fudan University, H.M. is supported by the Postdoctoral International Exchange Program of the China Postdoctoral Council (CPC). X.W. is supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number 1K01 DK111764.

Author information

Author notes

  1. These authors contributed equally: Honghui Ma, Xiaoyun Wang, Jiabin Cai.

Authors and Affiliations

  1. Liver Cancer Institute, Zhongshan Hospital, Fudan University, and Institute of Biomedical Sciences, Fudan University, Shanghai, China

    Honghui Ma, Ruitu Lv, Hao Chen, Yujiang Geno Shi, Fei Lan & Yang Shi

  2. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Honghui Ma, Qing Dai, Kai Chen, Zhike Lu & Chuan He

  3. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA

    Honghui Ma, Qing Dai, Kai Chen, Zhike Lu & Chuan He

  4. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA

    Xiaoyun Wang, Tao Pan & Chuan He

  5. Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion (Fudan University), Ministry of Education, Shanghai, China

    Jiabin Cai & Jia Fan

  6. Centre for Integrative Biology (CBI), Department of Integrated Structural Biology, Institute of Genetics and of Molecular and Cellular Biology (IGBMC), CNRS, Inserm, Université de Strasbourg, Illkirch, France

    S. Kundhavai Natchiar & Bruno P. Klaholz

  7. Endocrinology Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Yujiang Geno Shi

  8. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Yang Shi

Authors
  1. Honghui Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoyun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiabin Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qing Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. Kundhavai Natchiar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ruitu Lv
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kai Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhike Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yujiang Geno Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Fei Lan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jia Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Bruno P. Klaholz
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Tao Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Yang Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Chuan He
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.M., T.P., Y.S. and C.H. designed the experiments. H.M. purified the recombinant ZCCHC4 protein, established the ZCCHC4 knockout stable cell lines, and carried out the in vitro and in vivo enzyme activity experiments and rescue experiments. X.W. performed polysome profile sequencing and reporter assays. J.C. performed WB and IHC in liver cancer samples and tumor growth assay in nude mice. Q.D. and K.C. assisted with enzyme activity experiments. R.L. and Z.L. helped with PAR-CLIP analysis. H.C. and Y.G.S. helped measure in vivo enzyme activity. F.L. helped design the experiments. J.F. helped prepare patient samples and analyze data. S.K.N. and B.P.K. helped with cryo-EM analysis of the m6A4220 modification site. H.M., X.W., T.P., Y.S. and C.H. wrote the manuscript.

Corresponding authors

Correspondence to Tao Pan, Yang Shi or Chuan He.

Ethics declarations

Competing interests

Y.S. is a co-founder of Constellation Pharmaceuticals, Inc., and Athelas Therapeutics. C.H. is a scientific founder of Accent Therapeutics, Inc. and a member of its scientific advisory board.

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 1–10

Reporting Summary

Supplementary Dataset 1

PAR-CLIP showing ZCCHC4-bound RNAs in cells

Supplementary Dataset 2

ZCCHC4 KO affects translation

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ma, H., Wang, X., Cai, J. et al. N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation. Nat Chem Biol 15, 88–94 (2019). https://doi.org/10.1038/s41589-018-0184-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0184-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer