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:

Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation

Nature volume 443, pages 1008–1012 (2006)Cite this article

Abstract

DNA methylation has important functions in stable, transcriptional gene silencing, immobilization of transposable elements and genome organization1. In Arabidopsis, DNA methylation can be induced by double-stranded RNA through the RNA interference (RNAi) pathway, a response known as RNA-directed DNA methylation2. This requires a specialized set of RNAi components, including ARGONAUTE4 (AGO4)3,4,5,6. Here we show that AGO4 binds to small RNAs including small interfering RNAs (siRNAs) originating from transposable and repetitive elements, and cleaves target RNA transcripts. Single mutations in the Asp-Asp-His catalytic motif of AGO4 do not affect siRNA-binding activity but abolish its catalytic potential. siRNA accumulation and non-CpG DNA methylation at some loci require the catalytic activity of AGO4, whereas others are less dependent on this activity. Our results are consistent with a model in which AGO4 can function at target loci through two distinct and separable mechanisms. First, AGO4 can recruit components that signal DNA methylation in a manner independent of its catalytic activity. Second, AGO4 catalytic activity can be crucial for the generation of secondary siRNAs that reinforce its repressive effects.

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: A catalogue of AGO4-associated small RNAs.
Figure 2: AGO4 is a Slicer.
Figure 3: Slicer activity is not required for non-CpG methylation and silencing at the SUP locus.
Figure 4: Distinct effects of Slicer activity on DNA methylation and siRNA accumulation at endogenous repeats.

Similar content being viewed by others

References

  1. Chan, S. W., Henderson, I. R. & Jacobsen, S. E. Gardening the genome: DNA methylation in Arabidopsis thaliana. Nature Rev. Genet. 6, 351–360 (2005)

    Article  CAS  Google Scholar 

  2. Matzke, M. A. & Birchler, J. A. RNAi-mediated pathways in the nucleus. Nature Rev. Genet. 6, 24–35 (2005)

    Article  CAS  Google Scholar 

  3. Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, e104 (2004)

    Article  Google Scholar 

  4. Zilberman, D., Cao, X. & Jacobsen, S. E. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716–719 (2003)

    Article  ADS  CAS  Google Scholar 

  5. Chan, S. W. et al. RNA silencing genes control de novo DNA methylation. Science 303, 1336 (2004)

    Article  CAS  Google Scholar 

  6. Zilberman, D. et al. Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr. Biol. 14, 1214–1220 (2004)

    Article  CAS  Google Scholar 

  7. Herr, A. J., Jensen, M. B., Dalmay, T. & Baulcombe, D. C. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005)

    Article  ADS  CAS  Google Scholar 

  8. Onodera, Y. et al. Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120, 613–622 (2005)

    Article  CAS  Google Scholar 

  9. Li, C. et al. An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126, 93–106 (2006)

    Article  CAS  Google Scholar 

  10. Lippman, Z., May, B., Yordan, C., Singer, T. & Martienssen, R. Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification. PLoS Biol. 1, e67 (2003)

    Article  Google Scholar 

  11. Rohila, J. S., Chen, M., Cerny, R. & Fromm, M. E. Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J. 38, 172–181 (2004)

    Article  CAS  Google Scholar 

  12. Qi, Y., Denli, A. M. & Hannon, G. J. Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell 19, 421–428 (2005)

    Article  CAS  Google Scholar 

  13. Baumberger, N. & Baulcombe, D. C. Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl Acad. Sci. USA 102, 11928–11933 (2005)

    Article  ADS  CAS  Google Scholar 

  14. Hamilton, A., Voinnet, O., Chappell, L. & Baulcombe, D. Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671–4679 (2002)

    Article  CAS  Google Scholar 

  15. Singer, T., Yordan, C. & Martienssen, R. A. Robertson’s Mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene Decrease in DNA Methylation (DDM1). Genes Dev. 15, 591–602 (2001)

    Article  CAS  Google Scholar 

  16. Cao, X. & Jacobsen, S. E. Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc. Natl Acad. Sci. USA 99, (Suppl. 4)16491–16498 (2002)

    Article  ADS  CAS  Google Scholar 

  17. Girard, A., Sachidanandam, R., Hannon, G. & Carmell, M. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199–202 (2006)

    Article  ADS  Google Scholar 

  18. Jurka, J. et al. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet. Genome Res. 110, 462–467 (2005)

    Article  CAS  Google Scholar 

  19. Vaucheret, H., Vazquez, F., Crete, P. & Bartel, D. P. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18, 1187–1197 (2004)

    Article  CAS  Google Scholar 

  20. Grewal, S. I. & Moazed, D. Heterochromatin and epigenetic control of gene expression. Science 301, 798–802 (2003)

    Article  ADS  CAS  Google Scholar 

  21. Bao, N., Lye, K. W. & Barton, M. K. MicroRNA binding sites in Arabidopsis class III HD-ZIP mRNAs are required for methylation of the template chromosome. Dev. Cell 7, 653–662 (2004)

    Article  CAS  Google Scholar 

  22. Qi, Y. & Hannon, G. J. Uncovering RNAi mechanisms in plants: biochemistry enters the foray. FEBS Lett. 579, 5899–5903 (2005)

    Article  CAS  Google Scholar 

  23. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004)

    Article  ADS  CAS  Google Scholar 

  24. Rivas, F. V. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nature Struct. Mol. Biol. 12, 340–349 (2005)

    Article  CAS  Google Scholar 

  25. Jacobsen, S. & Meyerowitz, E. Hypermethylated SUPERMAN epigenetic alleles in Arabidopsis. Science 277, 1100–1103 (1997)

    Article  CAS  Google Scholar 

  26. Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005)

    Article  CAS  Google Scholar 

  27. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Ossowski and D. Weigel for help with the analysis of AGO1-associated RNAs; R. Sachidanandam for bioinformatics assistance; T. Mulligan for assistance with Arabidopsis culture; and members of the Hannon laboratory for discussion. This work was supported in part by grants from the NSF and the NIH (G.J.H.). G.J.H. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Yijun Qi

    Present address: National Institute of Biological Sciences, No. 7 Science Park Road, Zhongguanchun Life Science Park, 102206, Beijing, China

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Watson School of Biological Sciences and Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

    Yijun Qi, Xingyue He & Gregory J. Hannon

  2. Program in Genetics, Stony Brook University, Stony Brook, New York, 11794, USA

    Xingyue He

  3. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, 100101, Beijing, China

    Xiu-Jie Wang

  4. Genetic Information Research Institute, 1925 Landings Drive, Mountain View, California, 94043, USA

    Oleksiy Kohany & Jerzy Jurka

Authors
  1. Yijun Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xingyue He
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiu-Jie Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Oleksiy Kohany
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jerzy Jurka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gregory J. Hannon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gregory J. Hannon.

Ethics declarations

Competing interests

Sequences referred to in this study have been deposited in the GenBank database under accession numbers DQ927324–DQ972825. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1–10. (PDF 1737 kb)

Supplementary Figure Legends

This file contains text to accompany the above Supplementary Figures (DOC 30 kb)

Supplementary Tables

This file contains Supplementary Tables 1–10. (PDF 181 kb)

Supplementary Methods

This file contains additional details of the methods used in this study. (PDF 88 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Qi, Y., He, X., Wang, XJ. et al. Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443, 1008–1012 (2006). https://doi.org/10.1038/nature05198

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05198

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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