Nothing Special   »   [go: up one dir, main page]
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:

Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex

Nature volume 456, pages 921–926 (2008)Cite this article

This article has been updated

Abstract

Here we report on a 3.0 Å crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10–11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson–Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2′-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

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

Figure 1: Crystal structure of T. thermophilus Ago bound to 5′-phosphorylated 21-nucleotide guide DNA and 20-nucleotide target RNA.
Figure 2: Comparison of structural details between the binary Ago complex with bound guide DNA and the ternary complex with added target RNA.
Figure 3: Conformational changes within the bilobal Ago scaffold on proceeding from the binary (guide) complex to the ternary (guide + target) complex.
Figure 4: Target RNA cleavage activity of T. thermophilus Ago loaded with mismatched, bulge-containing or length-altered guide DNA strands.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structural coordinates of the ternary complex of T. thermophilus Ago bound to 5′-phosphorylated 21-nucleotide guide DNA and 20-nucleotide target RNA have been submitted to the Protein Data Bank under accession number 3F73.

Change history

  • 17 February 2008

    In the version of this article initially published online, the equation in the Methods section was incorrect. The correct equation is shown. The error has been corrected for all versions of the article.

References

  1. Hock, J. & Meister, G. The Argonaute protein family. Genome Biol. 9, 210 (2008)

    Article  Google Scholar 

  2. Filipowicz, W. The nuts and bolts of the RISC machine. Cell 122, 17–20 (2005)

    Article  CAS  Google Scholar 

  3. Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nature Rev. Mol. Cell Biol. 9, 22–32 (2008)

    Article  CAS  Google Scholar 

  4. Tolia, N. H. & Joshua-Tor, L. Slicer and the argonautes. Nature Chem. Biol. 3, 36–43 (2007)

    Article  ADS  CAS  Google Scholar 

  5. Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  7. Parker, J. S., Roe, S. & Barford, D. Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J. 23, 4727–4737 (2004)

    Article  CAS  Google Scholar 

  8. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 391, 806–811 (1998)

    Article  ADS  CAS  Google Scholar 

  9. Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001)

    Article  CAS  Google Scholar 

  10. Martinez, J. & Tuschl, T. RISC is a 5′-phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975–980 (2004)

    Article  CAS  Google Scholar 

  11. Schwarz, D. S., Tomari, Y. & Zamore, P. D. The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr. Biol. 14, 787–791 (2004)

    Article  CAS  Google Scholar 

  12. Tomari, Y. & Zamore, P. D. Perspective: machines for RNAi. Genes Dev. 19, 517–529 (2005)

    Article  CAS  Google Scholar 

  13. De Fougerolles, A., Vornlocher, H.-P., Maraganore, J. & Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nature Rev. Drug. Discovery 6, 443–453 (2007)

    Article  CAS  Google Scholar 

  14. Kim, D. H. & Rossi, J. J. Strategies for silencing human disease using RNA interference. Nature Rev. Genetics 8, 173–184 (2007)

    Article  CAS  Google Scholar 

  15. Filipowicz, W., Jaskiewicz, L., Kolb, F. A. & Pillai, R. S. Post-transcriptional gene silencing by siRNAs and miRNAs. Curr. Opin. Struct. Biol. 15, 331–341 (2005)

    Article  CAS  Google Scholar 

  16. Hall, T. M. Structure and function of argonaute proteins. Structure 13, 1403–1408 (2005)

    Article  CAS  Google Scholar 

  17. Parker, J. S. & Barford, D. Argonaute: a scaffold for the function of short regulatory RNAs. Trends Biochem. Sci. 31, 622–630 (2006)

    Article  CAS  Google Scholar 

  18. Patel, D. J. et al. Structural biology of RNA silencing and its functional implications. Cold Spring Harb. Symp. Quant. Biol. 71, 81–93 (2006)

    Article  CAS  Google Scholar 

  19. Yuan, Y. R. et al. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005)

    Article  CAS  Google Scholar 

  20. Wang, Y. et al. Structure of the guide-strand-containing Argonaute silencing complex. Nature 456, 209–213 (2008)

    Article  ADS  CAS  Google Scholar 

  21. Parker, J. S., Roe, S. M. & Barford, D. Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex. Nature 434, 663–666 (2005)

    Article  ADS  CAS  Google Scholar 

  22. Ma, J. B. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670 (2005)

    Article  ADS  CAS  Google Scholar 

  23. Ma, J. B., Ye, K. & Patel, D. J. Structural basis for overhang-specific small interfering RNA recognition by the Paz domain. Nature 429, 318–322 (2004)

    Article  ADS  CAS  Google Scholar 

  24. Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Nucleic acid 3′-end recognition by the Argonaute2 Paz domain. Nature Struct. Mol. Biol. 11, 576–577 (2004)

    Article  CAS  Google Scholar 

  25. Haley, B. & Zamore, P. D. Kinetic analysis of the RNAi enzyme complex. Nature Struct. Mol. Biol. 11, 599–606 (2004)

    Article  CAS  Google Scholar 

  26. Nowotny, M., Gaidamakov, S. A., Crouch, R. J. & Yang, W. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 121, 1005–1016 (2005)

    Article  CAS  Google Scholar 

  27. Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003)

    Article  CAS  Google Scholar 

  28. Chen, P. Y. et al. Strand-specific 5′-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity. RNA 14, 263–274 (2008)

    Article  CAS  Google Scholar 

  29. Jackson, A. L. et al. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12, 1197–1205 (2006)

    Article  CAS  Google Scholar 

  30. Dorsett, Y. & Tuschl, T. siRNAs: applications in functional genomics and potential as therapeutics. Nature Rev. Drug Discov. 3, 318–329 (2004)

    Article  CAS  Google Scholar 

  31. Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21, 635–637 (2003)

    Article  CAS  Google Scholar 

  32. Gunawardane, L. S. et al. A slicer-mediated mechanism for repeat-associated siRNA 5′-end formation in Drosophila . Science 315, 1587–1590 (2007)

    Article  ADS  CAS  Google Scholar 

  33. Martinez, J. et al. Single-stranded anti-sense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574 (2002)

    Article  CAS  Google Scholar 

  34. MacRae, I. J. et al. In vitro reconstitution of human RISC-loading complex. Proc. Natl Acad. Sci. USA 105, 512–517 (2008)

    Article  ADS  CAS  Google Scholar 

  35. Ameres, S. L., Martinez, J. & Schroeder, R. Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130, 101–112 (2007)

    Article  CAS  Google Scholar 

  36. Rana, T. M. Illuminating the silence: understanding the structure and function of small RNAs. Nature Rev. Mol. Cell Biol. 8, 23–26 (2007)

    Article  CAS  Google Scholar 

  37. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Meth. Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  38. McCoy, A. J. et al. Phaser crystallographic software. J. App. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  39. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  40. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  41. Jones, T. A., Zhou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

Download references

Acknowledgements

The research was supported by funds from the National Institutes of Health and the Starr Foundation to D.J.P. and T.T. We would like to thank the staff of NE-CAT beam line at the Advanced Photon Source, Argonne National Laboratory, supported by the US Department of Energy, for assistance with data collection.

Author Contributions Y.W. and G.S. expressed and purified T. thermophilus Ago, and grew crystals of the ternary complex. H.L. and Y.W. collected X-ray diffraction data on the micro-focus beam line, and Y.W. solved the structure of the ternary complex. The structural studies were undertaken with the supervision of D.J.P. S.J. was responsible for the cleavage assays on Ago with modified guide strands under the supervision of T.T. D.J.P. and T.T. were primarily responsible for writing the paper and all authors read and approved the submitted manuscript.

Author information

Authors and Affiliations

  1. Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA ,

    Yanli Wang, Haitao Li, Gang Sheng & Dinshaw J. Patel

  2. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA ,

    Stefan Juranek & Thomas Tuschl

Authors
  1. Yanli Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Juranek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haitao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gang Sheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas Tuschl
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dinshaw J. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Thomas Tuschl or Dinshaw J. Patel.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3 and Supplementary Figures 1-14 with Legends. (PDF 7679 kb)

Supplementary Movie 1

Supplementary Movie 1 shows the Ago ternary complex structure and the conformational transitions on proceeding from the binary to the ternary complex. (AVI 15949 kb)

Supplementary Movie 2

Supplementary Movie 2 shows the Ago ternary complex structure and the conformational transitions on proceeding from the binary to the ternary complex. (AVI 2923 kb)

Supplementary Movie 3

Supplementary Movie 3 shows the Ago ternary complex structure and the conformational transitions on proceeding from the binary to the ternary complex. (AVI 24460 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., Juranek, S., Li, H. et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008). https://doi.org/10.1038/nature07666

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07666

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