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Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex

Nature volume 434, pages 663–666 (2005)Cite this article

Abstract

RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes1,2. Argonaute proteins3, containing amino-terminal PAZ (for PIWI/Argonaute/Zwille) domains and carboxy-terminal PIWI domains, are core components of these mechanisms. Here we show the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a small interfering RNA (siRNA)-like duplex, which mimics the 5′ end of a guide RNA strand bound to an overhanging target messenger RNA. The structure contains a highly conserved metal-binding site that anchors the 5′ nucleotide of the guide RNA. The first base pair of the duplex is unwound, separating the 5′ nucleotide of the guide from the complementary nucleotide on the target strand, which exits with the 3′ overhang through a short channel. The remaining base-paired nucleotides assume an A-form helix, accommodated within a channel in the PIWI domain, which can be extended to place the scissile phosphate of the target strand adjacent to the putative slicer catalytic site. This study provides insights into mechanisms of target mRNA recognition and cleavage by an Argonaute–siRNA guide complex.

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Figure 1: General features of AfPiwi–siRNA interactions and distortion of the 5′ terminus of the RNA duplex.
Figure 2: Interactions between AfPiwi and the guide RNA strand.
Figure 3: Proposed model for a 19-nucleotide guide–target duplex bound to AfPiwi.

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References

  1. Mello, C. C. & Conte, D. Jr Revealing the world of RNA interference. Nature 431, 338–342 (2004)

    Article  ADS  CAS  Google Scholar 

  2. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004)

    Article  ADS  CAS  Google Scholar 

  3. Carmell, M. A., Xuan, Z., Zhang, M. Q. & Hannon, G. J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742 (2002)

    Article  CAS  Google Scholar 

  4. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001)

    Article  CAS  Google Scholar 

  5. Verdel, A. et al. RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676 (2004)

    Article  ADS  CAS  Google Scholar 

  6. Sontheimer, E. J. Assembly and function of RNA silencing complexes. Nature Rev. Mol. Cell Biol. 6, 127–138 (2005)

    Article  CAS  Google Scholar 

  7. Yan, K. S. et al. Structure and conserved RNA binding of the PAZ domain. Nature 426, 468–474 (2003)

    Article  ADS  Google Scholar 

  8. Song, J. J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nature Struct. Biol. 10, 1026–1032 (2003)

    Article  CAS  Google Scholar 

  9. 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 

  10. 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 

  11. 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 

  12. Parker, J. S., Roe, S. M. & 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 

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

    Article  ADS  CAS  Google Scholar 

  14. Nykanen, A., Haley, B. & Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001)

    Article  CAS  Google Scholar 

  15. Chiu, Y. L. & Rana, T. M. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol. Cell 10, 549–561 (2002)

    Article  CAS  Google Scholar 

  16. Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. & Tuschl, T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574 (2002)

    Article  CAS  Google Scholar 

  17. Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888 (2001)

    Article  CAS  Google Scholar 

  18. 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 

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

    Article  CAS  Google Scholar 

  20. Hutvagner, G. & Zamore, P. D. A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060 (2002)

    Article  ADS  CAS  Google Scholar 

  21. Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. & Burge, C. B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003)

    Article  CAS  Google Scholar 

  22. Stark, A., Brennecke, J., Russell, R. B. & Cohen, S. M. Identification of Drosophila MicroRNA targets. PLoS Biol. 1, 397–409 (2003)

    Article  CAS  Google Scholar 

  23. Doench, J. G. & Sharp, P. A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004)

    Article  CAS  Google Scholar 

  24. Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are MicroRNA targets. Cell 120, 15–20 (2005)

    Article  CAS  Google Scholar 

  25. Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Okamura, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655–1666 (2004)

    Article  CAS  Google Scholar 

  28. Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P. D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004)

    Article  ADS  CAS  Google Scholar 

  29. Collaborative Computational Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

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

    Article  Google Scholar 

  31. Ma, J.-B. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature doi:10.1038/nature03514 (this issue)

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Acknowledgements

We are grateful to staff at ESRF for help with data collection.The work was funded by Cancer Research–UK, the Wellcome Trust and the Institute of Cancer Research.

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Authors and Affiliations

  1. Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, SW3 6JB, London, UK

    James S. Parker, S. Mark Roe & David Barford

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  1. James S. Parker
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  2. S. Mark Roe
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  3. David Barford
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Corresponding author

Correspondence to David Barford.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

Shows the conformation of the siRNA duplex in the two copies of the AfPiwi-RNA complex per P1 unit cell (a), and a superimposition of the siRNA duplex onto an A-form RNA helix (b). (PPT 363 kb)

Supplementary Figure S2

Shows a multiple sequence alignment of the PIWI domains of AfPiwi, PfAgo and selected eukaryotic Argonautes. (PPT 1405 kb)

Supplementary Figure S3

Shows a superimposition of AfPiwi with the target RNA strand and PfAgo in the region of the putative slicer catalytic site. (PPT 639 kb)

Supplementary Table S1

Table of crystallographic data collection, processing and refinement statistics. (DOC 22 kb)

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Parker, J., Roe, S. & Barford, D. Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex. Nature 434, 663–666 (2005). https://doi.org/10.1038/nature03462

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