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Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes


The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5′-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg2+-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide–target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3′-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

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Figure 1: Crystal structure of T. thermophilus Ago(Asn 546) catalytic mutant bound to 5′-phosphorylated 21-nucleotide guide DNA and 12-nucleotide target RNA.
Figure 2: Crystal structure of T. thermophilus Ago(Glu 546) catalytic mutant bound to 5′-phosphorylated 21-nucleotide guide DNA and 15-nucleotide target RNA.
Figure 3: Crystal structure of T. thermophilus Ago(Asn 478) catalytic mutant bound to 5′-phosphorylated 21-nucleotide guide DNA and 19-nucleotide target RNA and identification of Mg 2+ binding sites within the catalytic pocket of the wild-type Ago complex.
Figure 4: Effect of complementarity and length on target DNA cleavage by T. thermophilus Ago.
Figure 5: Effect of sugar-phosphate backbone modifications on target DNA cleavage by T. thermophilus Ago.

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The structures of ternary complexes of T. thermophilus Ago have been submitted to the Protein Data Bank. The accession codes are: 3HO1 (mutant Ago(Asn 546)–12-nucleotide target RNA), 3HJF (mutant Ago(Glu 546)–15-nucleotide target RNA), 3HK2 (mutant Ago(Asn 478)–19-nucleotide target RNA), 3HM9 (wild-type Ago–19-nucleotide target RNA, 50mMMg), 3HVR (wild-type Ago–19-nucleotide target RNA, 80mM Mg), and 3HXM (second crystal form of wild-type Ago–20-nucleotide target RNA containing twomismatches31).


  1. Dykxhoorn, D. M., Novina, C. D. & Sharp, P. A. Killing the messenger: short RNAs that silence gene expression. Nature Rev. Mol. Cell Biol. 4, 457–467 (2003)

    Article  CAS  Google Scholar 

  2. Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. 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 

  6. 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 

  7. Kim, V. N., Han, J. & Siomi, M. C. Biogenesis of small RNAs in animals. Nature Rev. Mol. Cell Biol. 10, 126–139 (2009)

    Article  CAS  Google Scholar 

  8. Carthew, R. W. & Sontheimer, E. J. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655 (2009)

    Article  CAS  Google Scholar 

  9. 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 

  10. 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 

  11. 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 

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

    Article  ADS  CAS  Google Scholar 

  13. Rivas, F. V. et al. Purified Ago2 and an siRNA form recombinant human RISC. Nature Struct. Biol. 12, 340–349 (2005)

    Article  CAS  Google Scholar 

  14. 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 

  15. 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 

  16. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. 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 

  20. 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 

  21. 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 

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

    Article  CAS  Google Scholar 

  23. Parker, J. S. et al. Enhancement of the seed-target recognition step in RNA silencing by a PIWI/MID domain protein. Mol. Cell 33, 204–214 (2009)

    Article  CAS  Google Scholar 

  24. 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 

  25. 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 

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

    Article  ADS  CAS  Google Scholar 

  27. Jinek, M. & Doudna, J. A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405–412 (2009)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Castanotto, D. & Rossi, J. J. The promises and pitfalls of RNA-interference based therapeutics. Nature 457, 426–433 (2009)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  31. Wang, Y. et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008)

    Article  ADS  CAS  Google Scholar 

  32. Mi, S. et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′-terminal nucleotide. Cell 133, 116–127 (2008)

    Article  CAS  Google Scholar 

  33. Montgomery, T. A. et al. Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128–141 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Chi, S. W., Zang, J. B., Mele, A. & Darnell, R. B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479–486 (2009)

    Article  ADS  CAS  Google Scholar 

  36. 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 

  37. Nowotny, M. Retroviral integrase superfamily: the structural perspective. EMBO Rep. 10, 144–151 (2009)

    Article  CAS  Google Scholar 

  38. Turner, D. H. Thermodynamics of base pairing. Curr. Opin. Struct. Biol. 6, 299–304 (1996)

    Article  MathSciNet  CAS  Google Scholar 

  39. Freier, S. M. & Altmann, K. H. The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 25, 4429–4443 (1997)

    Article  CAS  Google Scholar 

  40. Verma, S. & Eckstein, F. Modified oligonucleotides: synthesis and strategy for users. Annu. Rev. Biochem. 67, 99–134 (1998)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  45. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  46. Tonelli, M. et al. Dynamic NMR structures of [RP]- and [SP]-phosphorothioated DNA-RNA hybrids: is flexibility required for RNase H recognition? Biophys. J. 85, 2525–2538 (2003)

    Article  ADS  CAS  Google Scholar 

  47. Thorogood, H., Grasby, J. A. & Connolly, B. A. Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease. A study using oligodeoxynucleotide phosphorothioates. J. Biol. Chem. 271, 8855–8862 (1996)

    Article  CAS  Google Scholar 

  48. Burgers, P. M. & Eckstein, F. Absolute configuration of the diastereomers of adenosine 5′-O-(1-thiotriphosphate): consequences for the stereochemistry of polymerization by DNA-dependent RNA polymerase from Escherichia coli . Proc. Natl Acad. Sci. USA 75, 4798–4800 (1978)

    Article  ADS  CAS  Google Scholar 

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The research was supported by funds from the National Institutes of Health (NIH) and the Starr Foundation to D.J.P., and from the NIH, Starr Foundation and the Howard Hughes Medical Institute (HHMI) to T.T. We would like to thank the staff of NE-CAT beam lines at the Advanced Photon Source (APS), Argonne National Laboratory, and the X-29 beamline at the Brookhaven National Laboratory, supported by the US Department of Energy, for assistance with data collection. We thank Z. Wang for assistance with X-ray data collection at the APS.

Author Contributions Y.W. and G.S. expressed and purified wild-type T. thermophilus Ago and its catalytic mutants, and also grew crystals of the various ternary complexes. H.L. collected X-ray diffraction data on the various NE-CAT beam lines, and Y.W. solved the structures of these ternary complexes. D.J.P. supervised the structural studies. S.J. was responsible for the cleavage assays on Ago with modified DNA and RNA target strands, and G.S.W. purified the phosphorothioate diastereomers and quality controlled oligonucleotides, under the supervision of T.T. D.J.P. and T.T. were primarily responsible for writing the structural and biochemical contents of the paper, respectively, and all authors read and approved the submitted manuscript.

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Correspondence to Thomas Tuschl or Dinshaw J. Patel.

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[COMPETING INTERESTS: T.T. is a cofounder and scientific advisor to Alnylam Pharmaceuticals and an advisor to Regulus Therapeutics.]

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Supplementary Tables 1-4 and Supplementary Figures 1-27 with Legends. (PDF 10468 kb)

Supplementary Movie 1

This movie shows interconversion between Ago binary complex containing guide DNA (in red) and Ago ternary complex containing added 12-nucleotide target RNA (in blue). (AVI 3932 kb)

Supplementary Movie 2

This movie shows interconversion between Ago ternary complexes containing guide DNA (in red) and added 12- and 15-nucleotide target RNAs (in blue). (AVI 3704 kb)

Supplementary Movie 3

This movie shows rotation of Ago ternary complex structure containing guide DNA (in red) and 19-nucleotide target RNA (in blue). (AVI 13088 kb)

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Wang, Y., Juranek, S., Li, H. et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754–761 (2009).

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