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Purified Argonaute2 and an siRNA form recombinant human RISC

Abstract

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5′ phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

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Figure 1: Ago2 and an siRNA form recombinant RISC.
Figure 2: The 5′ phosphate contributes to the formation and stability of active RISC.
Figure 3: Cleavage by recombinant RISC is accurate.
Figure 4: Position of the 5′ end of the siRNA in RISC.
Figure 5: The catalytic site of Ago2.
Figure 6: Comparison of active sites in RNase H–fold proteins.
Figure 7: ATP does not accelerate cleavage by recombinant RISC.
Figure 8: Kinetic analysis of recombinant RISC.

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References

  1. Hannon, G.J. RNA interference. Nature 418, 244–251 (2002).

    CAS  Article  Google Scholar 

  2. 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).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  4. Lee, Y.S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004).

    CAS  Article  Google Scholar 

  5. Hammond, S.M., Bernstein, E., Beach, D. & Hannon, G.J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000).

    CAS  Article  Google Scholar 

  6. Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  8. 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).

    CAS  Article  Google Scholar 

  9. 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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  Google Scholar 

  12. 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).

    CAS  Article  Google Scholar 

  13. Hockensmith, J.W., Kubasek, W.L., Vorachek, W.R., Evertsz, E.M. & von Hippel, P.H. Laser cross-linking of protein-nucleic acid complexes. Methods Enzymol. 208, 211–236 (1991).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

  19. Stuckey, J.A. & Dixon, J.E. Crystal structure of a phospholipase D family member. Nat. Struct. Biol. 6, 278–284 (1999).

    CAS  Article  Google Scholar 

  20. Gray, C.H., Good, V.M., Tonks, N.K. & Barford, D. The structure of the cell cycle protein Cdc14 reveals a proline-directed protein phosphatase. EMBO J. 22, 3524–3535 (2003).

    CAS  Article  Google Scholar 

  21. Cox, D.N. et al. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 12, 3715–3727 (1998).

    CAS  Article  Google Scholar 

  22. Yang, W. & Steitz, T.A. Recombining the structures of HIV integrase, RuvC and RNase H. Structure 3, 131–134 (1995).

    CAS  Article  Google Scholar 

  23. Davies, J.F. 2nd, Hostomska, Z., Hostomsky, Z., Jordan, S.R. & Matthews, D.A. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science 252, 88–95 (1991).

    CAS  Article  Google Scholar 

  24. Haruki, M., Tsunaka, Y., Morikawa, M., Iwai, S. & Kanaya, S. Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate. Biochemistry 39, 13939–13944 (2000).

    CAS  Article  Google Scholar 

  25. Kanaya, S., Oobatake, M. & Liu, Y. Thermal stability of Escherichia coli ribonuclease HI and its active site mutants in the presence and absence of the Mg2+ ion. Proposal of a novel catalytic role for Glu48. J. Biol. Chem. 271, 32729–32736 (1996).

    CAS  Article  Google Scholar 

  26. Kanaya, S. & Ikehara, M. Functions and structures of ribonuclease H enzymes. Subcell. Biochem. 24, 377–422 (1995).

    CAS  Article  Google Scholar 

  27. Steitz, T.A. & Steitz, J.A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. USA 90, 6498–6502 (1993).

    CAS  Article  Google Scholar 

  28. Beese, L.S. & Steitz, T.A. Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25–33 (1991).

    CAS  Article  Google Scholar 

  29. Goedken, E.R. & Marqusee, S. Co-crystal of Escherichia coli RNase HI with Mn2+ ions reveals two divalent metals bound in the active site. J. Biol. Chem. 276, 7266–7271 (2001).

    CAS  Article  Google Scholar 

  30. Katayanagi, K., Okumura, M. & Morikawa, K. Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 Å resolution: proof for a single Mg(2+)-binding site. Proteins 17, 337–346 (1993).

    CAS  Article  Google Scholar 

  31. Chapados, B.R. et al. Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication. J. Mol. Biol. 307, 541–556 (2001).

    CAS  Article  Google Scholar 

  32. Klumpp, K. et al. Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors. Nucleic Acids Res. 31, 6852–6859 (2003).

    CAS  Article  Google Scholar 

  33. Steiniger-White, M., Rayment, I. & Reznikoff, W.S. Structure/function insights into Tn5 transposition. Curr. Opin. Struct. Biol. 14, 50–57 (2004).

    CAS  Article  Google Scholar 

  34. Davies, D.R., Goryshin, I.Y., Reznikoff, W.S. & Rayment, I. Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. Science 289, 77–85 (2000).

    CAS  Article  Google Scholar 

  35. Steiniger-White, M., Bhasin, A., Lovell, S., Rayment, I. & Reznikoff, W.S. Evidence for “unseen” transposase-DNA contacts. J. Mol. Biol. 322, 971–982 (2002).

    CAS  Article  Google Scholar 

  36. Lovell, S., Goryshin, I.Y., Reznikoff, W.R. & Rayment, I. Two-metal active site binding of a Tn5 transposase synaptic complex. Nat. Struct. Biol. 9, 278–281 (2002).

    CAS  Article  Google Scholar 

  37. Bujacz, G. et al. Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity. J. Biol. Chem. 272, 18161–18168 (1997).

    CAS  Article  Google Scholar 

  38. Peterson, G. & Reznikoff, W. Tn5 transposase active site mutations suggest position of donor backbone DNA in synaptic complex. J. Biol. Chem. 278, 1904–1909 (2003).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  40. Ishizuka, A., Siomi, M.C. & Siomi, H. A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 16, 2497–2508 (2002).

    CAS  Article  Google Scholar 

  41. Tabara, H., Yigit, E., Siomi, H. & Mello, C.C. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–871 (2002).

    CAS  Article  Google Scholar 

  42. Tijsterman, M., Ketting, R.F., Okihara, K.L., Sijen, T. & Plasterk, R.H. RNA helicase MUT-14-dependent gene silencing triggered in C. elegans by short antisense RNAs. Science 295, 694–697 (2002).

    CAS  Article  Google Scholar 

  43. Tomari, Y. et al. RISC assembly defects in the Drosophila RNAi mutant armitage. Cell 116, 831–841 (2004).

    CAS  Article  Google Scholar 

  44. Motamedi, M.R. et al. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789–802 (2004).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  46. Haley, B., Tang, G. & Zamore, P.D. In vitro analysis of RNA interference in Drosophila melanogaster. Methods 30, 330–336 (2003).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  48. Jones, T.A. & Kjeldgaard, M. Electron-density map interpretation. Methods Enzymol. 277, 173–208 (1997).

    CAS  Article  Google Scholar 

  49. 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 

  50. Ryter, J.M. & Schultz, S.C. Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA. EMBO J 17, 7505–7513 (1998).

    CAS  Article  Google Scholar 

  51. Esnouf, R.M. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J. Mol. Graph. 15, 132–134 (1997).

    CAS  Article  Google Scholar 

  52. Bacon, D.J. & Anderson, W.F. A fast algorithm for rendering space-filling molecule pictures. J. Mol. Graph. 6, 219–220 (1988).

    Article  Google Scholar 

  53. Merritt, E.A. & Murphy, M.E.P. Raster3D version 2.0—a program for photorealistic molecular graphics. Acta Crystallogr. D 50, 869–873 (1994).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank members of the Hannon and Joshua-Tor laboratories for helpful discussions and A. Heroux (X26C) for support with data collection at the National Synchrotron Light Source (NSLS). C. Marsden and S. Smith provided technical support. P. Zamore kindly provided kinetic tutoring. The NSLS is supported by the US Department of Energy, Division of Material Sciences and Division of Chemical Sciences. F.V.R. is a fellow of the Jane Coffin Childs Memorial Fund. J.J.S. is a Bristol-Myers Squibb Predoctoral Fellow. This work was supported in part by a grant from the US National Institutes of Health (G.J.H.) and the Louis Morin Charitable Trust (L.J.).

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Correspondence to Gregory J Hannon or Leemor Joshua-Tor.

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Supplementary information

Supplementary Fig. 1

Slicer activity is intrinsic to recombinant Ago2. (PDF 3987 kb)

Supplementary Fig. 2

The 5′ phosphate contributes to the formation of active RISC. (PDF 704 kb)

Supplementary Fig. 3

Tungstate-binding sites. (PDF 628 kb)

Supplementary Fig. 4

Mn2+-bound PfAgo. (PDF 561 kb)

Supplementary Fig. 5

ATP does not accelerate cleavage by recombinant RISC. (PDF 595 kb)

Supplementary Fig. 6

Kinetic analysis of recombinant RISC. (PDF 917 kb)

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Rivas, F., Tolia, N., Song, JJ. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol 12, 340–349 (2005). https://doi.org/10.1038/nsmb918

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