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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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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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