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

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Article 16 April 2025 GTSF1 ACCELERATES TARGET RNA CLEAVAGE BY PIWI-CLADE ARGONAUTE PROTEINS Article Open access 30 June 2022 STRUCTURAL INSIGHTS INTO RNA CLEAVAGE BY PIWI ARGONAUTE Article

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molecular graphics. _Acta Crystallogr. D_ 50, 869–873 (1994). Article  CAS  Google Scholar  Download references 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.). AUTHOR INFORMATION Author notes * Fabiola V Rivas, Niraj H Tolia and Ji-Joon Song: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Cold

Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA Fabiola V Rivas, Niraj H Tolia, Ji-Joon Song, Juan P Aragon, Jidong

Liu, Gregory J Hannon & Leemor Joshua-Tor * W.M. Keck Structural Biology Laboratory, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA Niraj H Tolia, Ji-Joon Song & Leemor

Joshua-Tor Authors * Fabiola V Rivas View author publications You can also search for this author inPubMed Google Scholar * Niraj H Tolia View author publications You can also search for

this author inPubMed Google Scholar * Ji-Joon Song View author publications You can also search for this author inPubMed Google Scholar * Juan P Aragon View author publications You can also

search for this author inPubMed Google Scholar * Jidong Liu View author publications You can also search for this author inPubMed Google Scholar * Gregory J Hannon View author publications

You can also search for this author inPubMed Google Scholar * Leemor Joshua-Tor View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS

Correspondence to Gregory J Hannon or Leemor Joshua-Tor. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. 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) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 22 December

2004 * Accepted: 09 March 2005 * Published: 30 March 2005 * Issue Date: 01 April 2005 * DOI: https://doi.org/10.1038/nsmb918 SHARE THIS ARTICLE Anyone you share the following link with will

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