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Science 7 August 2009:
Vol. 325. no. 5941, pp. 750 - 753
DOI: 10.1126/science.1176325
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Reports

C3PO, an Endoribonuclease That Promotes RNAi by Facilitating RISC Activation

Ying Liu,1 Xuecheng Ye,1 Feng Jiang,1 Chunyang Liang,1 Dongmei Chen,2 Junmin Peng,2 Lisa N. Kinch,1,3 Nick V. Grishin,1,3 Qinghua Liu1,*

The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded small interfering RNA (siRNA) direct target mRNA cleavage. We reconstituted long double-stranded RNA– and duplex siRNA–initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We used this core reconstitution system to purify an RNAi regulator that we term C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a Mg2+-dependent endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products. These studies establish an in vitro RNAi reconstitution system and identify C3PO as a key activator of the core RNAi machinery.

1 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
2 Department of Human Genetics, Center for Neurodegenerative Diseases, Emory University, Atlanta, GA 30322, USA.
3 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

* To whom correspondence should be addressed. E-mail: qinghua.liu{at}utsouthwestern.edu

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THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.
D. M. Kenski, A. J. Cooper, J. J. Li, A. T. Willingham, H. J. Haringsma, T. A. Young, N. A. Kuklin, J. J. Jones, M. T. Cancilla, D. R. McMasters, et al. (2009)
Nucleic Acids Res.
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