C3PO, an Endoribonuclease That Promotes RNAi by Facilitating RISC Activation

Overview

Journal Science

Specialty Science

Date 2009 Aug 8

PMID 19661431

Citations 127

Authors

Ying Liu

Xuecheng Ye

Feng Jiang

Chunyang Liang

Dongmei Chen

Junmin Peng

Lisa N Kinch

Nick V Grishin

Qinghua Liu

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

A guide to the biogenesis and functions of endogenous small non-coding RNAs in animals.

Jouravleva K, Zamore P Nat Rev Mol Cell Biol. 2025; .

PMID: 39856370 DOI: 10.1038/s41580-024-00818-9.

Harnessing antiviral RNAi therapeutics for pandemic viruses: SARS-CoV-2 and HIV.

Bowden-Reid E, Moles E, Kelleher A, Ahlenstiel C Drug Deliv Transl Res. 2025; .

PMID: 39833468 DOI: 10.1007/s13346-025-01788-x.

RNA Interference in Insects: From a Natural Mechanism of Gene Expression Regulation to a Biotechnological Crop Protection Promise.

Ortola B, Daros J Biology (Basel). 2024; 13(3).

PMID: 38534407 PMC: 10968014. DOI: 10.3390/biology13030137.

N-terminal region of Drosophila melanogaster Argonaute2 forms amyloid-like aggregates.

Narita H, Shima T, Iizuka R, Uemura S BMC Biol. 2023; 21(1):78.

PMID: 37072852 PMC: 10114355. DOI: 10.1186/s12915-023-01569-3.

A Tale of Two Lobsters-Transcriptomic Analysis Reveals a Potential Gap in the RNA Interference Pathway in the Tropical Rock Lobster .

Banks T, Wang T, Fitzgibbon Q, Smith G, Ventura T Int J Mol Sci. 2022; 23(19).

PMID: 36233053 PMC: 9569428. DOI: 10.3390/ijms231911752.

References

Liu Q, Rand T, Kalidas S, Du F, Kim H, Smith D . R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science. 2003; 301(5641):1921-5. DOI: 10.1126/science.1088710. View

Carthew R, Sontheimer E . Origins and Mechanisms of miRNAs and siRNAs. Cell. 2009; 136(4):642-55. PMC: 2675692. DOI: 10.1016/j.cell.2009.01.035. View

Tomari Y, Matranga C, Haley B, Martinez N, Zamore P . A protein sensor for siRNA asymmetry. Science. 2004; 306(5700):1377-80. DOI: 10.1126/science.1102755. View

Suseendranathan K, Sengupta K, Rikhy R, DSouza J, Kokkanti M, Kulkarni M . Expression pattern of Drosophila translin and behavioral analyses of the mutant. Eur J Cell Biol. 2007; 86(3):173-86. DOI: 10.1016/j.ejcb.2006.11.005. View

Maiti M, Lee H, Liu Y . QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands. Genes Dev. 2007; 21(5):590-600. PMC: 1820900. DOI: 10.1101/gad.1497607. View

Pham J, Pellino J, Lee Y, Carthew R, Sontheimer E . A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell. 2004; 117(1):83-94. DOI: 10.1016/s0092-8674(04)00258-2. View

Okamura K, Ishizuka A, Siomi H, Siomi M . Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 2004; 18(14):1655-66. PMC: 478188. DOI: 10.1101/gad.1210204. View

Claussen M, Koch R, Jin Z, Suter B . Functional characterization of Drosophila Translin and Trax. Genetics. 2006; 174(3):1337-47. PMC: 1667053. DOI: 10.1534/genetics.106.063016. View

Wang J, Boja E, Oubrahim H, Chock P . Testis brain ribonucleic acid-binding protein/translin possesses both single-stranded and double-stranded ribonuclease activities. Biochemistry. 2004; 43(42):13424-31. DOI: 10.1021/bi048847l. View

10.

Liu J, Carmell M, Rivas F, Marsden C, Thomson J, Song J . Argonaute2 is the catalytic engine of mammalian RNAi. Science. 2004; 305(5689):1437-41. DOI: 10.1126/science.1102513. View

11.

Liu X, Jiang F, Kalidas S, Smith D, Liu Q . Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. RNA. 2006; 12(8):1514-20. PMC: 1524895. DOI: 10.1261/rna.101606. View

12.

Sugiura I, Sasaki C, Hasegawa T, Kohno T, Sugio S, Moriyama H . Structure of human translin at 2.2 A resolution. Acta Crystallogr D Biol Crystallogr. 2004; 60(Pt 4):674-9. DOI: 10.1107/S0907444904002549. View

13.

Rand T, Petersen S, Du F, Wang X . Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell. 2005; 123(4):621-9. DOI: 10.1016/j.cell.2005.10.020. View

14.

Paroo Z, Liu Q, Wang X . Biochemical mechanisms of the RNA-induced silencing complex. Cell Res. 2007; 17(3):187-94. DOI: 10.1038/sj.cr.7310148. View

15.

Nykanen A, Haley B, Zamore P . ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001; 107(3):309-21. DOI: 10.1016/s0092-8674(01)00547-5. View

16.

Tomari Y, Du T, Haley B, Schwarz D, Bennett R, Cook H . RISC assembly defects in the Drosophila RNAi mutant armitage. Cell. 2004; 116(6):831-41. DOI: 10.1016/s0092-8674(04)00218-1. View

17.

Siomi H, Siomi M . On the road to reading the RNA-interference code. Nature. 2009; 457(7228):396-404. DOI: 10.1038/nature07754. View

18.

Matranga C, Tomari Y, Shin C, Bartel D, Zamore P . Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell. 2005; 123(4):607-20. DOI: 10.1016/j.cell.2005.08.044. View

19.

Miyoshi K, Tsukumo H, Nagami T, Siomi H, Siomi M . Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 2005; 19(23):2837-48. PMC: 1315391. DOI: 10.1101/gad.1370605. View