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Structure of C3PO and mechanism of human RISC activation

Abstract

Assembly of the RNA-induced silencing complex (RISC) consists of loading duplex (guide–passenger) siRNA onto Argonaute (Ago2) and removing the passenger strand. Ago2 contributes critically to RISC activation by nicking the passenger strand. Here we reconstituted duplex siRNA-initiated RISC activity using recombinant human Ago2 (hAgo2) and C3PO, indicating that C3PO has a critical role in hAgo2-RISC activation. Consistently, genetic depletion of C3PO compromised RNA silencing in mammalian cells. We determined the crystal structure of hC3PO, which reveals an asymmetric octamer barrel consisting of six translin and two TRAX subunits. This asymmetric assembly is critical for the function of C3PO as an endonuclease that cleaves RNA at the interior surface. The current work supports a Dicer-independent mechanism for human RISC activation, in which Ago2 directly binds duplex siRNA and nicks the passenger strand, and then C3PO activates RISC by degrading the Ago2-nicked passenger strand.

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Figure 1: Reconstitution of human RISC activity.
Figure 2: C3PO is required for efficient RNAi in mammalian cells.
Figure 3: Crystal structure of C3PO.
Figure 4: Asymmetric octameric assembly of C3PO.
Figure 5: Catalytic center of C3PO.
Figure 6: C3PO cleaves ssRNA at the interior surface.
Figure 7: C3PO activates human RISC by degrading Ago2-nicked passenger strand.

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Acknowledgements

We thank N.B. Hecht (University of Pennsylvania School of Medicine) for his generous gift of TB-RBP/translin knockout MEF cells, Y. Tomari for his help and insightful discussion, M. Rosen, Y. Liu, B. Tu, N. Grishin and L. Kinch for reading the manuscript and Y. Zhang, A. Shen, M. Yoda, W. Gao and D. Chen for technical assistance. Results shown in this report are derived from work conducted at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. Y.L. is supported by a Sara and Frank McKnight student fellowship. The work is supported by a Welch grant (I-1608) and US National Institutes of Health grants awarded to Q.L. (GM084010 and GM091286).

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Authors

Contributions

X.Y. conducted C3PO purification, human RISC reconstitution, mutagenesis and genetic experiments. N.H., helped by C.H., determined the crystal structures of human C3PO in apo form and in complex with Mn2+. Z.P. generated human Dicer, TRBP and Ago2 baculoviruses. Y.L. helped with reconstitution studies, and P.L. participated in mutagenesis experiments. S.C. conducted mass spectrometric analyses. Q.L. and H.Z. supervised the study and wrote the manuscript together. All authors discussed the results and approved the manuscript.

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Correspondence to Qinghua Liu or Hong Zhang.

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The authors declare no competing financial interests.

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Supplementary Figures 1–8 (PDF 3741 kb)

Supplementary Movie 1

A movie in three stages illustrating the architecture and potential ssRNA binding and active site of C3PO complex. (MOV 2876 kb)

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Ye, X., Huang, N., Liu, Y. et al. Structure of C3PO and mechanism of human RISC activation. Nat Struct Mol Biol 18, 650–657 (2011). https://doi.org/10.1038/nsmb.2032

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