Article

Structure of C3PO and mechanism of human RISC activation

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 06/2011; 18(6):650-7. DOI: 10.1038/nsmb.2032
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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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Available from: Xuecheng Ye, Sep 22, 2014
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    • "Briefly, introduction of siRNA into the cell cytoplasm results in its association with protein components of the RNA-induced silencing complex (RISC) (Fig. 1). Once RISC is loaded with the siRNA, the passenger strand is nicked by the RISC component Argonaute2 (AGO2), and further degraded by component 3 promoter of RISC (C3PO) (Matranga et al., 2005; Miyoshi et al., 2005; Rand et al., 2005; Ye et al., 2011). This results in the exposure of nucleotide bases of the guide strand, enabling them to base pair with the complementary target mRNA sequence. "
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    Antiviral research 06/2015; 121. DOI:10.1016/j.antiviral.2015.06.019 · 3.94 Impact Factor
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    • "C3PO assembled on let-7 ssRNA-ChemiC films Structural studies of C3PO as dimers, tetramers, hexamers and octamers raised a fundamental question about what oligomeric state the native C3PO takes inside a cell (Parizotto et al., 2013; Tian et al., 2011), and how C3PO recruits short ssRNAs for degradation. Crystallographic studies were all conducted with high concentrations (0.2–0.4 mM) of proteins (Tian et al., 2011; Ye et al., 2011), orders of magnitude higher than the native concentration. "
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    • "More recent studies have indicated that in Drosophila and in mammals hetero-octamers consisting of Translin and TRAX subunits are involved in RNA silencing [24] [25] [26] [27] [28]. In particular, the TRAX subunits in these hetero-octamers were reported to have a ribonuclease activity that appears to activate RISC complexes by removing siRNA passenger strand cleavage products and thereby promoting the activity of the RNAi machinery [25] [26]. It has also been reported that complexes including Translin, or both Translin and TRAX, are involved in transport of specific mRNAs along dendrites in mammalian neurons [29]. "
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