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
Source: PubMed

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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    ABSTRACT: Current therapies for chronic hepatitis B virus infection (CHB) - nucleos(t)ide analogue reverse transcriptase inhibitors and interferons - result in low rates of functional cure defined as sustained off-therapy seroclearance of hepatitis B surface antigen (HBsAg). One likely reason is the inability of these therapies to consistently and substantially reduce the levels of viral antigen production. Accumulated evidence suggests that high serum levels of HBsAg result in exhaustion of the host immune system, rendering it unable to mount the effective antiviral response required for HBsAg clearance. New mechanistic approaches are required to produce high rates of HBsAg seroclearance in order to greatly reduce off-treatment disease progression. Already shown to be a clinically viable means of reducing gene expression in a number of other diseases, therapies based on RNA interference (RNAi) can directly target hepatitis B virus transcripts with high specificity, profoundly reducing the production of viral proteins. The fact that the viral RNA transcripts contain overlapping sequences means that a single RNAi trigger can result in the degradation of all viral transcripts, including all messenger RNAs and pregenomic RNA. Advances in the design of RNAi triggers have increased resistance to degradation and reduced nonspecific innate immune stimulation. Additionally, new methods to effectively deliver the trigger to liver hepatocytes, and specifically to the cytoplasmic compartment, have resulted in increased efficacy and tolerability. An RNAi-based drug currently in clinical trials is ARC-520, a dynamic polyconjugate in which the RNAi trigger is conjugated to cholesterol, which is coinjected with a hepatocyte-targeted, membrane-active peptide. Phase 2a clinical trial results indicate that ARC-520 was well tolerated and resulted in significant, dose-dependent reduction in HBsAg for up to 57days in CHB patients. RNAi-based therapies may play an important role in future therapeutic regimes aimed at improving HBsAg seroclearance and eliminating the need for lifelong therapy. This paper forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B." Copyright © 2015. Published by Elsevier B.V.
    Antiviral research 06/2015; 121. DOI:10.1016/j.antiviral.2015.06.019 · 3.94 Impact Factor
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    • "However, it is also reported that translational inhibition mediated by the other three Ago proteins (Ago1, 3, and 4) also significantly contributes to siRNA repression.6 In addition to different repression mechanisms, the loading of siRNA or miRNA can also differ between Ago2 and the other Ago proteins because Ago2 can cleave the passenger strand, which is removed by C3PO (component 3 promoter of RISC, a complex of Translin and Trax);7,8 while Ago1, 3, and 4 cannot cleave the passenger strand, and must load siRNA or miRNA using the “bypass mechanism”.9 Thus, Ago2 is distinct from the other three Ago proteins in its slicer function. "
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    ABSTRACT: Relatively large amounts of transfected siRNA can compete for Ago proteins and thus compromise endogenous miRNA function, potentially leading to toxicities. Here, we show that shRNA can also perturb endogenous miRNA function similarly. More importantly, we also show that the problem can be solved by designing shRNAs in the context of pre-miR-451 structure with completely complementary stem, which significantly improves the Ago2 specificity. This shRNA was shown to be Ago2-specific, and maintain target-silencing ability while avoiding competition with endogenous miRNAs by not competing for Agos 1, 3, and 4. We conclude that modified pre-miR-451 structure provides a general platform to design shRNAs that significantly reduce perturbation of miRNA function.
    Molecular Therapy - Nucleic Acids 07/2014; 3(7):e176. DOI:10.1038/mtna.2014.27 · 4.51 Impact Factor
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    • "In fact, minimal RISC that is capable of cleaving target RNA has been reconstituted using single-stranded siRNA and recombinant Ago2 (36). Furthermore, a recent experiment indicated that the recombinant human Ago2 alone can acquire a guide RNA strand by first associating with siRNA or miRNA duplex, leading to cleavage of target RNA in vitro (37), although recombinant Ago2 was initially thought to be unable to bind to siRNA duplex (36,38). Active RISCs are shown to be minimally formed by double-stranded siRNA and Ago in vitro, but Dcr and TRBP or PACT may be often considered to be essential for siRNA strand selection (37). "
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    ABSTRACT: Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5' proximal nucleotides, which include seed sequence (positions 2-8 from the 5' end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19-21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15-18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.
    Nucleic Acids Research 02/2014; 42(8). DOI:10.1093/nar/gku153 · 9.11 Impact Factor
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