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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    • "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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    ABSTRACT: Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and chemical linkage to introduce a high density of bioactive ligands onto nanometer-thick carbon films and enable selective enrichment of individual macromolecular complexes at subnanogram levels. The introduced ligands are physically separated. Ni-NTA, Protein G and DNA/RNA oligonucleotides were covalently linked to the carbon surface. They embody negligible mass and their stability makes the functionalized films able to survive long-term storage and tolerate variations in pH, temperature, salts, detergents, and solvents. We demonstrated the application of the new method to the electron microscopic imaging of the substrate-bound C3PO, an RNA-processing enzyme important for the RNA interference pathway. On the ssRNA-linked carbon surface, the formation of C3PO oligomers at subnanomolar concentrations likely mimics their assembly onto ssRNA substrates presented by their native partners. Interestingly, the 3D reconstructions by negative stain EM reveal a side port in the C3PO/ssRNA complex, and the 15 Å cryoEM map showed extra density right above the side port, which probably represents the ssRNA. These results suggest a new way for ssRNAs to interact with the active sites of the complex. Together our data demonstrate that the surface-engineered carbon films are suitable for selectively enriching low-abundance biological complexes at nanomolar level and for developing novel applications on a large number of surface-presented molecules.
    Journal of Structural Biology 01/2014; 185(3). DOI:10.1016/j.jsb.2014.01.006 · 3.23 Impact Factor
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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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    ABSTRACT: Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes (15)N and (14)N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.
    Biochimica et Biophysica Acta 12/2013; 1844(4). DOI:10.1016/j.bbapap.2013.12.016 · 4.66 Impact Factor
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