Localization of Double-stranded Small Interfering RNA to Cytoplasmic Processing Bodies Is Ago2 Dependent and Results in Up-Regulation of GW182 and Argonaute-2

Department of Physiology, Anatomy, and Genetics, University of Oxford, United Kingdom.
Molecular biology of the cell (Impact Factor: 5.98). 11/2008; 20(1):521-9. DOI: 10.1091/mbc.E08-08-0796
Source: PubMed

ABSTRACT Processing bodies (P-bodies) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and degradation. Key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2), miRNAs, and their cognate mRNAs, are localized to these structures; however, the precise role that P-bodies and their component proteins play in small interfering RNA (siRNA)-mediated RNAi remains unclear. Here, we investigate the relationship between siRNA-mediated RNAi, RNAi machinery proteins, and P-bodies. We show that upon transfection into cells, siRNAs rapidly localize to P-bodies in their native double-stranded conformation, as indicated by fluorescence resonance energy transfer imaging and that Ago2 is at least in part responsible for this siRNA localization pattern, indicating RISC involvement. Furthermore, siRNA transfection induces up-regulated expression of both GW182, a key P-body component, and Ago2, indicating that P-body localization and interaction with GW182 and Ago2 are important in siRNA-mediated RNAi. By virtue of being centers where these proteins and siRNAs aggregate, we propose that the P-body microenvironment, whether as microscopically visible foci or submicroscopic protein complexes, facilitates siRNA processing and siRNA-mediated silencing through the action of its component proteins.

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Available from: Aarti Jagannath, Jul 18, 2014
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    • "Ago-2 can recruit GW182 that has three Ago-2 binding sites (Takimoto et al. 2009; Han et al. 2004). Expression of GW182 and Ago-2 is upregulated after siRNA delivery (Jagannath and Wood 2009). These small molecules together with Ago-2 are rapidly localized to p-bodies, and then, Ago proteins are regulated by phosphorylation at specific residues as serine-387 phosphorylation mediated by p38 MAPK pathway which increases Ago-2 P-body localization (Rudel and Meister 2008; Rudel et al. 2011; Zeng et al. 2008). "
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    Functional & Integrative Genomics 11/2013; 14(1). DOI:10.1007/s10142-013-0344-1 · 2.69 Impact Factor
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    • "After initial encounter and endonucleolytic cleavage of the target mRNA by Ago2 at the rER , the sliced mRNA ( or silenced miRNP ) may likely be taken up by P - bodies to facilitate the degradation of the mRNA . This would be con - sistent with the repeated detection of siRNA as well as Ago2 and particularly Ago2 - GFP in P - bodies ( Liu et al , 2005 ; Sen and Blau , 2005 ; Leung et al , 2006 ; Ohrt et al , 2008 ; Jagannath and Wood , 2009 ) , as well as the notion that P - bodies , while clearly involved in RNAi , are rather a consequence than cause of silencing . Interestingly , life cell microscopy supports a transient and dynamic association of Ago2 - GFP , which is known to be primarily P - body associated , with the ER ( Supplementary Movie S1 ) . "
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    The EMBO Journal 03/2013; 32(8). DOI:10.1038/emboj.2013.52 · 10.75 Impact Factor
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    • "We have also shown that inhibition of HSP90 reduced the number of microscopic P-bodies. This is probably a consequence of the destabilization of the GW182 protein family, a key component of the P-bodies, rather than Ago2 because the presence of Ago2 is not required to form this cytoplasmic structure (Lian et al., 2007; Jagannath and Wood, 2009). Also, when we rescued Ago2 from HSP90 inhibition by transfecting increasing amounts of siRNA into the cells before geldanamycin treatment (Figure 4A), we did not observe a significant increase in the number or size of P-bodies in the geldanamycin-treated cells (Supplementary Figure 5, A and B), suggesting that the depletion of P-bodies is independent of the effect of HSP90 on Argonautes. "
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    ABSTRACT: Key components of the miRNA-mediated gene regulation pathway are localized in cytoplasmic processing bodies (P-bodies). Mounting evidence suggests that the presence of microscopic P-bodies are not always required for miRNA-mediated gene regulation. Here we have shown that geldanamycin, a well-characterized HSP90 inhibitor, abolishes P-bodies and significantly reduces Argonaute and GW182 protein levels but does not affect the miRNA level and the efficiency of miRNA-mediated gene repression; however, it significantly impairs siRNA loading and the efficacy of exogenous siRNA. Our data suggests that HSP90 protein chaperones Argonautes before binding RNA and may facilitate efficient loading of small RNA.
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