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: 4.47). 11/2008; 20(1):521-9. DOI: 10.1091/mbc.E08-08-0796
Source: PubMed


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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    • "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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    • "We can rule out the possibility that the cytoplasmic foci containing siRNA against the Ehpc4 gene were non-transfected vesicles or endosomes, since we used the soaking method to deliver the dsRNA-Cy3 probe to trophozoites. However, we cannot be sure that only the siRNA guide strand was located in the cytoplasmic foci, since it has been reported that siRNAs can localize in P-bodies as dsRNA [29]. "
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