Article

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

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.

Download full-text

Full-text

Available from: Aarti Jagannath, Jul 18, 2014
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding gene regulation mechanisms has been a serious challenge in biology. As a novel mechanism, small non-coding RNAs are an alternative means of gene regulation in a specific and efficient manner. There are growing reports on regulatory roles of these RNAs including transcriptional gene silencing/activation and post-transcriptional gene silencing events. Also, there are several known small non-coding RNAs which all work through RNA interference pathway. Interestingly, these small RNAs are secreted from cells toward targeted cells presenting new communication approach in cell-cell or cell-organ signal transduction. In fact, understanding cellular and molecular basis of these pathways will strongly improve developing targeted therapies and potent and specific regulatory tools. This study will review some of the most recent findings in this subject and will introduce a super-pathway RNA interference-based small RNA silencing network.
    Full-text · Article · Nov 2013 · Functional & Integrative Genomics
  • Source
    • "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 ) . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.
    Full-text · Article · Mar 2013 · The EMBO Journal
    • "Our earlier publications showed that siRNA (Jakymiw et al. 2005 ) and miRNA (Pauley et al. 2006 ) are highly enriched in GW bodies and, by extension, we suggested that they may be critical for RNA interference. Other investigators have shown that siRNA are localized to GW/P bodies and they showed that it is Ago2-dependent and transfection of siRNA results in upregulation of Ago2 and GW182 (Jagannath and Wood 2009 ) . Functional siRNA activity correlates with increases in both number and size of GW bodies (Lian et al. 2007 ) . "
    [Show abstract] [Hide abstract]
    ABSTRACT: The literature on human autoantibodies in systemic rheumatic diseases has clearly elucidated major autoimmune targets, many of which are nucleic acid-protein macromolecular complexes or subcellular particles (Tan et al. 1988). Well-documented examples of these include the ribonucleoprotein Sm/RNP complex comprised of key components of U-rich small nuclear ribonucleoproteins (UsnRNPs) that are critical for processes in mRNA splicing, and chromatin subunits composed of DNA, histones and high mobility group proteins. Although there are still unanswered questions about why these are the targets of the B-cell response, the current thinking is that these nucleic acid-protein complexes are preferred target autoantigens in systemic autoimmune diseases because of their interactions with toll-like receptors (TLR). TLR3, 7, and 9 are primarily located in the endosomes and are responsible for sensing of endogenous RNA and DNA ligands (Kawai and Akira 2009). Thus, endogenous nucleic acids-protein complexes have a higher tendency to stimulate a B-cell autoimmune response.
    No preview · Article · Jan 2013 · Advances in Experimental Medicine and Biology
Show more