LETTERS NATURE CELL BIOLOGY VOLUME 7 | NUMBER 12 | DECEMBER 2005 1267 Disruption of GW bodies impairs mammalian RNA interference Andrew Jakymiw 1 , Shangli Lian 1 , Theophany Eystathioy 2 , Songqing Li 1 , Minoru Satoh 3 , John C. Hamel 1 , Marvin J. Fritzler 2 and Edward K.L. Chan 1,4 The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs) 1 . GWBs are enriched in proteins that are involved in mRNA degradation 2 . Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures 3,4 . In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing. Gene regulation is becoming more intricate with the discovery of RNA interference (RNAi) or RNA silencing 5,6 . This process uses double- stranded RNA (dsRNA) to silence genes in a sequence-specific manner. The dsRNA is processed by Dicer into 21–26 nucleotide fragments that generate small interfering RNAs (siRNAs) or microRNAs. The siRNA and microRNA bind ribonucleoproteins to form the RNA-induced silencing complex (RISC), which then either cleaves or inhibits the translation of the targeted mRNA. Recent information indicates that one member of the Argonaute (Ago) protein family, Ago2, is the key catalytic component for RISC-related endonucleolytic cleavage of mRNA 7,8 . The role of the other proteins in the cascade event that leads to silencing activity is not well defined. The human GWB protein, GW182, is characterized by multiple gly- cine/tryptophan repeats and a classic RNA recognition motif 1 . With the discovery that proteins involved in mRNA decay were part of the GWBs 2 or cytoplasmic foci/P-bodies 9–11 , it was postulated that these structures, and perhaps the GW182 protein, were part of a selective mRNA degrada- tion network. In our quest to understand the functional role of the GW182 1 Departments of Oral Biology and Anatomy & Cell Biology, University of Florida, Gainesville, FL 32610, USA. 2 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1. 3 Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA. 4 Correspondence should be addressed to E.K.L.C. (email: echan@ufl.edu) Received 18 July 2005; accepted 24 September 2005; published online: 13 November 2005; DOI: 10.1038/ncb1334 protein and GWBs, we show data supporting a role for these structures in mRNA degradation that is intriguingly linked with RNA silencing. To explore the biology of GWBs, it was of interest to identify protein(s) that are associated with GW182, because this could provide some insight to its function. In an initial experiment, sonicated cell lysates from ~1 billion HeLa cells were harvested and fractionated in a 14–80% discon- tinuous sucrose gradient. Fractions that were highly enriched for GW182 (34.5% sucrose) were subsequently used for co-immunoprecipitation studies with the human prototype anti-GW182 serum. The immuno- precipitate was analysed by SDS–PAGE and silver-stained. Subsequently, several prominent bands were excised and submitted for analysis by mass spectroscopy (MALDI). An excised protein band with a relative molecular mass of ~100,000 was identified as Ago2. To independently confirm this data, we analysed the interaction of Ago2 with GW182 by immunoprecipitation using unfractionated HeLa cell lysates and a human anti-GW182 serum. We found that endogenous Ago2 co-immu- noprecipitated with GW182, whereas it did not co-immunoprecipitate with the negative control, normal human serum (Fig. 1A). This data indicated that, in human cells, Ago2 interacts with GW182. There is evidence that RISC activity occurs within the cytoplasm of HeLa cells 12 . Furthermore, endogenous Ago2 has been shown to local- ize to discrete cytoplasmic foci; however, no cellular marker was used to verify the specific location of this protein within the cell 3 . Based on the observation that Ago2 was capable of interacting with GW182, we next analysed whether the endogenous Ago2 was capable of localizing to GWBs. Using antibodies specific for Ago2, we demonstrated that Ago2 localized to discrete cytoplasmic foci in HEp-2 cells; these foci were determined to be GWBs by co-staining with a GW182 marker (Fig. 1B). The high enrichment of cellular Ago2 to GWBs was inde- pendently confirmed in HeLa cells using a green fluorescent protein (GFP)–Ago2 plasmid construct (Fig. 1C). These data are consistent with recently published observations that Ago2 concentrates in GWBs 3,4 . In mammalian cells, exogenously delivered 21-nucleotide siRNAs induce sequence-specific gene silencing and guide mRNA degrada- tion 13 . Duplex siRNAs subsequently unwind and the antisense strand Nature Publishing Group ©2005