6 G), which may represent large tubulin aggregates. Open in a separate window Figure 8. Lack of functional Ptgs1 GW protein leads to nuclear breakdown caused by defects in mitosis. colocalizes with GW when expressed in cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5C3 mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads DG051 to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development. Introduction The GW182 protein is a critical component of cytoplasmic RNP bodies that have been shown to function in mRNA degradation, storage, and, recently, microRNA (miRNA)- and siRNA-based gene silencing (Eystathioy et al., 2003; Yang et al., DG051 2004; Ding et al., 2005; Jakymiw et al., 2005; Liu et al., 2005a; Rehwinkel et al., 2005). GW182 was named for the presence of multiple glycine (G)Ctryptophan (W) amino acid pairs in the N-terminal region of a 182-kD protein with a predicted C-terminal RNA recognition motif (RRM). It localizes into cytoplasmic GW bodies (GWBs; Eystathioy et al., 2002; Maris et al., 2005) that also contain factors involved in 5C3 mRNA decay, including the exonuclease XRN1, decapping enzymes DCP1 and DCP2, and the LSm1C7 decapping activator, pointing to a role for GWBs in regulating mRNA stability (Ingelfinger et al., 2002; Eystathioy et al., 2003; Cougot et al., 2004). These bodies may participate in additional roles in mRNA regulation, as they also contain the m7G capCbinding protein eIF4E and the eIF4E transporter but no other components of translation machinery (Andrei et al., 2005; Kedersha et al., 2005). Importantly, intact GWBs are DG051 required for the functioning of the RNAi pathway in human cells potentially via direct interaction between GW182 (and the related TNRC6B protein) and Argonaute1 (Ago1) and 2 (Ago2; Jakymiw et al., 2005; Liu et al., 2005a,b; Meister et al., 2005). GWBs are thought to be analogous to cytoplasmic processing bodies (PBs). They are involved in mRNA decapping and 5C3 exonucleolytic decay DG051 (Sheth and Parker, 2003), and their integrity depends on the presence of nontranslating mRNAs (Sheth and Parker, 2003; Cougot et al., 2004; Teixeira et al., 2005). Both PBs and GWBs dissociate when polysomes are stabilized with drugs such as cycloheximide (Sheth and Parker, 2003; Cougot et al., 2004; Teixeira et al., 2005). However, despite similar compositions, there are functional differences between GWBs and PBs. GWBs increase in size and number in proliferating DG051 cells (Yang et al., 2004), whereas PBs increase in size and number during growth limitation and increased cell density (Teixeira et al., 2005). GWBs and PBs also differ in their responses to stress, as PBs increase in size and number in response to environmental stress. This is likely caused by decreased translation initiation because this response can be reproduced using a temperature-sensitive allele of Prt1p, a subunit of the eIF3 complex (Teixeira et al., 2005). In stressed mammalian cells, stalled preinitiation complex mRNAs are first targeted to stress granules (SGs), which may function as triage sites where mRNAs are sorted for future degradation, storage, or reinitiation of translation. Observation of interactions between SGs and GWBs in live cells suggest that transcripts may be exported from SGs to GWBs for degradation (Kedersha et al., 2005). We have characterized the role of (orthologue of the human GW182 gene family. GW localizes to punctate structures in the cytoplasm of embryos and cultured S2 cells. GWBs are electron-dense nonmembrane-bound cytoplasmic foci. These structures are targeted by human GW182 and its paralogues TNRC6B and TNRC6C in cells. Unlike what is seen in some mammalian cells, only some foci colocalize with the previously identified GWB components LSm4, the Xrn1 orthologue Pacman (PCM), and AGO2 (Ingelfinger et al., 2002; Eystathioy et al., 2003; Kedersha et al., 2005; Liu et al., 2005a; Sen and Blau, 2005). There is a requirement for the zygotic expression of full-length GW during early embryonic nuclear divisions. This suggests a critical role for GWB-based cytoplasmic RNA regulation in beginning with.