Data was analyzed by Student’sttest (**P<0

Data was analyzed by Student’sttest (**P<0.001). == Wnt signalling antagonizes SG assembly == As our studies clearly indicated a role for Dvl in SG dynamics, we wished to explore whether Wnt signalling regulates SG assembly. and reveal a novel role for Wnt/Dvl pathway in the modulation of mRNA functions. Keywords:Wnt, Dvl, RNA, Stress granule, G3BP == Introduction == Stress granules (SGs) and Processing bodies (P bodies) represent mRNA-containing, non-membranous, dynamic cytoplasmic foci defined by the presence of specific and common protein components (Anderson and Kedersha, 2006;Anderson and Kedersha, 2009;Balagopal and Parker, 2009;Parker and Sheth, 2007). P bodies are present at the stationary phase, whereas SGs are assembled during diverse stress conditions, as a result of aggregation of mRNAs stalled in a translational pre-initiation complex, a process mediated by RNA-binding proteins such as G3BP and TIA-1 (Gilks et al., 2004;Kedersha et al., 1999;Tourriere et al., 2003). The actively translating mRNAs (polysomes) are present mostly diffused in the cytoplasm. Although polysomes, P bodies and SGs represent mRNAs associated with different sets of cellular proteins present at distinct cytoplasmic locations, recent data suggests that the mRNAs and protein components may shuttle between these three pools (Balagopal and Parker, 2009). Thus, SGs are thought to be sites for mRNA triage, wherein the mRNAs could be further sorted into P bodies for degradation, returned to cytoplasm for translation re-initiation or stored in the SGs (Anderson and Kedersha, Rabbit Polyclonal to SNIP 2008). SKLB1002 Although these studies highlight the importance of post transcriptional regulation of mRNAs in achieving cell homeostasis, the mechanism by which cells modulate the assembly/disassembly of these mRNA containing structures and/or determine the fate of individual mRNA is far from clear. It is possible that diverse cell signalling pathways could operate through post transcriptional regulation of specific mRNAs. Wnt signalling is a conserved pathway that regulates cell fate determination, cell proliferation and cell polarization during growth and development of multi-cellular organisms (Boutros and Mlodzik, 1999;Clevers, 2006;Gao and Chen, 2010;Logan and Nusse, 2004;van Amerongen and Nusse, 2009;Wallingford and Habas, 2005). Wnt pathway is broadly categorized into canonical or non-canonical, based on the dependence on -catenin for the signalling process. In canonical Wnt signalling, binding of Wnt to the cognate receptors on the cell membrane leads to -catenin stabilization in the cytoplasm, through a mechanism involving Dishevelled (Dvl)-dependent inhibition of the -catenin degradation complex. The accumulated -catenin then enters the nucleus and activates transcription of Wnt responsive genes. However, SKLB1002 the non-canonical signalling operates independent of -catenin, but involves regulation of cytoskeleton dynamics by SKLB1002 Dvl-dependent activation of Rac and Rho GTPases. Alternatively, Wnt could activate Ca2+signalling, which also requires Dvl. Despite its demonstrated role in diverse cellular processes including Wnt signalling, the molecular function of Dvl is largely unknown. Mammalian cells express three isoforms of this protein, namely Dvl1, Dvl2 and Dvl3 (Klingensmith et al., 1996;Lee et al., 2008). An interesting feature of overexpressed Dvl is its ability to form cytoplasmic puncta, the nature of which remains mysterious (Axelrod et al., 1998;Kishida et al., 1999;Smalley et al., 1999). Endogenous Dvl also localizes to cytoplasmic granules (Chang et al., 2005;Miller et al., 1999;Yanagawa et al., 1995). Overexpression and live imaging studies have shown that these cytoplasmic puncta are Dvl protein assemblages, which are in dynamic equilibrium with the cytoplasmic Dvl pool (Schwarz-Romond et al., 2005;Smalley et al., 2005). Importantly, these structures are non-membranous and do not represent any known cytoplasmic vesicle compartments (Schwarz-Romond et al., 2005;Smalley et al., 2005). Owing to the structural similarities between Dvl and SGs/P bodies, we investigated the possible connection between these cytoplasmic structures. In this study, we demonstrate that Wnt/Dvl signalling negatively regulates SG assembly, mediated through a mechanism involving Rac1-dependent inhibition of RhoA. Moreover, Dvl2 physically interacts with G3BP and inhibits its SG-nucleating activity. Our findings functionally connect Dvl to stress response, and implicate a role for Wnt/Dvl signalling in the modulation of mRNA translation and degradation. == Results == == Dishevelled negatively regulates SG assembly in a DEP-dependent manner == To investigate the possible connection between Dvl and SGs or P bodies, NIH3T3 cells were transfected with full length or various deletion mutants of mouse Dvl2 (Fig. 1C), and were subjected to oxidative stress by treating with 0.5 mM sodium arsenite for 30 min. Ectopically expressed full length Dvl2 did not localize to SKLB1002 SGs (Fig. 1A) or P bodies (Fig..