RNA localization pathways direct numerous mRNAs to distinct subcellular regions and

RNA localization pathways direct numerous mRNAs to distinct subcellular regions and affect many physiological processes. polarity (Li et al., 2008; Nagaoka et al., 2012), migration (Shestakova et al., 2001), neuronal axon growth and pathfinding (Leung et al., 2006; Hengst et al., 2009), and mitotic spindle assembly (Blower et al., 2007). Defects in localization have been implicated in diseases such as mental retardation and cancer metastasis (Bassell and Warren, 2008; Vainer et al., 2008). We previously described a pathway that targets many RNAs to cellular protrusions (Mili et al., 2008). A central component of this pathway is usually the tumor suppressor protein adenomatous polyposis coli (APC; N?thke, 2004). At protrusive areas, and specifically at the plus-ends of detyrosinated microtubules, APC affiliates with multiple RNAs (such as Pkp4, Rab13, Kank2, and Ddr2) and proteins (such as FMRP and PABP1) to form APC-containing ribonucleoprotein complexes (APC-RNPs; Mili et al., 2008). This APC function might mediate effects on cell migration (Sansom et al., 2004; Kroboth et al., 2007; Harris and Nelson, 2010), and is usually distinct from its canonical function in the Wnt pathway where it regulates -catenin degradation (Kennell and Cadigan, 2009). APC-RNPs are concentrated in granules that likely contain many different transcripts (Mili et al., 2008). Several RNA granule types exist that share common components and are either constitutively present (such as neuronal transport granules and P-bodies) or form in response to stress (stress granules). They are sites where RNAs are silenced through translational Butenafine HCl supplier repression or decay (Anderson and Kedersha, 2008; Buchan and Parker, 2009). Other types of higher order RNACprotein assemblies are also formed by aggregation-prone RNA-binding protein such as Fus (fused in sarcoma) and TDP43 in neurodegenerative diseases (Lagier-Tourenne et al., 2010; Liu-Yesucevitz et al., 2011). Dominant mutations in Fus are found in amyotrophic lateral sclerosis (ALS) cases, and Fus is usually also the pathological protein in types of frontotemporal lobar degeneration (FTLD; Lagier-Tourenne et al., 2010; Mackenzie et al., 2010). The disease hallmark is usually Butenafine HCl supplier Fus-containing inclusions, which share components with stress granules, suggesting that alterations in RNA metabolism might underlie disease pathogenesis (Andersson et al., 2008; Bosco et al., 2010; Dormann et al., 2010). We show here that Fus is usually a component of APC-RNPs at cell protrusions and is usually required for their efficient translation. Using a metabolic labeling approach to mark newly synthesized Butenafine HCl supplier proteins, we show that Fus preferentially affects translation within protrusions. Cytoplasmic granules formed by either overexpression of wild-type Fus or by expression of ALS mutants of Fus preferentially recruit APC-RNPs. Strikingly, these granules are not translationally silent. Instead, we show that translation occurs within cytoplasmic Fus granules leading to local protein production from APC-RNPs. Results and discussion Fus is usually a component of APC-RNPs at cell protrusions To find additional APC-RNP components, we identified by mass spectrometry proteins that coimmunoprecipitate with APC Butenafine HCl supplier from mouse fibroblasts. One candidate was the RNA-binding protein Fus (Fig. S1 a). Indeed, endogenous Fus, but not hnRNPA2, affiliates with immunoprecipitated APC (Fig. 1 a). Additionally, immunoprecipitated GFP-Fus affiliates specifically with APC, but not with -catenin (Fig. 1 b), indicating that Fus is usually not part of the destruction organic in the Wnt pathway. Furthermore, Fus affiliates with RNAs that are present in APC-RNPs (Pkp4, CTCF Rab13, Kank2; Fig. 1 c; Mili et al., 2008). Consistent with the limited sequence specificity and large number of RNA targets described for Fus (Lagier-Tourenne et al., 2012; Rogelj et al., 2012), we find little specificity for Fus with regards to RNA binding. Interestingly, however, quantitation of the efficiency of binding revealed that Fus affiliates preferentially with RNAs enriched in protrusions (Pkp4, Rab13, Kank2) compared with RNAs not enriched in protrusions.