(Fig.5D),5D), but SecP43 was not (Fig. (Fig.5F).5F). Selenophosphate synthetase 2 (Sps2), which produces the Se-donor selenophosphate for selenoprotein translation, was detected in synaptosomes
(Fig. (Fig.5E).5E). Unlike Sepw1, none of the proteins involved in selenoprotein synthesis were altered in Sepp1−/− mice compared with wild-type controls. This is unexpected for Sps2, which is also a selenoprotein and thus predicted to be dependent on Sepp1 to supply Se. Figure 5 Several selenoprotein synthesis factors are present in synaptosomes. (A) To check for contaminating nuclear proteins, TBP was analyzed. TBP was clearly present in the S1 fractions (−Syn.) but #selleck chemical keyword# not in the synaptosome fractions (+Syn.). Both EFSec … To uncover a potential mechanism for translational regulation of Sepw1, we performed Inhibitors,research,lifescience,medical RNA immunoprecipitation (RIP) experiments using human SH-SY5Y neuroblastoma
cells. We immunoprecipitated using antibodies directed at the two paralogs of the RNA-binding protein Staufen, Stau1, and Stau2, which are involved in mRNA transport and localization in neurons (Duchaine et al. 2002). After normalizing to a synthetic RNA spiked into the samples, ~2% of Sepw1 mRNA was identified in the Stau2-containing mRNP relative to total RNA (Fig. (Fig.6A,6A, left). This amount corresponds to a significant ~1.5-fold enrichment compared with Inhibitors,research,lifescience,medical the Stau1-mRNP (t(4) = 6.701, Inhibitors,research,lifescience,medical P = 0.0026). Conversely Gpx4 mRNA had the opposite profile, with more mRNA found in the Stau1-containing mRNP, corresponding to ~1% of the total Gpx4 mRNA (Fig. (Fig.6A,6A, center). Sepp1 mRNA was undetectable in the RIP samples, despite detection in total RNA (Fig. (Fig.6A6A
and B, right). We also normalized the data to endogenous HPRT mRNA, which Inhibitors,research,lifescience,medical is a putative target of Stau2 in rat brain (Maher-Laporte and DesGroseillers 2010). Sepw1 mRNA associates with Stau2 ~150% more than HPRT mRNA, while associating with Stau1 ~33% less than HPRT mRNA (t(4) = 9.389, P = 0.0007) (Fig. (Fig.6B,6B, left). Both Stau proteins associate with Gpx4 mRNA at about 50% or less than their association with HPRT mRNA (Fig. (Fig.6B,6B, center). Together, Non-specific serine/threonine protein kinase these data argue that Sepw1 mRNA is a specific target of Stau2 in SH-SY5Y cells, and that Stau2-mediated translational regulation of Sepw1 may occur in neurons. Figure 6 Selenoprotein W (Sepw1) mRNA associates with Stau2 in SH-SY5Y neuroblastoma cells. (A) RT-qPCR was performed on Stau1- and Stau2-RNA immunoprecipitation (RIP) samples and Total RNA samples (n = 3) harvested and processed in parallel and normalized to … Discussion The results reported herein describe the first characterization of regional Sepw1 localization in mouse brain, as well as selenoprotein and synthesis factor expression in synaptosome preparations. Sepw1 is abundantly expressed in neuronal somata and neuropil, and is expressed along with several selenoprotein synthesis proteins in synaptosome fractions.