Fig. 5: Ataxin-2 binds Xbp1 mRNA during UPR activation.
From: Fbxo42 promotes the degradation of Ataxin-2 granules to trigger terminal Xbp1 signaling
a Immunofluorescence and RNA FISH of untreated ring gland cells (3rd instar larva). Endogenous Ataxin-2 is in red (anti-Ataxin-2 antibody), Xbp1 mRNA in green (Stellaris FISH probes against Xbp1) and the nuclei is in blue (DAPI). Scale bar = 10 μm. b Immunofluorescence and RNA FISH of ring gland cells treated with 5 mM DTT (to induce ER stress and UPR activation) for 4 h. Endogenous Ataxin-2 (red), Xbp1 mRNA (green) and nuclei (blue), as in (a). Arrows indicate Xbp1 mRNA in Ataxin-2 granules. Arrowheads indicate Xbp1 mRNA in the nucleus. c Quantification of the number of granules of Ataxin-2 protein only (red bar), Xbp1 mRNA only (green bar) or both Ataxin-2 protein and Xbp1 mRNA (yellow bar), in untreated ring gland cells (shown in (a)) and in ring gland cells treated with 5 mM DTT for 4 h (shown in (b)). The quantification was done in 2 biological replicates per condition and is presented in percentage (%) as mean ± SD. Two-way ANOVA coupled with Sidak’s multiple-comparison test, ****p < 0.0001. The number of granules scored in untreated ring glands was n = 125 (replicate 1) and n = 150 (replicate 2). The number of granules scored in ring gland cells treated with DTT was n = 123 (replicate 1) and n = 156 (replicate 2). d iCLIP results for Xbp1/Ataxin-2-HA. In green are depicted the peaks (sequencing reads) in Xbp1 from UV-irradiated S2 cells transfected with Ataxin-2-HA. In blue are depicted the peaks in Xbp1 of UV-irradiated cells transfected with Ataxin-2C244A-HA. The non-UV irradiated controls for Ataxin-2-HA and Ataxin-2C244A-HA samples are shown by the yellow and orange lines, respectively. To the right is a zoom of the Xbp1 3’UTR containing the AU-rich region.
