FIGURE 8:

RALY mutant retains FUS in the cytoplasm and alters its interaction with mRNAs.  (A) MUT RALY retains endogenous FUS in the cytoplasm and recruits it to cytosolic aggregates induced by arsenite treatment (arrowheads). Immunofluorescence images show WT or MUT RALY-GFP (green) and endogenous FUS (red) stained with anti-GFP and anti-FUS antibody, respectively. HeLa cells were transfected for 24 h before fixation. The scale bar corresponds to 20 μm. (B) MUT RALY significantly retains FUS in the cytoplasm. The graph reports the quantification of FUS nucleus/cytoplasm signal, obtained by high-content image analysis, in untreated and arsenite-treated HeLa cells. Bars indicate means ± SEM of five replicates, and p values were calculated with unpaired two-tailed Student’s t test to compare MUT RALY with WT RALY-overexpressing cells (*p < 0.05; ***p < 0.001). (C) RALY mutants impair FUS interaction with Dctn1, Sod1, Sncb, and Pink1 mRNAs. The graph shows the statistical analysis of five independent experiments, normalized on Gapdh. To compare all the experiments, the yield was set equal to 1 for FUS RIPs in cells expressing WT RALY; hence the yield for FUS RIPs in cells expressing MUT RALY was calculated proportionately. Bars indicate means ± SEM of five replicates, and p values were calculated with unpaired two-tailed Student’s t test to compare MUT RALY with WT RALY-overexpressing cells (*p < 0.05; **p < 0.01; ***p < 0.001).