Figure 5

The RGG domains modulate the incorporation of FUS into arsenite-induced stress granules. (A) Illustration of full length (FL) GFP-FUS R521G and constructs lacking the following sequences: Gln-Gly-Ser-Tyr-rich (∆QGSY), Gly-rich (∆GLY), RNA recognition motif (∆RRM), and Arg-Gly-Gly-rich (RGG) regions (∆RGG1 and ∆RGG2). (B) Confocal images of HeLa cells transfected with GFP-FUS R521G constructs (green) alone (−) or co-transfected (+) with MBP-M9M, the transportin-1 inhibitor. Note the increased levels of cytoplasmic GFP-FUS in co-transfected cells (compare columns 1 and 2). Confocal fluorescence images of co-transfected cells treated with 0.5 mM sodium arsenite for 1 hr were used to assess the ability of GFP-FUS R521G constructs (green; column 3) to associate with stress granules (G3BP; red; column 4). The greatest degree of GFP and G3BP co-localization was observed in cells expressing FL GFP-FUS R521G, whereas there was minimal co-localization in control cells expressing free GFP (compare panels in column 5). (C) Quantitative analysis (see Materials and methods) of (B) reveals that constructs lacking RGG domains (∆RGG1 and ∆RGG2) exhibit impaired localization to stress granules. Statistically significant comparisons include FL and ∆RGG1 (*P < 0.05), FL and GFP (**P < 0.01), and ∆QGSY and GFP (*P < 0.05; not shown on graph for clarity) by one-way ANOVA followed by a Dunnett’s post-hoc test on n=3 independent experiments. All error bars represent SEMs. (D) Western blot analysis of HeLa cells in (B) demonstrates equivalent expression levels for all GFP-FUS R521G constructs.