Figure 1.

ID-related DDX3X L556S mutation drives the assembly of stable ectopic stress granules under basal conditions

(A) Sanger sequencing validation of the C/T substitution found in the patient from the analyzed cohort.

(B) ATPase activity of 0.5 μM purified D1D2 domain of WT, L556S, and R376C DDX3X variants (N = 5 different experiments with different batches of proteins performed in triplicate).

(C) Images of SH-SY5Y cells transfected for 48 hr with WT, or mutant DDX3X L556S-GFP mutant DDX3X R376C-GFP (green) and immunostained for TIA-1 (red). Inserts show colocalization (yellow) of DDX3X-GFP-tagged granules (green) with the stress granule marker TIA-1 (red). Intensity map on the right represents relative gray scale intensity.

(D) Quantification of percentage of transfected SH-SY5Y cells containing WT or mutant GFP-tagged-DDX3X granules (N = at least 55 cells per group; one-way ANOVA; ns.: not statistically significant; ∗p < 0.05).

(E) FRAP analysis of DDX3X L556S-GFP granule. Granule (arrow) is shown prior to photobleaching, and at 0 s, 4 s, and 40 s after. Graph shows recovery curve as an average ± SEM at each respective time point. Error bars represent mean values ± SEM (N = at least 20 granules from multiple cells per experiment from 3 individual experiments). Scale bar = 10 μm.

(F) MTS assay showing the reduced cell viability of SH-SY5Y cells expressing full human DDX3X-L556S mutant protein. Error bars represent mean values ± SEM (N = 3 individual experiments in triplicate, Student t-test; ∗p < 0.05 when compared to DDX3X WT; ∗∗p < 0.05 when compared to DDX3X WT).

(G) Apoptosis in DDX3X-GFP-positive transfected cells evaluated by TUNEL staining. Error bars represent mean values ± SEM (N = 55 from 3 individual experiments were evaluated, one-way ANOVA; ∗p < 0.05 when compared with DDX3X WT).