Fig 2: In silico structural analysis of DDX3X mutations(A) 200-ns molecular dynamic simulations of R376C (upper graph) or L556S (lower graph) mutants and wild-type DDX3X. Simulations were performed in triplicates. The graph shows the Ca RMSF for each residue from domain 1 and 2. Lines represent mean, and vertical traces represent standard deviation.(B) Representative frames from MD simulations of R376 or R376C showing intradomain contacts observed during simulations.(C) Ramachandran plot of the first leucine from the wild-type sequence LLDLL and the serine from the mutated sequence SLDLL. Yellow contours represent the favored regions in Ramachandran plot (detailed information in STAR methods section).(D) Representative frames from MD simulations showing intradomain contacts of L556S.

Fig 3: Biophysical characterization of DDX3X protein and aggregation propensity induced by pathogenic mutations(A) Circular dichroism (CD).(B) Differential scanning fluorimetry (DSF) curves for D1D2 domain of WT-DDX3X and mutants during thermal denaturation. Tm values: WT - 51.6 ± 0.1°C, L556S monomer – 40.6 ± 0.1°C, L556S dimer – 44.5 ± 0.1°C, R376C – 40.1 ± 0.2°C.(C) ANS fluorescence at 25°C.(D) Rayleigh light scattering (RLS) at 350 nm of WT and mutants during incubation at 37°C.(E) ThT fluorescence of protein samples before and after incubation at 37°C. *p < 0.05 when compared with WT 0 hr **p < 0.005 when compared with WT 24 hr.(F) ANS fluorescence of proteins during incubation at 37°C.(G) CD of L556S mutant at 10°C after incubation at 37°C during 24 hr.(H) CD of R376C mutant before and after incubation at 37°C.(I) Negative staining image of L556S-DDX3X after 3-hr-incubation at 37°C.(J) Negative staining image of R376C-DDX3X after 3-hr-incubation at 37°C. Scale bar = 100 nm

Fig 4: 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).

Fig 5: BioID-based proteomic analysis of R-loop interactome at sites of transcription–replication conflicts. (A) Schematic representation of BioID screen of proteins associated with R-loops. The expression of RNH1(D210N)-BioID2-HA fusion protein in U-2 OS T-REx RNH1(D210N)-BioID2-HA cells is induced by doxycycline. CPT (camptothecin, 100 nM) promotes the formation of co-transcriptional R-loops. (B) Top panel: Experimental workflow of protein biotinylation by RNH1(D210N)-BioID2-HA in living cells. Bottom panel: Volcano plots showing significantly differentially abundant proteins for comparison of data from RNH1(D210N)-BioID2-HA-expressing cells treated with vehicle alone (DMSO) or 100 nM CPT for 90 min to control cells in which expression of RNH1(D210N)-BioID2-HA was not induced (control). The -log10 (permutation-based FDR corrected P value) is plotted against the log2 (fold change: DMSO/control) or log2 (fold change: CPT/control). Each point represents the difference in abundance of particular protein between the two conditions plotted against the level of statistical significance. Data were processed using the Perseus program with false discovery rate (FDR) of 0.1 (black lines). Significantly enriched proteins are in the right upper quadrant of the volcano plot, depicted in black. DEAD/DEAH box helicases DDX17, DDX5, DHX15, DHX9 and DDX3X helicases are depicted in pink. (C) Venn diagram showing overlap of significantly enriched proteins in DMSO and CPT samples. (D) Significantly enriched proteins from BioID screen clustered into individual classes according to gene ontology molecular function (GO MF) annotations and protein domains. The x-axis indicates statistical significance of overrepresentation.