Fig. 2

Protein variants of DHX30 affect ATPase and helicase activity. a, b ATPase assays were performed for DHX30-WT, novel DHX30 missense variants (a), and two common polypmorphisms, p.(Val556Ile) and p.(Glu948Lys) (b) in the presence of exogenous RNA. ATPase activity was calculated by subtracting phosphate values obtained with GFP alone from those obtained with GFP-tagged DHX30-WT and mutants. These figures were then normalized on precipitated protein amounts using the intensities of the GFP signal in the western blot. Means ± standard deviation values are based on 3 replications. **,***: significantly different from DHX30-WT, ns: not significantly different from DHX30-WT (**p< 0.01;***p< 0.001; n=3; One-Way ANOVA, followed by Dunnett’s multiple comparisons test). Values were normalized on DHX30-WT ATPase activity obtained in the presence of RNA. c Increasing amounts of His₆-SUMO-tagged DHX30 WT protein were incubated with a ³²P-labeled RNA substrate in the presence (lane 3–7) or absence (lane 8) of ATP and analyzed by native PAGE. The position of the RNA duplex and the single-stranded RNA are indicated in the first and second lanes, respectively. Their schematic representation is shown at the right side. d Helicase assay was repeated for selected DHX30 missense variants affecting either conserved motifs within the helicase core region (lane 4–8) or the auxiliary RL domain (lane 9)