Fig 2: DDX17 promotes replication restart following R-loop-mediated fork stalling. (A) Western blot analysis of the lysates of U-2 OS cells transfected with indicated siRNAs. Note that DDX17 exists in two isoforms, p72 and p82, which result from alternative translation initiation sites. (B) Left panel: Experimental workflow of DNA fiber assays. U-2 OS cells were transfected with indicated siRNAs for 72 h followed by pulse-labeling with CldU and IdU. During the IdU labeling, cells were treated with 100 nM CPT or DMSO. Where indicated, 10 µM PARP inhibitor olaparib (PARPi) was added 2 h before labeling and was also present during the labeling. Right panel: Representative immunofluorescence images of replication tracts on DNA fibers of U-2 OS cells transfected with control siRNA (siLuc) and treated as indicated. Scale bar, 10 µm. (C) Effect of the depletion of DDX17, DHX9, DDX5, DHX15 and DDX3X respectively, on the rescue of CPT-induced replication fork slowing by PARP inhibition (PARPi). Scatter plot of the values of IdU/CldU tract length ratio obtained for indicated conditions (n = 300) is shown. Red horizontal lines indicate mean value. ns, non-significant (P > 0.05); **P = 0.01; ****P = 0.0001 (Mann–Whitney test). (D) Effect of the depletion of DDX17, DHX9, DDX5, DHX15 and DDX3X, respectively, on replication fork velocity in U-2 OS cells (n = 300). Red horizontal lines indicate mean value. ns, non-significant (P > 0.05); **P = 0.01; ****P = 0.0001 (Mann–Whitney test). (E) Western blot analysis of the lysates of U-2 OS cells transfected with indicated siRNAs. (F) DDX17 is required for the rescue of CPT-induced replication fork slowing by ZRANB3 depletion in U-2 OS cells. Top panel: Experimental workflow of DNA fiber assay. U-2 OS cells were transfected with indicated siRNAs for 72 h followed by pulse-labeling with CldU and IdU. During the second labeling, cells were treated with 100 nM CPT or DMSO. Bottom panel: Scatter plot of the values of IdU/CldU tract length ratio obtained for indicated conditions (n = 300). Red horizontal lines indicate mean value. ns, non-significant (P > 0.05); ****P = 0.0001 (Mann–Whitney test). (G) Western blot analysis of the lysates of U-2 OS T-REx [RNH1(WT)-GFP] cells transfected with indicated siRNAs. (H) Experimental workflow of DNA fiber assays with U-2 OS T-REx [RNH1(WT)-GFP] cells for (I) and (J). Expression of RNH1(WT)-GFP was induced by addition of doxycycline (DOX; 1 ng/ml) to eliminate R-loops. (I) Loss of DDX17 induces R-loop-dependent sister fork asymmetry. Top panel: representative images of symmetric and asymmetric replication tracts of sister forks. Scale bar, 10 µm. Bottom panel: scatter plot of the values of sister IdU tract length ratio (sister fork ratio; shorter IdU tract/longer IdU tract) measured for the indicated conditions (n = 125). Red horizontal lines indicate mean value. ns, non-significant (P > 0.05); ****P = 0.0001 (Mann–Whitney test). (J) Loss of DDX17 increases the frequency of stalled replication forks in an R-loop-dependent manner. Top panel: representative immunofluorescence images of replication tracts corresponding to ongoing (CldU tract is followed IdU tract) and stalled (CldU tract is not followed IdU tract) replication. Scale bar, 10 µm. Bottom panel: quantification of the replication fork stalling events. Data are mean ± SD, n = 5. ns, non-significant (P > 0.05); *P = 0.05 (one-way ANOVA).

Fig 3: DHX15–CMTr1 interactions. (a) Domain structure of human CMTr1 and DHX15 proteins, showing boundaries of structural domains in full-length proteins and in CMTr1 variants used in the assay shown in (b–d). Patterned boxes indicate domains responsible for mutual interactions. Co-immunoprecipitation of CMTr1 with endogenous (b) and transfected (c) DHX15. (b) HEK293 lysates were precipitated with anti-DHX15 antibody; the complexes were captured on protein A-conjugated beads and blotted with anti-CMTr1 antibody. (c) FLAG-DHX15 vector was transfected into HEK293 cells. Cell lysates were precipitated with anti-FLAG antibody-conjugated agarose and blotted with anti-CMTr1 antibody. (d) FLAG-CMTr1 vectors were transfected into HEK293 cells. Cell lysates were precipitated with anti-FLAG antibody-conjugated agarose and blotted with anti-DHX15 antibody. (c,d) The left panels show western blotting controls from whole cell lysates from transfected cells to show the levels of expression of the transfected bait proteins. The middle and right panels show western blots with the indicated antibodies before (input) and after immunoprecipitation (IP) using an anti-FLAG antibody, respectively. IgGs (b) and empty anti-FLAG agarose beads (c,d) were used as negative controls. MW, molecular weight markers.

Fig 4: CMTr1 methyltransferase activity on RNA substrates with different types of secondary structure at 5′ ends. (a) Schematic of RNA substrates, with emphasis on the presence of a long base-paired region (other potential structures are not shown); canonical Watson–Crick base pairs (A-U and G-C) are indicated and wobble pairs G-U in the 5′-terminal hairpin are denoted as ‘○’. Sequences of RNA substrates and their predicted secondary structures are shown in electronic supplementary material, table S2 and figure S2. (b) CMTr1 methyltransferase activity, expressed as a level of tritium-labelled methyl group transfer onto different RNA substrates (shown in a). (c) Relative CMTr1 methyltransferase activity on different substrates in the absence or presence of DHX15, at 1 : 1 and 1 : 5 molar ratios. Data were normalized to CMTr1 activity on individual substrates as shown in (b) (set as 100% in each case) to illustrate the different relative effect of DHX15 on CMTr1 activity depending on the substrate. Electronic supplementary material, figure S3 shows the same data normalized to values obtained for RNA64. (d) The time-course of CMTr1 enzymatic activity on the RNA68 substrate in the presence (black squares) or absence (white circles) of DHX15 helicase (at 1 : 5 molar ratio). At the indicated time-points reactions were stopped by freezing in liquid nitrogen. In (b–d), results from three independent experiments are shown and error bars indicate standard deviations.

Fig 5: CMTr1 deletion mutant (CMTr1Δ135) activity towards the capped RNA68 substrate in comparison with the full-length protein (CMTr1) in the presence or absence of DHX15 helicase at 1 : 1 and 1 : 5 ratios. Data were normalized to CMTr1 activity. Results from three independent experiments are shown and error bars indicate standard deviations.