Loss of WRNIP1 or its UBZ domain results in DNA damage accumulation and enhanced chromosomal instability upon MRS

(A) Schematic representation of human WRNIP1 protein structure. Western blot analysis showing the expression of the WRNIP1 protein in wild-type cells (shWRNIP1WT), WRNIP1-deficient cells (shWRNIP1) and WRNIP1 ATPase mutant (shWRNIP1T294A) or WRNIP1 UBZ mutant (shWRNIP1D37A) cells. MRC5SV40 fibroblasts were used as a positive control. The membrane was probed with an anti-FLAG or anti-WRNIP1 antibody. GAPDH was used as a loading control.

(B) Analysis of DNA damage accumulation evaluated by alkaline Comet assay. MRC5SV, shWRNIP1, shWRNIP1D37A and shWRNIP1T294A cells were treated or not with 0.4 µM Aph for 24 h, then subjected to Comet assay. The graph shows data presented as mean tail moment ± SE from three independent experiments. Horizontal black lines represent the mean (ns, not significant; **, P < 0.01; ****, P < 0.0001; two-tailed Student’s t test). Representative images are given.

(C) Analysis of chromosomal aberrations in the indicated cell lines treated as shown in the experimental scheme. Dot plot shows the number of chromosomal aberrations per cell ± SE from three independent experiments. Horizontal black lines represent the mean (ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-tailed Student’s t test). Representative images are given. Insets show enlarged metaphases for a better visualization of chromosomal aberrations.

(D) Evaluation of DNA damage accumulation by alkaline Comet assay. Cells were treated as shown in the experimental scheme, then subjected to Comet assay. Dot plot shows data presented as mean tail moment ± SE from three independent experiments. Horizontal black lines represent the mean (ns, not significant; ****, P < 0.0001; two-tailed Student’s t test). Representative images are given.

Loss of WRNIP1 or its UBZ domain results in R-loop accumulation upon MRS

(A) Evaluation of R-loop accumulation by immunofluorescence analysis in shWRNIP1WT and shWRNIP1 cells treated as reported in the experimental design after transfection with GFP-tagged RNaseH1 or empty vector. Cells were fixed and stained with anti-RNA-DNA hybrid S9.6 monoclonal antibody. Representative images are given. Nuclei were counterstained with DAPI. Box plot shows nuclear S9.6 fluorescence intensity. Box and whiskers represent 20-75 and 10-90 percentiles, respectively. The line represents the median value. Data are presented as means of three independent experiments. Horizontal black lines represent the mean. Error bars represent standard error (***, P < 0.001; ****, P < 0.0001; two-tailed Student’s t test).

(B) Dot blot to confirm R-loop accumulation. Genomic DNA isolated from shWRNIP1WT and shWRNIP1 cells, treated as reported in the experimental design and processed as described in “Materials and Methods”, was spotted onto nitrocellulose membrane. The membrane was probed with anti-RNA-DNA hybrid S9.6 monoclonal antibody. Treatment with RNase H was used as a negative control. Representative gel images of at least three replicates are shown.

Loss of WRNIP1 or its UBZ domain leads to R-loop-dependent accumulation upon MRS

(A) Analysis of DNA damage accumulation by alkaline Comet assay. MRC5SV and shWRNIP1 cells were treated or not with Aph or HU, as reported by the experimental scheme after transfection with GFP-tagged RNaseH1 or empty vector (-),then subjected to Comet assay. The graph shows data presented as mean tail moment ± SE from three independent experiments (ns, not significant; **** P < 0.0001; two-tailed Student’s t test). Representative images are given.

(B) Immunofluorescence analysis to determine R-loop levels in MRC5SV, shWRNIP1, shWRNIP1D37A and shWRNIP1T294A cells treated or not with 0.4 µM Aph for 24 h. Cells were fixed and stained with anti-RNA-DNA hybrid S9.6 monoclonal antibody. Representative images are given. Nuclei were counterstained with DAPI. Box plot shows nuclear S9.6 fluorescence intensity. Box and whiskers represent 20-75 and 10-90 percentiles, respectively. The line represents the median value. Data are presented as means of three independent experiments. Horizontal black lines represent the mean. Error bars represent SE (ns, not significant; * P < 0.05; ** P < 0.01; ***, P < 0.001; two-tailed Student’s t test).

(C) Analysis of the effect of R-loop resolution on DNA damage accumulation evaluated by alkaline Comet assay. Cells were treated as reported in the experimental scheme after transfection with GFP-tagged RNaseH1 or empty vector, then subjected to Comet assay. Dot plot shows data presented as mean tail moment ± SE from three independent experiments. Horizontal black lines represent the mean (ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-tailed Student’s t test). Representative images are given.

Loss of WRNIP1 or its UBZ domain promotes R-loop-dependent TRCs accumulation

(A) Detection of TRCs by fluorescence-based PLA assay in MRC5SV, shWRNIP1 and shWRNIP1D37A cells treated as reported in the experimental scheme after transfection with GFP-tagged RNaseH1 or empty vector. Cells were fixed and stained with antibodies against PCNA and RNA pol II. Representative images are given. Each red spot represents a single interaction between proteins. No spot has been revealed in cells stained with each single antibody (negative control). Nuclei were counterstained with DAPI. Dot plot shows the number of PLA spots per nucleus. Data are presented as means of three independent experiments. Horizontal black lines represent the mean ± SE (ns, not significant; ****, P < 0.0001; Mann-Whitney test).

(B and C) Analysis of localization of WRNIP1 near/at the transcription and replication machineries by PLA. Cells were treated or not with 0.4 µM Aph for 24 h, fixed and stained with antibodies against WRNIP1 and RNA pol II (B) or WRNIP1 and PCNA (C) to visualize the interaction between WRNIP1 and replication or transcription machinery, respectively. Each red spot represents a single interaction between proteins. Representative images are given. Nuclei were counterstained with DAPI. Dot plots show the number of PLA spots per nucleus. Data are presented as means of three independent experiments. Horizontal black lines represent the mean ± SE (****, P < 0.0001; Anova one-way test).

(D and E) Detection of physical interaction between WRNIP1 and R-loops (D) or R-loops and RAD51(E). Cells were treated or not with 0.4 µM Aph for 24 h, followed by the PLA assay described in “Supplementary Materials and Methods”. Cells were stained with antibodies against RNA-DNA hybrid (anti-S9.6) and WRNIP1 (D) or RNA-DNA hybrid (anti-S9.6) and RAD51 (E). Representative images are given. Each red spot represents a single interaction between R-loops and the respective proteins (WRNIP1 or RAD51). No spot has been revealed in cells stained with each single antibody (negative control). Nuclei were counterstained with DAPI. Dot plot shows the number of PLA spots per nucleus. Horizontal black lines represent the mean ± SE (ns, not significant; ** P < 0.01****, P < 0.0001; Anova one-way test).

R-loops affects DNA replication in cells lacking WRNIP1 or its UBZ domain upon MRS

Experimental scheme of dual labelling of DNA fibers in MRC5SV, shWRNIP1 and shWRNIP1D37A cells under unperturbed conditions (A) or upon MRS (B). After transfection with GFP-tagged RNaseH1 or empty vector (-), cells were pulse-labelled with CldU, treated or not with 0.4 µM Aph, then subjected to a pulse-labelling with IdU. Representative DNA fiber images are shown. The graph shows the analysis of replication fork velocity (fork speed) in the cells. The length of the green tracks was measured. Mean values are represented as horizontal black lines (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Mann-Whitney test). (C and D) The graph shows the percentage of red (CldU) tracts (stalled forks) or green (IdU) tracts (restarting forks) in the cells. Error bars represent standard error (ns, not significant; *, P < 0.05; ** P < 0.01; *** P < 0.001; two tailed Student t-test).

FANCD2 pathway activation in cells lacking WRNIP1 or its UBZ domain upon MRS

(A) Western blot analysis showing FANCD2 ubiquitination in MRC5SV, shWRNIP1 and shWRNIP1D37A cells. The membrane was probed with an anti-FANCD2 antibody. LAMIN B1 was used as a loading control.

(B) Detection of physical interaction between FANCD2 and R-loops by PLA. MRC5SV and shWRNIP1D37A cells treated with or not with 0.4 µM Aph for 24h. Cells were stained with antibodies against RNA-DNA hybrid (anti-S9.6) and FANCD2. Representative images are given. Each red spot represents a single interaction between R-loops and FANCD2. No spot has been revealed in cells stained with each single antibody (negative control). Nuclei were counterstained with DAPI. Dot plot shows the number of PLA spots per nucleus. Data are presented as means of three independent experiments. Horizontal black lines represent the mean ± SE (ns, not significant; ****, P < 0.0001; Anova one-way test).

(C) Evaluation of DNA damage accumulation by alkaline comet assay in MRC5SV, shWRNIP1 and shWRNIP1D37A transfected with control siRNAs (siCTRL) or siRNAs targeting FANCD2 (siFANCD2). After 48 h, cells were treated with 0.4 µM Aph for 24h, then subjected to Comet assay. Dot plot shows data presented as the mean tail moment ± SE from three independent experiments.

Horizontal black lines represent the mean (ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-tailed Student’s t test). Representative images are given. Western blot shows FANCD2 depletion in the cells. The membrane was probed with an anti-FANCD2 and LAMIN B1 was used as a loading control.