WRNIP1 prevents transcription-associated genomic instability
- Section of Mechanisms Biomarkers and Models, Department of Environment and Health, Istituto Superiore di Sanita, Italy
Figures
Figure 1 with 2 supplements
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 shows WRNIP1 protein expression in wild-type cells (shWRNIP1WT), WRNIP1-deficient cells (shWRNIP1) and WRNIP1 …
-
Figure 1—source data 1
PDF containing original scans of the western blot (anti-WRNIP1, anti-FLAG, and anti-GAPDH) and related original TIFF images.
- https://cdn.elifesciences.org/articles/89981/elife-89981-fig1-data1-v1.zip
Figure 1—figure supplement 1
Loss of WRNIP1 or its UBZ domain increases DNA damage upon MRS.
Evaluation of DNA damage accumulation by alkaline Comet assay. MRC5SV, shWRNIP1, shWRNIP1D37A, and shWRNIP1T294A cells were treated according to the experimental scheme (0.4 µM Aph and 50 µM Cordy), …
Figure 1—figure supplement 2
Loss of WRNIP1 or its UBZ domain increases levels of γH2AX.
MRC5SV, shWRNIP1, shWRNIP1D37A, and shWRNIP1T294A cells were treated according to the experimental scheme (0.4 µM Aph and 50 µM DRB), and then immunostained for γ-H2AX. The graph displays data …
Figure 2
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, following treatment as reported in the experimental design post-transfection with GFP-tagged …
-
Figure 2—source data 1
PDF containing original scans of the dot blot (anti-S9.6, anti-dsDNA) and related original TIFF images used for the analysis.
- https://cdn.elifesciences.org/articles/89981/elife-89981-fig2-data1-v1.zip
Figure 3 with 1 supplement
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, post-transfection with GFP-tagged RNaseH1 or empty vector (-), were treated or not with Aph or HU, …
Figure 3—figure supplement 1
RNase III digestion significantly reduces the amount of dsRNA.
Immunofluorescence analysis to determine dsRNA signal in MRC5SV, shWRNIP1, shWRNIP1D37A, and shWRNIP1T294A cells treated or not with 0.4 µM Aph for 24 hr. Cells were then fixed, subjected or not to …
Figure 4
Loss of WRNIP1 or its UBZ domain promotes R-loop-dependent TRCs accumulation.
Detection of TRCs by fluorescence-based PLA assay in MRC5SV, shWRNIP1 and shWRNIP1D37A cells. Post-transfection with GFP-tagged RNase H1 or empty vector, cells treated according to the experimental …
Figure 5
Analysis of localization of WRNIP1 or UBZ mutant by PLA assay.
(A and B) Analysis of the localization of WRNIP1 near/at the transcription and replication machineries by PLA. Cells were treated or not with 0.4 µM Aph for 24 hr, fixed, and stained with antibodies …
Figure 6 with 1 supplement
R-loops affects DNA replication in cells lacking WRNIP1 or its UBZ domain upon MRS.
Experimental scheme for dual labeling 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 …
Figure 6—figure supplement 1
Transcription affects DNA replication in cells lacking WRNIP1 or its UBZ domain upon MRS.
Experimental scheme of dual labeling of DNA fibers in MRC5SV, shWRNIP1 and shWRNIP1D37A cells under unperturbed conditions (A) or upon MRS (B). After transcription inhibition by DRB, cells were …
Figure 7 with 2 supplements
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) …
-
Figure 7—source data 1
PDF containing original scans of the western blot (anti-FANCD2, anti-LAMINB1) and related original TIFF images.
- https://cdn.elifesciences.org/articles/89981/elife-89981-fig7-data1-v1.zip
-
Figure 7—source data 2
PDF containing original scans of the western blot (anti-FANCD2, anti-LAMINB1) and related original TIFF images.
- https://cdn.elifesciences.org/articles/89981/elife-89981-fig7-data2-v1.zip
Figure 7—figure supplement 1
Loss of WRNIP1 or its UBZ domain results in FANCD2 pathway activation upon MRS.
Evaluation of FANCD2 activation by immunofluorescence analysis in MRC5SV, shWRNIP1 and shWRNIP1D37A cells treated or not with 0.4 µM Aph for 24 hr and 50 µM DRB for 3 hr. The graph displays data …
Figure 7—figure supplement 2
Analysis of the dependency of FANCD2 activation on RAD18.
MRC5SV cells were depleted of RAD18, and 48 hr later, cells were fixed and immunostained for FANCD2. The graph shows data presented as the percentage of FANCD2-positive cells. Representative images …
-
Figure 7—figure supplement 2—source data 1
PDF containing original scans of the western blot (anti-RAD18, anti-LAMINB1) and related original TIFF images.
- https://cdn.elifesciences.org/articles/89981/elife-89981-fig7-figsupp2-data1-v1.zip
Figure 8
Evaluation of FANCD2 depletion on R-loop accumulation and replication dynamic.
(A) Immunofluorescence analysis to determine R-loop levels in shWRNIP1WT, shWRNIP1, and shWRNIP1D37A cells depleted or not of FANCD2 under untreated conditions or after MRS. Cells were fixed, …
Figure 9
Working model for the potential role of WRNIP1 in R-loop accumulation.
Upon replication fork stalling, WRNIP1 binds to R-loops and protects stalled forks, promoting genomic integrity. In the absence of WRNIP1, R-loops accumulate, and TRCs are enhanced, leading to …
Author response image 1
Author response image 2
