The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

Abstract

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway lead to R-loop accumulation, which contributes to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export.

Keywords: CP: Molecular biology; DNA damage; FANCD2; NXF1; R-loops; RNA; SRSF1; mRNA export; monoubiquitination.

Figure 1.. Knockdown of the splicing factor SRSF1 results in an FA-like cellular phenotype

(A) Immunoblot analysis of HeLa cells transfected with siRNA scrambled (siCTRL) or siRNA against SRSF1 (siSRSF1), untreated or treated with 1 μM MMC for 24 h. A rescue experiment was conducted in lanes 5 and 6 by co-transfection with siSRSF1 and SRSF1-GFP. Ku86 was used as a loading control (left). Densitometry analysis of the immunoblot shows the ratio of ubiquitinated to non-ubiquitinated FANCD2 (ub-FANCD2/FANCD2). The data represent the mean and SEM of three independent experiments. Data were analyzed using the unpaired t test (right). (B) FANCD2 mRNA levels were measured by RT-qPCR after transfection with siCTRL or siSRSF1. The data represent the mean and SEM of three independent experiments, normalized to siCTRL. Data were analyzed using the unpaired t test. (C) Immunofluorescence of MMC-induced FANCD2 foci in HeLa cells after transfection with siCTRL, siSRSF1 (left), or co-transfection of siSRSF1 and SRSF1-GFP (center). The graph shows the FANCD2 focus quantification. Data (mean and SEM) are representative of three independent analyses of at least 50 cells per slide. Data were analyzed using the unpaired t test (right). (D) HeLa cells were treated with the indicated concentrations of MMC, and cell survival was analyzed by crystal violet after incubation at 37°C for 4 days. The percentages of surviving cells were normalized to an untreated control and are shown as the mean and SEM of three independent experiments. Data were analyzed using two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, non-significant. *, compared with siCTRL, #, compared with siSRSF1). See also Figure S1.

Figure 2.. SRSF1 physically interacts with the ID2 complex

(A) FA-D2+FANCD2 and FA-D2+K561R cells were left untreated or treated with 0.5 μM MMC overnight. Whole-cell extracts were prepared and immunoprecipitated with FANCD2 antibody. The immunoprecipitation products were analyzed for FANCD2 interaction with SRSF1 antibody. (B) Whole-cell extracts from FA-D2 mutant cells and FA-D2+FANCD2-corrected cells were prepared and immunoprecipitated with immunoglobulin G (IgG) or SRSF1 antibody. The immunoprecipitation products were analyzed for SRSF1 interaction with FANCD2 antibody (above). HEK293T cells were transfected with mock or FLAG-SRSF1 plasmid and treated or not with 1 μM MMC for 24 h. Whole-cell extracts were treated with benzonase (50 units/mL) and immunoprecipitated with FLAG-M2 agarose. The immunoprecipitation products were analyzed for SRSF1 interaction with FANCD2 antibody (bottom). (C) Purified FANCI-His/FANCD2-FLAG (ID2-FLAG) and His-SRSF1 were analyzed by SDS-PAGE and Coomassie blue (top). The ID2-FLAG protein complex was incubated alone or with His-SRSF1 and treated with benzonase. Protein complexes were captured on FLAG resin, and the different fractions were analyzed by SDS-PAGE. S, supernatant; W, wash; E, SDS eluate of the FLAG resin (bottom). See also Figure S2.

Figure 3.. Binding of SRSF1 to RNA stimulates FANCD2 monoubiquitination

(A) His-tagged SRSF1 (100–400 nM) was incubated with radiolabeled ssRNA or ssDNA. The mobility shift of the RNA and DNA was analyzed by EMSA (left). Graph shows the quantification of the shifted nucleic acid substrate. The error bars represent the mean and SEM of data from three independent experiments. (B) In vitro ubiquitination reaction of recombinant ID2 with either ssDNA or ssRNA substrates and with or without SRSF1 (200 nM and 400 nM) (left). The graph shows the ratio of ubiquitinated to non-ubiquitinated FANCD2 (ub-FANCD2/FANCD2). Each nucleic acid has been normalized to its nucleic acid control without SRSF1 (3 and 4 to 2; 6 and 7 to 5). The error bars represent the mean and ± SEM of data from three independent experiments. Statistics: unpaired t test (right). *p < 0.05, **p < 0.01, ***p < 0.00, ****p < 0.0001. See also Figure S3.

Figure 4.. SRSF1 and FANCD2 colocalize at DNA damage within an actively transcribed genomic site prone to R-loop formation

(A) Genomic DNA (0.6 mg) from FA-D2, FA-D2+FANCD2, and FA-D2+K561R cells was extracted, left untreated or treated with RNase H at 37°C for 1 h, and analyzed with slot blot assay using the S9.6 antibody. Methylene blue staining was used as a loading control (left). Densitometry of the slot blot shows the S9.6 intensity normalized to FA-D2+FANCD2 (right). (B) HeLa cells transfected with siCTRL, siSRSF1, siFANCD2, or siSRSF1+siFANCD2 double knockdown were subjected to immunofluorescence to visualize RNA:DNA hybrids using the S9.6 antibody. RNAseH1 treatment was used as a control for S9.6 antibody specificity. Samples were co-stained with nucleolin to subtract the nucleolar S9.6 signal (top). The scatterplot shows the quantification of S9.6 intensity per nucleus after subtraction of the nucleolar signal. At least 65 cells per slide were analyzed (bottom). (C) Genomic DNA from samples in (A) (0.3 or 0.6 μg) was left untreated or treated with RNAseH1 at 37°C for 1 h and analyzed with S9.6 slot blot. Methylene blue staining was used as a loading control (left). Densitometry of the slot blot shows the S9.6 intensity normalized to siCTRL (right). (D) Schematic of the DART system (left). Immunostaining of SRSF1 was done in four U2OS TRE cell lines expressing different fusion effector proteins in a DART assay (center). Quantification of the average SRSF1 foci intensity of at least 50 cells per condition was performed (right). (E) TA-KR-transfected U2OS TRE cells showing the colocalization of FANCD2 and SRSF1. (F) siRNA depletion of FANCD2, SRSF1, or AQR increased S9.6 focus intensity in a DART assay (left). Shown is quantification of the average S9.6 focus intensity of at least 50 cells per condition (right). Data represent the mean and SEM of three independent experiments. Statistics were performed using the unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.00, ****p < 0.0001. See also Figure S4.

Figure 5.. RNase H1 overexpression rescues the MMC sensitivity observed in SRSF1-depleted cells

(A) Immunoblot of HeLa cells co-transfected with siCTRL or siSRSF1 as well as RNase H1-GFP or GFP-N1 empty vector and untreated or treated with 1 μM MMC for 24 h. Ku86 was used as a loading control (left). Densitometry of the immunoblot shows ub-FANCD2/FANCD2. The data represent the mean and SEM of three independent experiments. Data were analyzed using the unpaired t test (right). (B) Same cells from (A) were subjected to immunofluorescence using FANCD2 antibody. The graph shows the quantification of FANCD2 foci. Data (mean and SEM) are representative of three independent analyses of at least 50 cells per slide. Data were analyzed using the unpaired t test. (C) HeLa cells were treated with the indicated concentrations of MMC, and cell survival was analyzed by crystal violet staining after incubation at 37°C for 4 days. The percentages of surviving cells were normalized to the untreated control and are shown as the mean and SEM of three independent experiments. Data were analyzed using two-way ANOVA. *, compared with siCTRL; #, compared with siSRSF1+RNAseH1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S5.

Figure 6.. FANCD2 monoubiquitination is necessary for NXF1-SRSF1-mediated mRNA export

(A) HeLa cells were transfected with mock or FLAG-SRSF1 and treated or not with RNAse A. Whole-cell extracts were immunoprecipitated with FLAG-M2 agarose, and immunoprecipitation products were analyzed for SRSF1 interaction with FANCD2 and NXF1 antibodies. (B) Whole-cell extracts from FA-D2 mutant cells, FA-D2+FANCD2, and FA-D2+K561R cells were prepared and immunoprecipitated with IgG or SRSF1 antibody. The immunoprecipitation products were analyzed for SRSF1 interaction with NXF1 antibody. (C) Whole-cell extracts from FA-D2+FANCD2 cells were treated or not with increasing concentrations of MMC for 24 h and immunoprecipitated with IgG or SRSF1 antibody. The immunoprecipitation products were analyzed for SRSF1 interaction with NXF1 antibody. (D) Schematic of the approach followed to detect binding of FANCD2, NXF1, and SRSF1 to mature RNA by appending MS2 trap (6× MS2 stem loops) at the end of the 3′ UTR of targets, creating a downstream chimeric RNA luciferase (left). Immunoblotting was used to detect RNA binding proteins (RBPs) by FLAG-MS2-BP immunoprecipitation. β-Actin was used as a control for loading and to assess specificity of the immunoprecipitation (IP). (E) Immunoblot showing FLAG-MS2-BP immunoprecipitation in FA-D2 mutant, FA-D2+FANCD2, and FA-D2+K561R cells. β-Actin was used as control for loading and to assess the specificity of the IP. (F) FA-D2 mutant, FA-D2+FANCD2, and FA-D2+K561R cells showing the translocation of poly(A) RNAs detected by RNA FISH (oligo-dT probe, red). Using image analyses, the poly(A) signal in the nucleus and cytoplasm was quantified to calculate the nuclear/cytoplasmic (N/C) ratio. Each circle in the graph represents the mean of the poly(A) N/C ratio of at least 15 cells, and the mean ( ± SEM) is also indicated. The data correspond to three independent experiments and were analyzed using the unpaired t test (right). (G) Graph showing the N/C ratio of the mRNA targets identified by RNA-seq in FA-D2, FA-D2+FANCD2, and FA-D2+K561R cells. Each circle in the graph represents the N/C value of a specific mRNA target. Data were analyzed using unpaired t test compared with FA-D2+FANCD2 wild-type cells. *p < 0.05, **p < 0.01, ***p < 0.00, ****p < 0.0001. See also Figure S6.

Figure 7.. SRSF1 cancer-associated mutants are defective for FANCD2 interaction and monoubiquitination

(A) Immunoblot of HeLa cells co-transfected with siCTRL or siSRSF1 and mock, SRSF1-GFP wild type, or the indicated mutants and left untreated or treated with 1 μM MMC for 24 h. Ku86 was used as a loading control (left). Densitometry of the immunoblot shows ub-FANCD2/FANCD2. The data represent the mean and SEM of three independent experiments. Data were analyzed using the unpaired t test (right). (B) The same cells from (A) were subjected to immunofluorescence using FANCD2 antibody. The graph shows the FANCD2 focus quantification. Data (mean and SEM) are representative of three independent analyses of at least 50 cells per slide. Data were analyzed using the unpaired t test compared with MMC-treated siCTRL. (C) HeLa cells co-transfected as in (A) were treated with the indicated concentrations of MMC, and cell survival was analyzed by crystal violet after incubation at 37°C for 4 days. The percentages of surviving cells were normalized to the untreated control and are shown as the mean and SEM of three independent experiments. Data were analyzed using two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. *, #, ^, ~, compared with siCTRL). (D) HeLa cells were co-transfected with mock, FLAG-SRSF1 wild type, or mutants and treated with 1 μM MMC for 24 h. Whole-cell extracts were prepared and immunoprecipitated with FLAG M2 agarose. The immunoprecipitation products were analyzed for SRSF1 interaction with FANCD2 or NXF1 antibodies. (E) Purified His-SRSF1 wild type or E60D or P89S mutants were incubated alone or with FANCI-His/FANCD2-FLAG protein complex and treated with benzonase. Protein complexes were captured on FLAG resin, and the different fractions were analyzed by SDS-PAGE. (F) In vitro ubiquitination reaction of recombinant ID2 with ssRNA substrate and with His-SRSF1 wild type and E60D and P89S mutants (400 nM) (left). The graph shows ub-FANCD2/FANCD2 normalized to the control without SRSF1 (lane 2). The error bars represent the mean and ± SEM of data from three independent experiments. Statistics: unpaired t test (right). (G) HeLa cells co-transfected with siCTRL, siNXF1, or siSRSF1 and mock, FLAG-SRSF1 wild type, or the indicated mutants, showing the translocation of poly(A) RNAs detected by RNA FISH (oligo(dT) probe, red) (left). Using image analyses, the poly(A) signal in the nucleus and cytoplasm was quantified to calculate the N/C ratio. Each circle in the graph represents the mean of the poly(A) N/C ratio of at least 15 cells, and the mean ( ± SEM) is also indicated. The data correspond to three independent experiments and were analyzed using the unpaired t test compared with siCTRL (right). (H) Genomic DNA (0.6 μg) from HeLa cells co-transfected with siCTRL or siSRSF1 and mock, FLAG-SRSF1 wild type, or the indicated FLAG-SRSF1 mutants was extracted, treated with RNAseH1 at 37°C for 1 h or left untreated, and analyzed with S9.6 slot blot assay. Methylene blue was used as a loading control (left). Densitometry analysis of the slot blot shows the S9.6 intensity normalized to siCTRL. Results represent the mean and SEM of three independent experiments. Data were analyzed using unpaired t test, compared to siCTRL (right). *p < 0.05, **p < 0.01, ***p < 0.00, ****p < 0.0001. See also Figure S7.