Fig 2: Involvement of hnRNP UL1 in the DDR in human nucleoli. (A) The DNA damage sensitivity of HEK UL1 KO cells compared to HEK WT cells was tested after 2.5 h of treatment with the genotoxic reagents ETO and CPT. After this time, the cells were harvested at the following time points: 18 h, 24 h, 30 h, 48 h, 72 h and 80 h. Cell viability was assessed by Trypan blue staining, and the percentage of survival was calculated. The results were normalized to those for the control cells treated with DMSO. (B) DNA damage was assessed by Comet Assay in HEK UL1 KO cells compared to HEK WT cells after DNA damage induced with ETO and CPT. Cells treated with DMSO were used as negative controls. For the assay, 50 cells per sample were analyzed. The error bars represent the SDs of three biological replicates. The p values were calculated using Student's t test, and the statistical significance is represented as follows: *P = 0.05; **P = 0.01; ***P = 0.001. (C) IP was performed using an anti-hnRNP UL1 antibody and protein extracts from the NO and CN fractions of HEK WT cells. After elution, the immunoprecipitated proteins were identified by western blotting followed by immunostaining. Four independent biological replicates from each fraction were used for the experiment. For the input, 5% of the total volume applied to the beads was used; for the negative controls, beads without antibody were used; for immunostaining, anti-hnRNP UL1, anti-pChk1, and anti-pRPA32 antibodies were used; and actin was used as a loading control. (D) IP was performed using magnetic beads conjugated with an anti-FLAG antibody and protein extracts from the NO and CN fractions of HEK UL1 OE cells. Three independent biological replicates from each fraction were used for the experiment. For the input, 5% of the total volume applied to the beads was used. For immunostaining, anti-53BP1, anti-RAD50, anti-FLAG, and anti-XRCC1 were used, and an anti-actin antibody was used as a control. * - nonspecific signal.

Fig 3: DDX5 is an essential regulator of splicing in undifferentiated spermatogonia. a Gene ontology (GO) term enrichment analysis of data obtained from DDX5 immunoprecipitation followed by mass spectrometry using wildtype murine cultured undifferentiated spermatogonia (n = 3 independent experiments). b DDX5 immunoprecipitation followed by western blot in wildtype cultured spermatogonia confirming the interaction of DDX5 with proteins involved in pre-mRNA splicing (SRSF3), maintenance of mRNA stability (ELAVL1, PABP1, ILF3, ILF2, IGF2BP3), mRNA export (PABP1, SRSF3, ILF2, ILF3, IGF2BP3) and translation (IGF2BP3). Representative of n = 2 independent experiments. c Summary of differential splicing analysis performed between control and Ddx5-ablated cultured spermatogonia. Numbers of predicted alternative splicing events in each category upon Ddx5 deletion are indicated. d Visualisation of differential splicing analysis of RNA-sequencing data comparing control and Ddx5-ablated spermatogonia. Tracks are shown for selected candidate genes (left). Red arrows indicate differentially spliced exon. Schematics of alternative splicing events are shown (blue and yellow rectangles) (middle). Change in “percent spliced in” between conditions is shown as a value below splicing schematics (?PSI) and in bar charts (right). #: changes meet analysis cut-offs (?PSI >0.20, Bayes Factor =10). PSI ± 95% confidence interval shown. e RNA-immunoprecipitation using DDX5 antibody in cultured wildtype spermatogonia followed by PCR and gel electrophoresis for differentially spliced candidates depicted in d. Representative of n = 3 independent samples per condition. f PCR validation of differentially spliced candidates identified in d. CTL vehicle-treated control spermatogonia, KO Ddx5-ablated cultured spermatogonia. Differentially spliced exons are depicted by “?exon number”. Representative of n = 3 independent samples. Position of the PCR primers used are depicted in d in orange beneath schematic showing gene structure. g Bar chart of RNA-sequencing analysis for selected candidates. Fold change in Ddx5-ablated spermatogonia relative to vehicle-treated control. # denotes FDR <0.05. Mean ± SEM shown; n = 4 independent biological replicates per condition. h RAD50 protein levels by western blot in vehicle-treated control (Control) versus Ddx5-ablated cultured spermatogonia (Ddx5TAM-KO). *P < 0.05; two-tailed unpaired t-test; mean ± SEM shown; n = 4 independent biological replicates per condition. Representative western blot shown on right. i RAD50 immunofluorescence of vehicle-treated control (top) and Ddx5-ablated (bottom) cultured spermatogonia at D2 post-treatment. Representative of n = 4 independent biological replicates per condition. Scale bars = 100 µm

Fig 4: Interactions of nGRB2 with DNA damage repair factors.(A) MS identifies DDR protein associated with GRB2. Mean of coverage and unique peptides from three independent data sets are shown. (B) Strep-GRB2 precipitated from HEK293T cells followed by immunoblotting with indicated antibodies. (C) Strep-GRB2 expressed HEK293T cells treated with or without 5-Gy IR were lysed immediately followed by Strep-Tactin precipitation and immunoblotting with indicated antibodies. (D) HEK293T cells were cotransfected with red fluorescent protein (RFP)–GRB2 and green fluorescent protein (GFP)–NBS1 or GFP alone as control, precipitated with GFP-trap beads, and immunoblotted with indicated antibodies. (E) HEK293T cells expressing Flag-RAD50 or Flag-Ctrl were cotransfected with RFP-GRB2 for 24 hours. Unperturbed cells were immunoprecipitated with Flag-M2 beads and immunoblotted with indicated antibodies. (F) HEK293T cells expressing Flag-MRE11 were cotransfected with Strep-GRB2 or Strep-Ctrl for 24 hours. Unperturbed or IR-treated (5 Gy) cells were immunoprecipitated with Flag-M2 beads and immunoblotted with indicated antibodies. (G) Strep-Tactin precipitation of WT and K109R mutant Strep-GRB2 from HEK293T cells that were unperturbed or IR treated (5 Gy) and lysed, followed by Western blot detection with the indicated antibodies. (H) MST isotherms of 100 nM Atto488–labeled WT, K109R, or K109A mutant to titrating concentrations of human MRE11 (residues 1 to 411). All data are representative of three independent experiments.

Fig 5: EPAS1 promotes TRF1, TRF2, and RAD50 transcription