Fig 2: PRMT5-Dependent Methylation of RUVBL1 at R205 Regulates 53BP1 Removal and Is Epistatic with TIP60(A) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and cell viability was assessed by colony survival. Results are expressed normalized to the respective non-treatment control (mean ± SD; n = 3).(B and C) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (B) or CENPF/RPA (C) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3).(D and E) HeLa-shCTRL or RUVBL1-R205K cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (D) or CENPF/RPA (E) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3).(F) Validation of siTIP60 knockdown is shown.(G) Cells were transfected with si53BP1 and the number of mitosin-positive cells with >10 RAD51 foci was quantified (mean ± SEM; n = 3).(H) Cells were transfected with si53BP1 and the number of CENPF-positive cells with >10 RPA foci was quantified (mean ± SEM; n = 3).(I) Validation of 53BP1 siRNA SMARTpool. See also Figure S6.

Fig 3: PRMT5-Dependent Methylation of RUVBL1 Regulates TIP60 Acetyltransferase Activity(A and B) HeLa shCTRL or RUVBL1-R205K cells were siRNA transfected and treated with TSA (0.5 µM) for 16 hr before exposure to 3Gy IR. The number of cells with >10 53BP1 foci 24 hr after damage (A) and the number of CENPF-positive cells with >10 RPA foci 8 hr after damage (B) were quantified (mean ± SEM; n = 3).(C) In vitro TIP60 histone acetyltransferase assay. 293T cells were transfected with the TIP60 piccolo complex (TIP60/EPC1/ING3) and wild-type or methyl-deficient RUVBL1. TIP60 activity was determined by immunoblotting for recombinant H4 acetylation.(D) HeLa shCTRL, RUVBL1-WT, or -R205K cells were siRNA transfected, and acetylated histone marks were determined by immunoblotting.(E–G) ChIP performed with H4K16Ac (E), 53BP1 (F), and RUVBL1 (G) antibodies in the presence of mCherry-Lac1-Fok1-DD-induced DSBs, in U20S-265-RUVBL1 wild-type or RUVBL1-R205K-expressing cells. Data represent the average percentage input across the four primer pairs expressed relative to RUVBL1 WT (mean ± SEM; n = 3).(H) Model of how PRMT5-dependent methylation of RUVBL1 impacts on HR repair. PRMT5 symmetrically dimethylates RUVBL1 at R205, which stimulates TIP60 HAT activity enabling H4K16 acetylation and the removal of 53BP1 from DNA breaks. After end resection, RPA loading, and subsequent displacement by RAD51, homology searching and strand invasion can proceed enabling error-free HR-mediated repair of DSBs. Removal of PRMT5 results in hypomethylated RUVBL1 and ineffective TIP60 activity, causing reduced H4K16 acetylation and 53BP1 retention. Consequently, end resectioning is greatly impaired during late S/G2 phase, resulting in defective RPA and RAD51 recruitment, inappropriate error-prone NHEJ, and genomic instability. See also Figures S6 and S7.

Fig 4: Methylation of RUVBL1 Is Required for HR, but Not ATM Signaling(A and B) HeLa-RUVBL1-WT and HeLa-RUVBL1-R205K cells were transfected with siRUVBL1, exposed to 3Gy IR, and harvested at the time points indicated. CENPF/mitosin-positive cells were scored for RPA (A) and RAD51 (B) foci formation (mean ± SEM; n = 3).(C) HeLa-RUVBL1-WT and HeLa-RUVBL1-R205K cells were transfected with siRUVBL1, exposed to 10Gy IR, and harvested at the time points indicated for immunoblot analysis.(D and E) HeLa-RUVBL1-WT and HeLa-RUVBL1-R205K cells were transfected with siRUVBL1, treated with increasing doses of CPT (D) or Olaparib (E), and survival was assessed by colony survival assay (mean ± SEM; n = 3). Scale bar, 10 µm. See also Figure S5.

Fig 5: The negative correlation expression of RuvBL1 and c-Jun in lung adenocarcinoma (A) The meta-analysis of RuvBL1 and c-Jun expression in Oncomine. Hou Lung dataset contains 65 adjacent normal lung tissue samples and 91 lung tumor samples. (B) Regression analysis of the correlation between RuvBL1 and c-Jun expression in lung adenocarcinoma from Hou Lung dataset. (C) Kaplan Meier plotter for RuvBL1 or c-Jun expression on the survival rate of patients with lung adenocarcinoma. (D) Kaplan Meier plotter for the expression pattern of RuvBL1 versus c-Jun. The expression pattern of RuvBL1/c-Jun at high ratio is significantly associated with a worse prognosis. P < 0.05.