Fig. 4: 53BP1 stability is controlled by PRMT5.

a In vitro methylation of 53BP1 by PRMT5. Recombinant GST-53BP1-Tudor (focus forming region containing tandem Tudor domains, aa 1220–1711)-wild type (WT), −5RK, and −∆GAR were incubated with myc-PRMT5 purified from HEK293T and 10 μM cold AdoMet 37 °C for 1 h in vitro. Samples were analyzed by immunoblotting. b, c Symmetric dimethylation of GAR motif in 53BP1 by PRMT5. HEK293T cells were transfected with WT or 5RK GFP-53BP1 (b) or co-transfected with GFP-53BP1 and siPRMT5 (c), and then subjected to immunoprecipitation using GFP antibody. d–i Regulation of 53BP1 protein level by PRMT5. U2OS cells were transfected with siPRMT5 for 3 days (d, e), treated with 10 μM EPZ015666 for 2 days (f, g) or overexpressed with WT or Y324F myc-PRMT5 for 2 days (h, i). The intensity of protein bands was quantified using image processing software (n = 3). j–m Analysis of 53BP1 protein stability. (j, k) U2OS cells were treated with DMSO or 10 μM EPZ015666 for 16 h, and then treated with 50 μg/ml cycloheximide for the indicated times. l, m U2OS cells were transfected with WT or Y324F myc-PRMT5 for 1 day, and then co-treated with 50 μg/ml cycloheximide for the indicated times. Band intensities were quantitated using image processing software. Error bars indicate standard deviation of 3 independent replicates. n–p IF Analysis of 53BP1 foci formation. U2OS cells overexpressing empty vector (EV), WT or Y324F myc-PRMT5 were treated with 10 μM etoposide for 2 h and then assessed by co-immunostaining for 53BP1 (green) and γH2AX (red), followed by DAPI staining. n Representative pictures are shown. Scale bar: 5 μm. o The fluorescence intensity of 53BP1 was quantified by image analysis software (n = 3). p Quantification of the percentage of cells with ≥5 53BP1 foci (n = 3). *P < 0.05 and **P < 0.01. The immunoblots in (a–d, f, h, j, l) are representative of three independent experiments with similar results, respectively.