Figure 3.

PRMT5 promotes the arginine methylation of ALKBH7 to regulate RNA m6A modification of BRCA1 and DOX sensitivity

(A) Silver staining of proteins associated with PRMT5. MDA-MB-231 cells stably expressing FLAG-PRMT5 were treated with DOX and immunoprecipitated with an anti-FLAG antibody, and the MDA-MB-231 cells with same treatment were set for control (Ctrl). Samples were resolved by SDS-PAGE and visualized by silver staining. The bands of interest were cut out and analyzed with a mass spectrometer. (B) IP of FLAG-PRMT5 and MYC-ALKBH7 in PRMT5-or ALKBH7-overexpressing MDA-MB-231 breast cancer cells treated with or without 0.4 μg/mL DOX for 24 h. (C) Wild-type (WT) ALKBH7 and mutant ALKBH7^3RK with arginine-to-lysine mutation were used for protein methylation detection. HEK293 cells were transfected with HA-ALKBH7, HA-ALKBH7^3RK or FLAG-PRMT5 as indicated. IP with anti-HA or IgG antibodies and western blotting with an anti-symmetric dimethylarginine (SDMA) antibody were performed to assess the methylation of ALKBH7 at the ¹⁹⁴RGRR site. (D) In vitro methylation assay with recombinant FLAG-tagged PRMT5, unlabeled S-adenosylmethionine (SAM), and the substrates His-tagged WT or methylation site-mutant (3RK) ALKBH7. The same blot was stripped and re-probed with an anti-ALKBH7 antibody, showing the amount of substrate in each reaction. H4 was used as a positive control. (E) Protein stability analysis by western blotting of MDA-MB-231 cells stably expressing WT ALKBH7 or ALKBH7^3RK treated with 100 μg/mL cycloheximide (CHX) for the indicated times. (F) HEK293 cells transfected with V5-ubiqutin, FBW7α, MYC-ALKBH7, and MYC-ALKBH7^3RK as indicated and then treated with 100 μM MG132 for 6 h. ALKBH7 and ALKBH7^3RK were immunoprecipitated from whole-cell lysates with an anti-MYC antibody. The ubiquitination of ALKBH7 and ALKBH7^3RK in the immunoprecipitants and levels of ALKBH7, ALKBH7^3RK, and FBW7α in whole-cell lysates (Input) were determined by western blotting as indicated. (G) Proliferation analysis (MTT) of PRMT5 overexpression with ALKBH7-specific siRNA knockdown in MDA-MB-231 cells treated with 0.4 μg/mL DOX for the indicated times. (H) Dot-blot assay of RNA m6A methylation in MDA-MB-231 cells overexpressing PRMT5 with ALKBH7-specific siRNA transfection treated with 0.4 μg/mL DOX for 24 h i, RT-PCR analysis following m6A-IP of the BRCA1 mRNA 3′-UTR in MDA-MB-231 cells overexpressing PRMT5 with ALKBH7-specific siRNA transfection treated with DOX for 24 h ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.