Fig. 4

METTL3 interacted with the microprocessor protein DGCR8 and modulated the pri-miR221/222 process in an m6A-dependent manner. a. m6A dot blot assays of EJ and T24 cells with knockdown or overexpression of METTL3. Methylene blue (MB) stain as loading control. It was obvious that the methylation of RNA decreased significantly after METTL3 knockdown, while increased significantly after METTL3 overexpression. b. Co-immunoprecipitation of the METTL3-interacting protein DGCR8. Cells were UV-cross-linked before the immunoprecipitation. Western blot using the DGCR8 and METTL3 antibodies and IgG used as control for the IP. c. Immunoprecipitation of DGCR8, METTL3 and associated RNAs from control cells or METTL3 overexpression cells. Cells were UV-cross-linked before the immunoprecipitation. Western blot or Immunoblot were conducted using the antibodies described above. d. The expression of miR221/222 were verified by qRT-PCR in METTL3 knockdown and overexpression cells. e. The expression of pri-miR221/222 were verified by qRT-PCR in METTL3 knockdown and overexpression cells. f. A moderate positive correlation between the expression of METTL3 and miR221/222 was showed in bladder cancer tissues by qRT-PCR. g. Detection of pri-miRNA binding to DGCR8 by immunoprecipitation of DGCR8-associated RNA from control and METTL3 overexpression cells followed by qRT-PCR. h. The detection of pri-miRNAs m6A modification level by immunoprecipitation of m6A modified miRNA in control or METTL3 overexpression cells followed by qRT-PCR. Data represented the mean ± SD from three independent experiments, *P < 0.05, **P < 0.01