Figure 4.

METTL3-Dependent m⁶A Methylation Regulates the Processing of miR34a

(A) Coimmunoprecipitation of the METTL3-interacting protein DGCR8 in VSMCs. An IgG antibody was used as a control for immunoprecipitation. (B) Immunoprecipitation of DGCR8, METTL3, and associated RNA from control VSMCs or METTL3-deficient VSMCs. (C) Poly(A)⁺ RNA isolated from METTL3-deficient or METTL3-overexpression VSMCs was used in RNA m⁶A dot blot analyses with an m⁶A antibody. (D and E) quantitative real-time PCR analysis to detect the expression of miR34a (D) and pri-miR34a (E) in sh-METTL3 cells, METTL3-overexpressing cells and corresponding control cells. (F) Immunoprecipitation of DGCR8-associated RNA from METTL3-knockdown VSMCs or control cells followed by quantitative real-time PCR analysis of pri-miR34a binding to DGCR8. Pri-let-7e was used as a positive control (n = 3). (G) Immunoprecipitation of m⁶A-modified RNA in METTL3-knockdown VSMCs or control cells followed by quantitative real-time PCR analysis of pri-let-7e (left) and m⁶A modification site1 of pri-miR34a (right) m⁶A modification levels. (H) Immunoprecipitation of m⁶A-modified RNA in METTL3-knockdown VSMCs or control cells followed by quantitative real-time PCR analysis of m⁶A modification site 2 (left) and m⁶A modification site 3 (right) of pri-miR34a m⁶A modification levels. (I and J) quantitative real-time PCR analysis of the expression of miR34a (I) and pri-miR34a (J) in human AAA tissues and control aortic tissues. The data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01.