FIGURE 7.

The SUMOylation of DGCR8 is critical for its binding to pri-miRNA. A and B, Overexpression of USP36 promotes DGCR8 binding to the tested pri-miRNAs. H1299 cells transfected with Flag-DGCR8 together with or without V5-USP36 were subjected to RNA-IP with anti-Flag, followed by qRT-PCR detection of the indicated pri-miRNAs. Shown are fold changes of immunoprecipitated pri-miRNA, determined by comparing IgG control in empty vector transfected cells normalized to input, in one representative experiment from three independent experiments (A). Data were presented as mean ± SD of three technical replicates. P values shown were calculated by Student t test. ***, P < 0.001. The expression of Flag-DGCR8 and V5-USP36 assayed by IB is shown in B. C and D, Abolishing DGCR8 SUMOylation impairs its binding to the tested pri-miRNAs. H1299 cells transfected with control or Flag-DGCR8^3KR mutant plasmid were subjected to RNA-IP with anti-Flag followed by qRT-PCR analysis. Shown are fold changes of immunoprecipitated pri-miRNAs, determined by comparing IgG control in empty vector transfected cells normalized to input, in one representative experiment from three independent experiments (C). Data were presented as mean ± SD of three technical replicates. P values shown were calculated by Student t test. ***, P < 0.001. The expression of Flag-DGCR8 proteins assayed by IB was shown in D.