1. Acetylation status of critical lysine residues in dsRBD affects the binding of DGCR8 to primary miRNA transcripts.
Acetylation status of critical lysine residues in dsRBD affects the binding of DGCR8 to primary miRNA transcripts. (A) We generated recombinant wild‐type DGCR8M protein and D1H2 and D2H2 mutants, in which K561/K562/K565 and K669/R670/K673 were, respectively, substituted with Ala, in Escherichia coli. Purified DGCR8M proteins (0–4.0 μM) were incubated with radiolabelled pri‐miR‐106b transcript (0.25 nM) in EMSA buffer and subjected to SDS–PAGE to separate the DGCR8M/pri‐miRNA complexes (B) and free probes (F). Before the binding reaction, DGCR8M proteins were either untreated or treated with p300 (67 ng/ml) with or without subsequent incubation with HDAC1 (8 ng/ml) in appropriate buffers. The effects of p300 and HDAC1 were monitored by immunoblotting with anti‐acetylated lysine antibody (supplementary Fig S3B online). (B) Signal intensities of the DGCR8M/pri‐miRNA complexes (bound) and free probes were quantified using the Scion Image software and are shown as a ratio of (bound)/[(bound)+(free)] on y axes. Means±s.d. (bars) of three independent experiments are shown. (C) We overexpressed either wild‐type or mutant (D2H2) DGCR8 in HEK293 cells, and examined the effects of KAT2B (HAT) overexpression on the expression levels of mature miRNAs using RT–PCR. Lower panel: the y axis indicates relative expression against untreated HEK293 cells. The means±s.d. (bars) of three independent experiments of individual miRNAs are shown. Upper panel: the results of miR‐106b, miR‐25, miR‐330, miR‐29a and miR‐101 were accumulated and evaluated by paired Student's t‐test. Equal expression of the introduced genes was confirmed by immunoblotting (supplementary Fig S3E online). (D) Schematic representation of the mechanisms by which HDACs regulate the cellular gene expression programs. See Discussion for the detail. dsRBD, double‐stranded RNA‐binding domain; HAT, histone acetyltransferase; HDAC, histone deacetylase; EMSA, electrophoretic mobility shift assay; miRNA, micro RNA; Pol II, RNA polymerase II; pri‐miR, primary transcript; pre‐miR, precursor transcript.
Taeko Wada et al. EMBO Rep. 2012;13:
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