Fig. 4.

Acetylation of AGO2 increases miR-19b biogenesis. a The expression of miR-19b in A549-shAGO2 cells re-expressing Flag-AGO2-WT was much more than that in cells re-expressing Flag-AGO2-3KR. Total RNA was extracted from A549-stable cell lines, the miR-19b expression were detected by Northern blotting. See Figure S4A for the expression levels of AGO2-WT, AGO2-3KR were comparable. Also see Supplementary Data S1 for the results of small RNA high-throughput deep sequencing. b–d Serum stimulates miR-19b biogenesis. 293T cells (b), A549 cells (c) or 293T cells overexpressing indicated plasmids (d) were serum-starvated for 24 h and then stimulated by 20% serum as indicated times. The total RNA were extracted and followed by northern blotting analysis. See Figure S5 for the expression levels of pri-miR-19b1, pre-miR-19b1 and miR-19b by QRT-PCR analysis. e, f P300/CBP increases miR-19b maturation from pre-miR-19b1 mediated by AGO2 while HDAC7 decreases it. 293T cells were transfected with indicated plasmids, 48 h later the total RNA were extracted and followed by northern blotting analysis. g Acetyl-mutants K493R and K720R, but not K355R of AGO2 decrease miR-19 biogenesis in a DICER dependent manner. DICER-knocked down 293T stable cells were transfected with indicated plasmids, 48 h later, the total RNA were extracted and followed by northern blotting analysis. See Figure S4B–D for miR-19b maturation from pre-miR-19b1 is highly sensitive to AGO2 in a DICER dependent manner