Fig. 3
m6A modification is a major mechanism for METTL3-promoted pri-miR-25 maturation. a Left: Identification of m6A by m6A individual-nucleotide resolution cross-linking and immunoprecipitation sequencing (miCLIP-seq). The m6A residues were detected by cross-linking induced mutation sites (CIMS) and cross-linking induced truncation sites (CITS) in pri-miR-25. Red tracks of miCLIP-seq are unique tag coverage and blue tracks represent C>T transition or truncation, respectively. Filled purple circles denote miCLIP-called m6A and the horizontal blue bars indicate transcript models of pre-miR-25. Right: The sequences of pre-miR-25 and miR-25-3p are highlighted, respectively, by different colors, and the m6A motif (GGACU) is located at the putative splicing site. b PDAC had significantly higher levels of [m6A]pri-miR-25 than paired non-tumor tissues. [m6A]pri-miR-25 was detected by immunoprecipitation followed by qRT-PCR analysis. ***P < 0.001 by Wilcoxon rank-sum test. c, d Effects of CSC exposure (100 µg/ml) and overexpression or knockdown of METTL3 on the levels of [m6A]pri-miR-25 in PDAC cells. Data represent mean ± S.D. from three independent experiments. e, f m6A modification substantially enhanced pri-miR-25 processing and maturation in an in vitro reaction system containing starting materials of pri-miR-25 or [m6A]pri-miR-25 and the whole cellular lysates of 293T cells transfected with plasmids carrying DROSHA and DGCR8. Pri-miR-1-1 which has no methylation site was included as a control. e Northern blot detection of starting materials (left panel) and the levels of resultants pre-miR-25 and pre-miR-1-1 in the reaction. f Quantification of pre-miR-25, miR-25-3p, and pri-miR-25 in the reaction mixture. Data represent fold change ± S.D. relative to pri-miR-25 as starting material. g, h Mutation of [m6A]pri-miR-25 at METTL3-recognizing site abolished pri-miR-25 processing and maturation in the in vitro reaction system. g Northern blot detection of starting materials (left panel) and the levels of resultants pre-miR-25 and pre-miR-1-1 in the reaction. h Quantification of pre-miR-25, miR-25-3p, and pri-miR-25 in the reaction mixture. Data represent fold change ± S.D. relative to [m6A]pri-miR-25 WT as starting material from three experiments. All statistic analyses in this figure are Student t test unless specified. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding control
