Fig. 4
Identification of NKAP as a mediator of m6A in pri-miR-25 maturation. a Schematic of RNA pulldown and immunoprecipitation experiment and proteomic screening by mass spectrometry (MS) for the identification of [m6A]pri-miR-25 reader. b Western blot analysis of potential m6A readers obtained from proteomic screening as indicated in (a) shows specific association of NKAP with both [m6A]pri-miR-25 and DGCR8. c RNA immunoprecipitation assays with m6A antibody show association of NKAP (FLAG-tagged) with pri-miR-25 in PDAC cells. Data from three independent experiments represent RNA levels associated with NKAP relative to input. Antibody against DGCR8 or DROSHA was included as positive control and IgG served as negative control. d Electrophoretic mobility shift assays of recombinant NKAP with unmethylated or methylated pri-miR-25 probes. The probes were maintained constantly while a gradient of 0–10 μM recombinant NKAP was added to the reactions. e, f Reciprocal immunoprecipitation assays show interaction among DGCR8, DROSHA, and NKAP (FLAG-tagged) in PANC-1 cells in the presence of RNase A. g Confirmation of binding of DGCR8 to NKAP by truncation mapping test. Upper panel shown are the schematic of the domain structures of DGCR8 and NKAP protein. Lower panel: Western blot analysis of constructs for His-tagged DGCR8 (wild-type versus domain truncation mutants) and FLAG-tagged NKAP or for FLAG-tagged NKAP (wild-type versus domain truncation mutants) and His-tagged DGCR8 that were co-transfected into PANC-1 cells, respectively. h Knockdown of NKAP expression in PDAC cells substantially decreased pre-miR-25 and miR-25-3p levels but substantially increased the level of pri-miR-25. i RNA immunoprecipitation assays show significantly decreased enrichment of pri-miR-25 with NKAP (left panel) or DGCR8 (right panel) in PDAC cells with METTL3 knockdown. j RNA immunoprecipitation assays show significantly decreased enrichment of pri-miR-25 with DGCR8 in PDAC cells with NKAP knockdown. k NKAP knockdown affected the expression of miR-25-3p in PDAC cells exposed to CSC. Values of (h–k) are the mean ± S.D. from three independent experiments. All statistic analyses in this figure are Student t test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding control
