Fig 2: Schematic diagram of molecular mechanisms for the current study.The current study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m6A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. In addition, m6A methylation suppressed the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC.

Fig 3: m6A methylation inhibits the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA in OSCC.(A) Potential binding site of DGCR8 in KRT4 mRNA. (B–D) Quantification of KRT4 pre-mRNA containing junctions of intron 2-exon 3 (KRT4-E(2)3, (B), exon 3-intron 3 (KRT4-E(3)4, (C) and exon 5-intron 5 (KRT4-E(5)6, (D) in KRT4 pre-mRNA by qRT-PCR following RIP performed by DGCR8 antibody in HN6 cells. E, exon. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: NCL expression affects specific miRNA levels. (A,B) Levels of the indicated miRNAs relative to U6 snRNA measured by qRT-PCR in PASMCs transfected with control or si-NCL for 24 h (A) or nucleofected with the control or NCL-expressing plasmid for 24 h (B). The data represent the mean ± S.E. of triplicate measurements of three independent experiments. (C,D) Immunoprecipitation assays from PASMC lysates were performed using an anti-NCL antibody or IgG. The presence of DGCR8, Ago2, and NCL in the pull-down and input (2%) samples was monitored by immunoblotting. Input: immunoblots showing the abundance of DGCR8, Ago2, or NCL in the total protein extracts. IP: IP products precipitated by the anti-NCL, anti-DGCR8, anti-Ago2 antibody, or IgG. (E,F) Immunoprecipitation assays from PASMC lysates were carried out using an anti-DGCR8 antibody, anti-Ago2 antibody, or IgG. The presence of NCL, DGCR8, and Ago2 in the immunoprecipitates and input (2%) samples was monitored by immunoblotting. Statistical analyses were performed using two-way ANOVA Sidak’s multiple comparisons test (A,B) and two-tailed unpaired Student’s t-test (C,F). **, p < 0.005; ***, p < 0.0005; ****, p < 0.0001.