FIGURE 3.

Methylation sites involved in m⁶A‐regulated ZNF677. (A) Schematic representation of positions of DMMPs within ZNF677 mRNA. (B) The m⁶A enrichment in 5′UTR, CDS or 3′UTR of ZNF677 in control or Mettl3 overexpression OSRC cells were analysed by MeRIP‐qPCR using fragmented RNA. (C) The relative luciferase activity of F‐Luc/R‐Luc of pmirGLO‐ZNF677‐3′UTR‐WT, or pmirGLO‐3′UTR‐MUT in control and Mettl3‐overexpressing OSRC cells. (D) Schematic representation of mutation in CDS of ZNF677 to investigate the m⁶A roles on ZNF677 expression. (E) The relative luciferase activity of F‐Luc/R‐Luc of pmirGLO‐ZNF677‐CDS‐WT, or pmirGLO‐ZNF677‐CDS‐MUT‐1/‐2/‐3/‐4 in control and Mettl3‐overexpressing OSRC cells. (F) Western blot analysis of ZNF677 expression in OSRC cells co‐expressing exogenous Mettl3 and pmirGLO‐ZNF677‐CDS‐WT or pmirGLO‐ZNF677‐CDS‐MUTs. (G and H) pmirGLO‐ZNF677‐CDS‐WT (G) or pmirGLO‐ZNF677‐CDS‐MUT4 (H) was transfected into control or Mettl3‐overexpressing OSRC cells for 24 h and then further treated with Act‐D for the indicated times. The mRNA of ZNF677 was checked by RT‐qPCR. NS, not significant; *p < .05 or **p < .01 indicates a significant difference between the indicated groups