Fig. 5. RBM15 regulates YES1 in an IGF2BP1-m⁶A-dependent pattern.

a, b Global m⁶A level of RNA extracted from RBM15-knockdown Huh7 and HCC-LM3 cells was investigated by m⁶A dot blot assays. The intensity of dot immunoblotting indicated the m⁶A level of total RNAs, while methylene blue staining was applied to measured input RNA; c a Venn diagram was generated for predicting potential m⁶A motif of YES1 in 3’ UTR from SRAMP and RMBase databases. Two candidate m⁶A motifs were selected; d, e showed m⁶A modification of YES1 by MeRIP-qPCR analyses in HCC-LM3 and MHCC97H cells. Knockdown of RBM15 induced a decreased m⁶A abundance on YES1 compared with the control group (**p < 0.01; t test); f construction pattern of luciferase reporters. The wild-type or mutant type (m⁶A motif mutated) sequence of YES1 3’ UTR was inserted into a pcDNA 3.1 vector, which contained Firefly and Renilla elements; g relative luciferase activity of the wild-type or mutant group was determined in RBM15-silenced HCC-LM3 and MHCC97H cells (normalized to Renilla activity; **p < 0.01; t test); h relative luciferase activity of the wild-type or mutant group was determined in RBM15 overexpression HCC-LM3 and MHCC97H cells (normalized to Renilla activity; **p < 0.01; t test); i YES1 mRNA expression was decreased after knockdown of IGF2BP1 in HCC-LM3 and MHCC97H cells (**p < 0.01, ***p < 0.001, ****p < 0.0001; t test). j RIP-qPCR validated that YES1 mRNA could bind to IGF2BP1 protein (***p < 0.001; t test); k the upregulation YES1 mRNA induced by RBM15 overexpression was reduced by knockdown of IGF2BP1 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; t test). The data are presented as mean ± SD.