Fig. 4

MeRIP-seq analysis identifies PRDM15 as a downstream target of METTL16. A (a) Venn diagram displays genes identified by MeRIP-seq from METTL16 knockout (KO, by guide RNA #2) and control cells (WT). b Gene ontology analysis of the 80 identified genes from A. c Integrative genomics viewer (IGV) plots show m6A peaks at PRDM15, SETD5, KMT2D, and NSD2 mRNAs in MeRIR-seq of HuCCT1 and CCLP1 cells. Ranges of reads are indicated. (d) MeRIP-qPCR analysis of m6A level in PRDM15, SETD5, KMT2D, and NSD2 mRNAs. B, C Depletion of METTL16 by CRISPR/Cas9 (B) or siRNA (C) decreases PRDM15 protein expression in CCLP1 and HuCCT1 cells as determined by Western blotting analysis. D Depletion of YTHDF1 reduces PRDM15 protein expression in CCA cells. CCLP1 and HuCCT1 cells were transfected with control or YTHDF1/YTHDF3 siRNAs for 48 h. The protein level of PRDM15 was measured by immunoblotting. E Depletion of METTL16 decreases the interaction between YTHDF1 protein and PRDM15 mRNA. YTHDF1 protein was immunoprecipitated from control or METTL16 knockout CCLP1 cells and analyzed by immunoblotting (left). The enrichment of PRDM15 mRNA in YTHDF1-RIP over IgG in control or METTL16 knockout CCLP1 cells was analyzed by RIP-qPCR (right). F Depletion of YTHDF1 decreases ribosome-bound PRDM15 mRNA. Immunoblotting analysis of immunoprecipitated RPL22-FLAG in CCLP1 cells transfected with control or YTHDF1 siRNA (left). Ribosomal immunoprecipitation assay was performed in siYTHDF1 and siNC CCLP1 cells transfected with RPL22-FLAG plasmid. **P < 0.01, ***P < 0.001, ****P < 0.0001. d, E, F Mean ± SD, Two-way ANOVA test