Fig. 2
METTL3 inhibits NR4A1 expression via m6A-dependent manner. A, B Direct RNA-seq analysis of the m6A abundance and sites in shR-NC and shR-METTL3 HeLa cells. C Volcano plot displaying the differentially expressed genes base on direct RNA-seq. D Venn diagram showed the candidate target gene from TCGA, GSE39001 and Direct RNA-seq. E, F RT-qPCR and western blot assays of the mRNA (E) and protein (F) expression of NR4A1 in CC cells with overexpression and knockdown of METTL3. G m6A RIP-qPCR analysis of NR4A1 mRNA in control and METTL3-silenced HeLa cells. H, The stability of NR4A1 mRNA in control and METTL3-silenced HeLa cells. Transcription was inhibited by act-D at 5 µg/ml during indicated times. I Left: Cells were incubated with 100 µg/ml CHX at the indicated times, the protein stability of NR4A1 was detected by western blot assay. Right: Quantification of protein stability. J Co-IP assay for the interactions between METTL3 and NR4A1. K Schematic depiction of mutated (GGACA to GGCCA) m6A site in CDS region in NR4A1. L RT-qPCR analysis showed the expression of EGFP from cells transfected with the indicated plasmids, and normalized to the corresponding β-actin (left) and RFP (right) values. All experiments were performed at least 3 independent times, and data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001