Methylation of adenosine at the N⁶ position post-transcriptionally regulates hepatic P450s expression

The methylation of adenosines at the N⁶ position (m⁶A formation) is the most prevalent type of RNA modification in humans. This modification is mediated by methyltransferase like 3 (METTL3)-METTL14 complex, and the methyl group can be removed by RNA demethylases including fat mass and obesity-associated (FTO) and AlkB homolog 5. The formed m⁶A is recognized by reader proteins such as members of the YT521-B homology (YTH) family, resulting in changes in the splicing, nuclear export, and decay of RNA or translation. In this study, we examined the impact of m⁶A modification on the expression of drug-metabolizing P450 isoforms. By treatment with 3-deazaadenosine, an inhibitor of RNA methylation, CYP1A2, CYP2B6, and CYP2C8 levels were significantly increased (1.6-fold, 2.2-fold, and 2.7-fold, respectively) in HepaRG cells. In subsequent experiments, we focused on CYP2C8, which showed the largest increase. Consistent with the increase in the mRNA level, CYP2C8 protein level and activity were significantly increased by treatment with 3-deazaadenosine. The CYP2C8 expression levels and activities in HepaRG and Huh-7 cells were increased by knockdown of METTL3/14, whereas they were decreased by knockdown of FTO, suggesting that m⁶A modification downregulates CYP2C8 expression. With an RNA immunoprecipitation assay using an anti-m⁶A antibody, it was revealed that the adenosines in the 5'-UTR and the last exon of CYP2C8 are methylated in HepaRG cells and human liver samples. It was demonstrated that YTHDC2, which is known to degrade m⁶A-containing mRNA, downregulates CYP2C8 expression. In conclusion, we found a novel post-transcriptional regulation mechanism in which the YTHDC2 promotes CYP2C8 mRNA degradation via recognizing the m⁶A in CYP2C8 mRNA, which is installed by METTL3/14 and removed by FTO.