RESUMO
Methylation of adenosine at the N6 position to form N6-methyladenosine (m6A) is the most prevalent epitranscriptomic modification of mammalian mRNA. This modification is catalyzed by a methyltransferase-like 3 (METTL3)-METTL14 complex and is erased by demethylases such as fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5). m6A modification regulates mRNA stability, nuclear export, splicing, and/or protein translation via recognition by reader proteins such as members of YT521-B homology (YTH) family. Carboxylesterase 2 (CES2) is a serine esterase responsible for the hydrolysis of drugs and endogenous substrates, such as triglycerides and diacylglycerides. Here, we examined the potential regulation of human CES2 expression by m6A modification. CES2 mRNA level was significantly increased by double knockdown of METTL3 and METTL14 but was decreased by knockdown of FTO or ALKBH5 in HepaRG and HepG2 cells, leading to changes in its protein level and hydrolase activity for 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), suggesting that m6A modification negatively regulates CES2 expression. Consistent with the changes in CES2 expression, lipid accumulation in the cells was decreased by double knockdown of METTL3 and METTL14 but was increased by knockdown of FTO or ALKBH5. RNA immunoprecipitation assays using an anti-m6A antibody showed that adenosines in the 5'-untranslated region (UTR) and the last exon of CES2 are methylated. Luciferase assays revealed that YTHDC2, which degrades m6A-containing mRNA, downregulates CES2 expression by recognition of m6A in the 5'-UTR of CES2. Collectively, we demonstrated that m6A modification has a great impact on the regulation of CES2, affecting pharmacokinetics, drug response and lipid metabolism.
Assuntos
Adenosina/análogos & derivados , Carboxilesterase/metabolismo , Adenosina/genética , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Carboxilesterase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Irinotecano/farmacologia , Metabolismo dos Lipídeos/genética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The pregnane X receptor (PXR) is one of the major transcription factors that regulate the expression of different drug-metabolizing enzymes and transporters. Adenosine-to-inosine RNA editing, the most frequent nucleotide conversion on RNA, which is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, may modulate gene expression and function. Here, we investigated the potential regulation of human PXR expression by adenosine-to-inosine RNA editing. Knockdown of ADAR1 increased PXR mRNA level, and the knockdown of ADAR1 or ADAR2 significantly increased PXR protein level in HepaRG cells. In HepG2 cells, the knockdown of ADAR1 or ADAR2 significantly increased PXR mRNA and protein levels. The increase in the PXR protein by ADAR1 knockdown resulted in increased cytochrome P450 3A4 (CYP3A4) transactivity and CYP3A4 and UDP-glucuronosyltransferase 1A1 (UGT1A1) expression. A reporter assay revealed that the 3'-untranslated region (UTR) of PXR mRNA, especially from +3371 to +3440, is responsible for the ADAR-mediated post-transcriptional control of PXR expression, despite the lack of RNA edited sites in this region. Collectively, we found that PXR is negatively regulated by ADAR1 via an indirect mechanism, which facilitates the degradation of PXR mRNA. We could demonstrate that ADAR1 can cause interindividual variability in hepatic drug metabolism potencies.
Assuntos
Adenosina Desaminase/metabolismo , Receptor de Pregnano X/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Células Hep G2 , Humanos , Receptor de Pregnano X/metabolismoRESUMO
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.