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RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100601

1083-351X

Yuji Omata, Tomoaki Yamauchi, Akito Tsuruta, Naoya Matsunaga, Satoru Koyanagi, Shigehiro Ohdo,

RNA Interference and Gene Delivery

The expression and function of some xenobiotic transporters vary according to the time of the day, causing the dosing time-dependent changes in drug disposition and toxicity. P-glycoprotein (P-gp), encoded by the ABCB1 gene, is highly expressed in the kidneys and functions in the renal elimination of various drugs. The elimination of several P-gp substrates was demonstrated to vary depending on administration time, but the underlying mechanism remains unclear. We found that adenosine deaminase acting on RNA (ADAR1) was involved in the circadian regulation of P-gp expression in human renal proximal tubular epithelial cells (RPTECs). After synchronization of the cellular circadian clock by dexamethasone treatment, the expression of P-gp exhibited a significant 24-h oscillation in RPTECs, but this oscillation was disrupted by the downregulation of ADAR1. Although ADAR1 catalyzes adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA substrates, no significant ADAR1-regulated editing sites were detected in the human ABCB1 transcripts in RPTECs. On the other hand, downregulation of ADAR1 induced alternative splicing in intron 27 of the human ABCB1 gene, resulting in the production of retained intron transcripts. The aberrant spliced transcript was sensitive to nonsense-mediated mRNA decay, leading to the decreased stability of ABCB1 mRNA and prevention of the 24-h oscillation of P-gp expression. These findings support the notion that ADAR1-mediated regulation of alternative splicing of the ABCB1 gene is a key mechanism of circadian expression of P-gp in RPTECs, and the regulatory mechanism may underlie the dosing time-dependent variations in the renal elimination of P-gp substrates. The expression and function of some xenobiotic transporters vary according to the time of the day, causing the dosing time-dependent changes in drug disposition and toxicity. P-glycoprotein (P-gp), encoded by the ABCB1 gene, is highly expressed in the kidneys and functions in the renal elimination of various drugs. The elimination of several P-gp substrates was demonstrated to vary depending on administration time, but the underlying mechanism remains unclear. We found that adenosine deaminase acting on RNA (ADAR1) was involved in the circadian regulation of P-gp expression in human renal proximal tubular epithelial cells (RPTECs). After synchronization of the cellular circadian clock by dexamethasone treatment, the expression of P-gp exhibited a significant 24-h oscillation in RPTECs, but this oscillation was disrupted by the downregulation of ADAR1. Although ADAR1 catalyzes adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA substrates, no significant ADAR1-regulated editing sites were detected in the human ABCB1 transcripts in RPTECs. On the other hand, downregulation of ADAR1 induced alternative splicing in intron 27 of the human ABCB1 gene, resulting in the production of retained intron transcripts. The aberrant spliced transcript was sensitive to nonsense-mediated mRNA decay, leading to the decreased stability of ABCB1 mRNA and prevention of the 24-h oscillation of P-gp expression. These findings support the notion that ADAR1-mediated regulation of alternative splicing of the ABCB1 gene is a key mechanism of circadian expression of P-gp in RPTECs, and the regulatory mechanism may underlie the dosing time-dependent variations in the renal elimination of P-gp substrates. The daily variations in biological functions are thought to be important factors affecting the efficacy and/or toxicity of drugs; a large number of drugs cannot be expected to have the same potency at different administration times (1Ohdo S. Koyanagi S. Matsunaga N. Hamdan A. Molecular basis of chronopharmaceutics.J. Pharm. Sci. 2011; 100: 3560-3576Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 2Dallmann R. Okyar A. Lévi F. Dosing-time makes the poison: Circadian regulation and pharmacotherapy.Trends Mol. Med. 2016; 22: 430-445Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Dosing time-dependent differences in the therapeutic effects of drugs are, at least in part, due to diurnal changes in drug disposition such as absorption, distribution, metabolism, and elimination (3Ohdo S. Koyanagi S. Matsunaga N. Chronopharmacological strategies focused on chrono-drug discovery.Pharmacol. Ther. 2019; 202: 72-90Crossref PubMed Scopus (18) Google Scholar). ABC transporters are widely known as energy-dependent efflux pumps that extrude cytotoxic substances from cells. They are expressed in epithelial cells of several organs, such as the brain, liver, intestine, and kidneys (4Sissung T.M. Goey A.K.L. Ley A.M. Strope J.D. Figg W.D. Pharmacogenetics of membrane transporters: A review of current approaches.Methods Mol. Biol. 2014; 1175: 91-120Crossref PubMed Scopus (12) Google Scholar) and function in the biliary, intestinal, and renal elimination of drugs (5Shitara Y. Horie T. Sugiyama Y. Transporters as a determinant of drug clearance and tissue distribution.Eur. J. Pharm. Sci. 2006; 27: 425-446Crossref PubMed Scopus (408) Google Scholar). The expression and function of several ABC transporters exhibit diurnal variation, resulting in dosing time-dependent differences in drug disposition (6Hamdan A.M. Koyanagi S. Wada E. Kusunose N. Murakami Y. Matsunaga N. Ohdo S. Intestinal expression of mouse Abcg2/breast cancer resistance protein (BCRP) gene is under control of circadian clock-activating transcription factor-4 pathway.J. Biol. Chem. 2012; 287: 17224-17231Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7Yu F. Zhang T. Zhou C. Xu H. Guo L. Chen M. Wu B. The circadian clock gene Bmal1 controls intestinal exporter MRP2 and drug disposition.Theranostics. 2019; 9: 2754-2767Crossref PubMed Scopus (30) Google Scholar, 8Kato M. Tsurudome Y. Kanemitsu T. Yasukochi S. Kanado Y. Ogino T. Matsunaga N. Koyanagi S. Ohdo S. Diurnal expression of MRP4 in bone marrow cells underlies the dosing-time dependent changes in the oxaliplatin-induced myelotoxicity.Sci. Rep. 2020; 10: 13484Crossref PubMed Scopus (4) Google Scholar). We previously demonstrated that the expression and function of P-glycoprotein (P-gp), encoded by the ABCB1 gene, exhibit diurnal oscillation in the small intestine of mice and cynomolgus monkeys (9Murakami Y. Higashi Y. Matsunaga N. Koyanagi S. Ohdo S. Circadian clock-controlled intestinal expression of the multidrug-resistance gene mdr1a in mice.Gastroenterology. 2008; 135: 1636-1644Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 10Iwasaki M. Koyanagi S. Suzuki N. Katamune C. Matsunaga N. Watanabe N. Takahashi M. Izumi T. Ohdo S. Circadian modulation in the intestinal absorption of P-glycoprotein substrates in monkeys.Mol. Pharmacol. 2015; 88: 29-37Crossref PubMed Scopus (27) Google Scholar). In both rodents and monkeys, the oscillation of P-gp expression was associated with the dosing time-dependent differences in the intestinal absorption of its substrate drugs. The efflux pump activity of P-gp was also suggested to exhibit diurnal oscillation in humans because of the dosing time-dependent differences in renal clearance of substrate drugs (11Choi J. Park H. Circadian changes in pharmacokinetics of cyclosporin in healthy volunteers.Res. Commun. Mol. Pathol. Pharmacol. 1999; 130: 109-112Google Scholar, 12Ferrazzini G. Sohl H. Robieux I. Johnson D. Giesbrecht E. Koren G. Diurnal variation of methotrexate disposition in children with acute leukaemia.Eur. J. Clin. Pharmacol. 1991; 41: 425-427Crossref PubMed Scopus (10) Google Scholar), but diurnal variations in the expression and function of P-gp in human renal cells remain unclear. In general, protein levels are regulated at nearly all stages of the gene expression process. In addition to their synthesis, mRNA and proteins are both also subject to degradation, which influences the abundance of membrane proteins. The circadian expression of clock and clock-controlled proteins occurs at the transcriptional, posttranscriptional, translational, and posttranslational levels (13Fustin J.M. Doi M. Yamaguchi Y. Hida H. Nishimura S. Yoshida M. Isagawa T. Morioka M.S. Kakeya H. Manabe I. Okamura H. RNA-methylation-dependent RNA processing controls the speed of the circadian clock.Cell. 2013; 155: 793-806Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 14Tsurudome Y. Koyanagi S. Kanemitsu T. Katamune C. Oda M. Kanado Y. Kato M. Morita A. Tahara Y. Matsunaga N. Shibata S. Ohdo S. Circadian clock component PERIOD2 regulates diurnal expression of Na+/H+ exchanger regulatory factor-1 and its scaffolding function.Sci. Rep. 2018; 8: 9072Crossref PubMed Scopus (5) Google Scholar, 15Okazaki H. Matsunaga N. Fujioka T. Okazaki F. Akagawa Y. Tsurudome Y. Ono M. Kuwano M. Koyanagi S. Ohdo S. Circadian regulation of mTOR by the ubiquitin pathway in renal cell carcinoma.Cancer Res. 2014; 74: 543-551Crossref PubMed Scopus (39) Google Scholar). Among posttranscriptional regulation mechanisms, adenosine-to-inosine (A-to-I) RNA editing is the most prevalent nucleotide conversion in mammals (16Nishikura K. Functions and regulation of RNA editing by ADAR deaminases.Annu. Rev. Biochem. 2010; 79: 321-349Crossref PubMed Scopus (752) Google Scholar). Inosine, an analog of guanosine, forms a base pair with cytidine, altering the amino acid sequence, microRNA targeting, and splicing of RNA (16Nishikura K. Functions and regulation of RNA editing by ADAR deaminases.Annu. Rev. Biochem. 2010; 79: 321-349Crossref PubMed Scopus (752) Google Scholar). A-to-I RNA editing is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. They convert adenosines in the double-stranded RNA (dsRNA) structure to inosines by hydrolytic deamination. There are two functional members of the ADAR family in humans: ADAR1 and ADAR2. ADAR1 has two isoforms: ADAR1-p110 (110-kDa protein) and ADAR1-p150 (150-kDa protein). ADAR1-p110 is constitutively expressed in the nucleus, whereas ADAR1-p150 has an interferon-inducible promoter and is predominantly localized in the cytoplasm (16Nishikura K. Functions and regulation of RNA editing by ADAR deaminases.Annu. Rev. Biochem. 2010; 79: 321-349Crossref PubMed Scopus (752) Google Scholar, 17Patterson J.B. Samuel C.E. Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: Evidence for two forms of the deaminase.Mol. Cell. Biol. 1995; 15: 5376-5388Crossref PubMed Scopus (394) Google Scholar). Recently, A-to-I RNA editing is demonstrated to exhibit diurnal oscillation in the liver of mice, suggesting that A-to-I RNA editing generates diurnal rhythmicity of a wide range of mRNAs (18Terajima H. Yoshitane H. Ozaki H. Suzuki Y. Shimba S. Kuroda S. Iwasaki W. Fukada Y. ADARB1 catalyzes circadian A-to-I editing and regulates RNA rhythm.Nat. Genet. 2017; 49: 146-151Crossref PubMed Scopus (47) Google Scholar). ADAR1-mediated RNA editing is also reported to regulate the expression of drug metabolism enzymes (19Nakano M. Nakajima M. Significance of A-to-I RNA editing of transcripts modulating pharmacokinetics and pharmacodynamics.Pharmacol. Ther. 2018; 181: 13-21Crossref PubMed Scopus (8) Google Scholar). For example, ADAR1 creates microRNA targeting sites of aryl hydrocarbon receptor (AhR) mRNA, thereby inducing the downregulation of AhR in human liver cells (20Nakano M. Fukami T. Gotoh S. Takamiya M. Aoki Y. Nakajima M. RNA editing modulates human hepatic aryl hydrocarbon receptor expression by creating MicroRNA recognition sequence.J. Biol. Chem. 2016; 291: 894-903Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The downregulation of AhR interferes with drug-inducible expression of Cytochrome 1A1. During the analysis of the role of ADAR1 in the regulation of renal expression of xenobiotic transporters, we found that the levels of P-gp were reduced by the downregulation of ADAR1 in human renal proximal tubular epithelial cells (RPTECs). The expression of P-gp exhibited significant 24-h oscillation in circadian clock-synchronized cultured RPTECs, but this oscillation was disrupted in ADAR1-knockdown (KD) RPTECs. Therefore, we investigated the mechanism by which ADAR1 regulates the circadian expression of P-gp in human RPTECs. In the first set of experiments, we used nonsynchronized human RPTECs to investigate which renal transporters are under the control of ADAR1. To achieve this, we prepared ADAR1-KD human RPTECs by stable expression of small hairpin RNA (shRNA). Although no notable bands derived from ADAR1-p150 were detectable in RPTECs, the expression levels of the ADAR1-p110 isoform were significantly reduced to 40% in anti-ADAR1 shRNA-transduced cells (p < 0.01, Fig. 1A). Therefore, we used the ADAR1-KD RPTECs to assess the mRNA levels of xenobiotic transporters that are known to regulate renal drug elimination (21Giacomini K.M. Huang S.M. Tweedie D.J. Benet L.Z. Brouwer K.L.R. Chu X. Dahlin A. Evers R. Fischer V. Hillgren K.M. Hoffmaster K.A. Ishikawa T. Keppler D. Kim R.B. Lee C.A. et al.Membrane transporters in drug development.Nat. Rev. Drug Discov. 2010; 9: 215-236Crossref PubMed Scopus (2320) Google Scholar). As results of quantitative real-time RT-PCR analysis, the mRNA levels of the solute carrier (SLC) group of membrane transport proteins, SLC15A1, SLC15A2, SLC22A4, SLC22A5, SLC47A1, SLC47A2, and SLCO4C1, were not significantly different between mock-transduced and ADAR1-KD RPTECs (Fig. 1B). The transport proteins encoded by SLC22A2, SLC22A6, and SLC22A8 genes also regulate tubular secretion of various drugs, but their mRNA expression was undetectable in RPTECs. On the other hand, the mRNA levels of ABCB1 and ABCC2 in ADAR1-KD RPTECs decreased and increased, respectively, whereas ABCG2 mRNA levels in ADAR1-KD RPTECs were not significantly different from those in mock-transduced cells (Fig. 1C). Furthermore, downregulation of ADAR1 reduced the expression of P-gp encoded by the ABCB1 gene, but not of MRP2 encoded by the ABCC2 gene, in RPTECs (Fig. 1D). This suggests that ADAR1 positively regulates the expression of ABCB1 mRNA and P-gp in tubular cells of the human kidney. Due to the stability of MRP2 protein (22Jones B.R. Li W. Cao J. Hoffman T.A. Gerk P.M. Vore M. The role of protein synthesis and degradation in the post-transcriptional regulation of rat multidrug resistance-associated protein 2 (Mrp2, Abcc2).Mol. Pharmacol. 2005; 68: 701-710Crossref PubMed Scopus (39) Google Scholar), ADAR1-induced expression of ABCC2 mRNA might not be reflected to its protein levels. The elimination rate of several P-gp substrates is changed depending on their administration time of the day (11Choi J. Park H. Circadian changes in pharmacokinetics of cyclosporin in healthy volunteers.Res. Commun. Mol. Pathol. Pharmacol. 1999; 130: 109-112Google Scholar, 12Ferrazzini G. Sohl H. Robieux I. Johnson D. Giesbrecht E. Koren G. Diurnal variation of methotrexate disposition in children with acute leukaemia.Eur. J. Clin. Pharmacol. 1991; 41: 425-427Crossref PubMed Scopus (10) Google Scholar), suggesting that efflux pump function of P-gp varies during the day due to changes in its expression level. Indeed, we observed diurnal oscillations in the protein levels of ADAR1 and P-gp in the kidney of cynomolgus monkeys (Fig. 2A) kept under the light/dark cycle (ZT, zeitgeber time; ZT0, lights on; ZT12, lights off). This prompted us to explore the possibility that ADAR1 is also involved in circadian regulation of P-gp expression in the human kidney. We therefore investigated the temporal expression profiles of ADAR1 and P-gp in cultured human RPTECs after synchronization of their circadian clock. Treatment of mock-transduced RPTECs with 100 nM dexamethasone (DEX) for 2 h induced significant 24-h oscillations in the mRNA expression of the major circadian clock genes PERIOD2 and BMAL1 (p < 0.01, respectively, Fig. 2B). Similar 24-h oscillations of circadian gene expression were also observed in DEX-treated ADAR1-KD RPTECs (p < 0.01, respectively, Fig. 2B), although BMAL1 mRNA levels increased in ADAR1-KD RPTECs. Synchronization of the circadian clock persisted for up to 52 h after DEX treatment. In this in vitro culture model, the oscillation in the expression of clock genes after 24 h following synchronization is thought to be generated by cell-autonomous circadian machinery (23Balsalobre A. Damiola F. Schibler U. A serum shock induces circadian gene expression in mammalian tissue culture cells.Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1487) Google Scholar, 24Balsalobre A. Brown S.A. Marcacci L. Tronche F. Kellendonk C. Reichardt H.M. Schütz G. Schibler U. Resetting of circadian time in peripheral tissues by glucocorticoid signaling.Science. 2000; 289: 2344-2347Crossref PubMed Scopus (1286) Google Scholar), which constitute fundamental properties that characterize circadian rhythms. Therefore, we investigated the expression of ADAR1 and P-gp from 28 h to 48 h after DEX treatment. A significant 24-h oscillation in the protein levels of ADAR1 was observed in mock-transduced RPTECs (p < 0.01, Fig. 2C), but the oscillation of ADAR1 protein levels was dampened in ADAR1-KD cells. The expression of P-gp in circadian clock-synchronized mock-transduced RPTECs also exhibited significant 24-h oscillation (p < 0.05, Fig. 2D), accompanying significant time-dependent differences in the intracellular accumulation of digoxin, a typical substrate of P-gp (p < 0.05, Fig. 2E). Low intracellular accumulation of digoxin was observed around the peak time of P-gp expression, demonstrating that the levels of P-gp and its efflux pump activity increased at the time after the elevation of ADAR1 levels. In contrast, the expression levels of P-gp in ADAR1-KD RPTECs decreased at all examined time points, and the amplitude of P-gp oscillation was reduced by the downregulation of ADAR1 (Fig. 2D). Consequently, the time dependency of digoxin accumulation was not observed in ADAR1-KD RPTECs (Fig. 2E) due to the increased digoxin accumulation at both examined time points. This suggests that ADAR1 is involved in circadian regulation of P-gp expression in human RPTECs. To investigate the underlying mechanism of disruption of the circadian expression of P-gp in ADAR1-KD RPTECs, the temporal expression profiles of ABCB1 mRNA were assessed in circadian clock-synchronized RPTECs. A significant 24-h oscillation in the mRNA levels of ABCB1 was observed in mock-transduced RPTECs after DEX treatment (p < 0.01, Fig. 3A). The mRNA levels of ABCB1 decreased in DEX-treated ADAR1-KD RPTECs, although still exhibited significant 24-h oscillation (p < 0.01, Fig. 3A). This suggests that downregulation of ADAR1 alters the circadian expression of ABCB1 mRNA in human RPTECs, but disruption of the circadian expression of P-gp in ADAR1-KD RPTECs is not completely dependent on the alternation of the ABCB1 mRNA rhythm. Next, we investigated the mechanism by which ABCB1 mRNA expression exhibits 24-h oscillation in circadian clock-synchronized RPTECs. For this purpose, we constructed a reporter vector in which the human ABCB1 gene 5′-flanking region around the transcription start site spanning from −2091 to +701 (the number is the distance in base pairs from the putative transcription start site, +1) was inserted upstream of the luciferase gene (ABCB1 [2.8kb]::Luc) because the 5′-flanking region contains clock gene response elements (10Iwasaki M. Koyanagi S. Suzuki N. Katamune C. Matsunaga N. Watanabe N. Takahashi M. Izumi T. Ohdo S. Circadian modulation in the intestinal absorption of P-glycoprotein substrates in monkeys.Mol. Pharmacol. 2015; 88: 29-37Crossref PubMed Scopus (27) Google Scholar). Luciferase activity from ABCB1 [2.8kb]::Luc-transfected RPTECs was assessed at 32 and 44 h after DEX treatment, which were decreasing and increasing times of ABCB1 mRNA expression in circadian clock-synchronized cells, respectively (Fig. 3A). There was no significant time-dependent variation in luciferase activity driven by ABCB1 [2.8kb]::Luc in DEX-treated RPTECs (Fig. 3B), suggesting that the 24-h oscillation in ABCB1 mRNA expression is not dependent on its transcriptional level. Circadian oscillation of gene expression is not only caused by the transcription process, but also induced by posttranscriptional regulation (13Fustin J.M. Doi M. Yamaguchi Y. Hida H. Nishimura S. Yoshida M. Isagawa T. Morioka M.S. Kakeya H. Manabe I. Okamura H. RNA-methylation-dependent RNA processing controls the speed of the circadian clock.Cell. 2013; 155: 793-806Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 18Terajima H. Yoshitane H. Ozaki H. Suzuki Y. Shimba S. Kuroda S. Iwasaki W. Fukada Y. ADARB1 catalyzes circadian A-to-I editing and regulates RNA rhythm.Nat. Genet. 2017; 49: 146-151Crossref PubMed Scopus (47) Google Scholar). Thus, we also investigated the time dependence of the stability of ABCB1 mRNA in circadian clock-synchronized RPTECs. Cells were treated with 5 μM actinomycin D (ActD) at 32 or 44 h after synchronization of their circadian clock, and ABCB1 mRNA levels were then assessed until 6 h after the initiation of ActD treatment. The stability of ABCB1 mRNA at 32 h after synchronization of the circadian clock was significantly lower than that at 44 h (p < 0.05, Fig. 3C). This suggests that the 24-h oscillation of ABCB1 mRNA levels in circadian clock-synchronized RPTECs is caused by the time-dependent differences in their stability, but not due to the time-dependent variation in transcriptional activity. In the circadian clock-synchronized RPTECs, ABCB1 mRNA was stabilized around the time when ADAR1 protein levels increased (Figs. 2C and 3C). Therefore, we investigated whether ADAR1 is involved in the regulation of ABCB1 mRNA expression. As described above, mRNA levels of ABCB1 in mock-transduced and ADAR1-KD RPTECs were assessed after treatment with ActD. The reduction of mRNA levels of ABCB1 in ActD-treated ADAR1-KD RPTECs was significantly greater than that in mock-transduced cells (Fig. 4A). ADAR1 had negligible effects on the transcriptional activity of the human ABCB1 gene because the luciferase activity driven by ABCB1 [2.8kb]::Luc was not significantly different between mock-transduced and ADAR1-KD RPTECs (Fig. 4B). The stability of mRNA is often regulated via its 3′ untranslated region (3′ UTR) (25Fabian M.R. Sonenberg N. Filipowicz W. Regulation of mRNA translation and stability by microRNAs.Annu. Rev. Biochem. 2010; 79: 351-379Crossref PubMed Scopus (2021) Google Scholar). Thus, we also constructed reporter vector in which the human ABCB1 mRNA 3′ UTR was inserted downstream of the luciferase gene (ABCB1 3′ UTR::Luc) and then transfected it into cells. The luciferase activity of reporter vector was reduced by insertion of the 3′ UTR of the ABCB1 gene, but the reporter activity of ABCB1 3′ UTR::Luc in ADAR1-KD RPTECs was not significantly different from that in mock-transduced cells (Fig. 4C). This suggests that the decreased stability of ABCB1 mRNA in ADAR1-KD RPTECs is not due to 3′ UTR-mediated regulation by ADAR1. To investigate the underlying mechanism of ADAR1-mediated regulation of ABCB1 gene expression, we conducted a comprehensive analysis of RNA editing events in the human ABCB1 gene transcript using REDIPortal, a searching database of A-to-I RNA editing sites (26Picardi E. D'Erchia A.M. Lo Giudice C. Pesole G. REDIportal: A comprehensive database of A-to-I rna editing events in humans.Nucleic Acids Res. 2017; 45: D750-D757Crossref PubMed Scopus (129) Google Scholar). A total of 47 A-to-I RNA editing sites were registered in the human ABCB1 gene pre-mRNA in the database. Among them, 33 were identified in intron 27 (Fig. 5A). As A-to-I RNA editing by ADAR1 is often observed in the long regions of dsRNA (27Eggington J.M. Greene T. Bass B.L. Predicting sites of ADAR editing in double-stranded RNA.Nat. Commun. 2011; 2: 319Crossref PubMed Scopus (201) Google Scholar, 28Nishikura K. Yoo C. Kim U. Murray J.M. Estes P.A. Cash F.E. Liebhaber S.A. Substrate specificity of the dsRNA unwinding/modifying activity.EMBO J. 1991; 10: 3523-3532Crossref PubMed Scopus (129) Google Scholar), we also performed in silico prediction analysis for the secondary structure of pre-mRNA of ABCB1 gene (29Lorenz R. Bernhart S.H. Höner zu Siederdissen C. Tafer H. Flamm C. Stadler P.F. Hofacker I.L. ViennaRNA package 2.0.Algorithms Mol. Biol. 2011; 6: 26Crossref PubMed Scopus (2087) Google Scholar). The results indicated that intron 27 forms stable dsRNA of approximately 280 bp in length (Fig. 5B), whereas the other introns involving putative A-to-I editing sites failed to show stable form of dsRNA structure (Fig. S1). However, the results of direct sequence analysis revealed that no significant editing sites were detected in the intron 27 region of the ABCB1 gene pre-mRNA prepared from mock-traduced, ADAR1-KD, and ADAR1-overexpressing RPTECs (Figs. S2 and S3 and Table S1). During the analysis of transcript variants of the human ABCB1 gene, we noted that ABCB1 transcripts are subjected to alternative splicing that generates the transcripts of retained intron 27 (registered as ENST00000491360.1). Retention of intron 27 results in a premature stop codon, suggesting the cause of nonsense-mediated mRNA decay (NMD) (Fig. 6A). To address this possibility, we constructed minigene plasmid vectors in which the DNA fragment of the human ABCB1 gene from exon 27 to exon 28 was inserted into the multicloning site of pcDNA3.1 (ex27–ex28 minigene) and then transfected them into mock-transduced and ADAR1-KD RPTECs. The amount of splicing variants from the ex27–ex28 minigene were assessed at 32 and 44 h after synchronization of their circadian clock by DEX treatment. The percentage of retained intron 27 transcripts among the sum of all transcripts and normal splicing variant (intron 27 removal transcript) in mock-transduced cells was significantly higher at 32 h than at 44 h (p < 0.05, Fig. 6B). The generation of retained-intron 27 transcripts from the ex27–ex28 minigene in ADAR1-KD cells increased at both time points. The regulation of gene function by ADAR1 depends on both its editing activity and binding capacity to dsRNA (30Sakurai M. Shiromoto Y. Ota H. Song C. Kossenkov A.V. Wickramasinghe J. Showe L.C. Skordalakes E. Tang H.-Y. Speicher D.W. Nishikura K. ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay.Nat. Struct. Mol. Biol. 2017; 24: 534-543Crossref PubMed Scopus (65) Google Scholar). Although no significant editing sites were found in the intron 27 region of the ABCB1 gene pre-mRNA (Figs. S2 and S3), the results of RNA immunoprecipitation (RIP) analysis using RNA extracted from the minigene-transfected RPTECs indicated that ADAR1 can bind to intron 27 region of pre-mRNA of the ABCB1 gene (p < 0.01, Fig. 6C). The NMD inhibitor NMDI-14 (31Martin L. Grigoryan A. Wang D. Wang J. Breda L. Rivella S. Cardozo T. Gardner L.B. Identification and characterization of small molecules that inhibit nonsense-mediated rna decay and suppress nonsense p53 mutations.Cancer Res. 2014; 74: 3104-3113Crossref PubMed Scopus (68) Google Scholar) significantly increased the percentage of retained-intron 27 transcripts in ex27–ex28 minigene-transfected cells (p < 0.01, Fig. 6D), suggesting that the retention of intron 27 produces NMD-sensitive transcripts. The stability of retained-intron 27 transcripts was significantly lower than that of normal splicing variants (Fig. 6E). Lower expression levels of ABCB1 mRNA and P-gp in ADAR1-KD RPTECs were increased by treatment with NMDI-14 (Fig. 6, F and G). Furthermore, the NMD inhibition in RPTECs by NMDI-14 also decreased intracellular accumulation of digoxin (Fig. 6H). These findings suggest that ADAR1 prevents the generation of retained-intron 27 transcripts from the ABCB1 gene and increases the stability of ABCB1 mRNA. The ADAR1-dependent maturation of ABCB1 mRNA may also function in the production of P-gp in RPTECs. The circadian clock machinery controls many downstream events through transcription, translation, or degradation processes. In this study, we demonstrated that the 24-h oscillation of P-gp expression in circadian clock-synchronized RPTECs is generated through a posttranscriptional mechanism, by which ADAR1-mediated regulation of alternative splicing is associated with the maturation and stabilization of ABCB1 mRNA. ADAR1 time-dependently prevented the production of aberrant spliced transcripts of the ABCB1 gene, resulting in circadian expression of P-gp (Fig. 7). To evaluate the circadian profiles of gene expression, it is necessary to collect tissue samples every few hours. Therefore, experimental animals are often used for chronopharmacological and chronopharmacokinetic studies. Diurnal expression of drug metabolism enzymes and xenobiotic transporters has been identified in mice and rats (6Hamdan A.M. Koyanagi S. Wada E. Kusunose N. Murakami Y. Matsunaga N. Ohdo S. Intestinal expression of mouse Abcg2/breast cancer resistance protein (BCRP) gene is under control of circadian clock-activating transcription factor-4 pathway.J. Biol. Chem. 2012; 287: 17224-17231Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 32Matsunaga N. Ikeda M. Takiguchi T. Koyanagi S. Ohdo S. The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver.Hepatology. 2008; 48: 240-251Crossref PubMed Scopus (62) Google Scholar). In contrast to experimental animals, our understanding about circadian characteristics of human pharmacokinetics regulators is limited because of restrictions on frequent sampling from tissues. In mammals, nearly all cells contain circadian oscillators organized in a hierarchical fashion, with a master pacemaker located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus (33Hastings M.H. Reddy A.B. Maywoo