Translational regulation of rat multidrug resistance-associated protein 2 expression is mediated by upstream open reading frames in the 5 ' untranslated region

Multidrug resistance-associated protein 2 (Mrp2/Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo translational regulation. Transcription of rat hepatic Mrp2 mRNA is initiated at multiple sites (-213, -163, -132, and -98 nucleotides relative to the Mrp2 ATG) and contains potential upstream open reading frames (uORFs) in the 5' untranslated region (UTR) starting at -213, -149 and -109 nucleotides. Ribonuclease protection assays demonstrated that transcription of the Mrp2 gene at the various initiation sites was tissue- specific, with the major initiation site in the liver and kidney being -98 and -132 nucleotides, respectively. In the jejunum, the primary and secondary initiation sites were -98 and -132 nucleotides, respectively, with the converse true in the ileum. The relative abundance of these Mrp2 transcripts expressed in tissues varied with age from birth to the adult. HepG2 transient expression assays and in vitro translation assays in which the 5'UTRs were fused with a luciferase reporter showed that the 5'UTR without any uORF (-98 nucleotide) expressed maximal luciferase activity compared with those with one (-132 nucleotides), two (-163 nucleotides), or three (-213 nucleotides) uORFs. Disruption of the uORF by site- directed mutagenesis at nucleotide -109 enhanced luciferase activity 2- to 3- fold, whereas disruption of the uORF at nucleotide -149 had little effect. We conclude that among the uORFs in the Mrp2 5'UTR, the uORF starting at nucleotide -109 probably plays an important role in the regulation of Mrp2 protein expression.