The 5′-Untranslated Region of Multidrug Resistance Associated Protein 2 (MRP2; ABCC2) Regulates Downstream Open Reading Frame Expression through Translational Regulation

Abstract

MRP2 (ABCC2), a member of the ATP binding cassette superfamily of efflux transporters that mediates the apical efflux of organic anions from hepatocytes, enterocytes, and renal epithelial cells, is postulated to undergo post-transcriptional regulation. The MRP2 5'-untranslated region (5'UTR) contains seven upstream start codons and six upstream open reading frames (uORFs). Ribonuclease protection assays in human liver, placenta, kidney, small intestine, and HepG2 cells identified multiple MRP2 transcription initiation sites. We investigated MRP2 5'UTRs [-247 (-247 to -1), -204 (-204 to -1), or -99 (-99 to -1)] for their effects on regulation of gene expression with the use of transient gene expression in HepG2 cells and in vitro translation assays. In HepG2 cells transfected with SV40-MRP2-5'UTR-Luciferase cassettes, luciferase activities of constructs -247 and -204 were significantly lower than that of -99. Disruption of the uORFs at -105 and -74 nucleotides by mutation of ATGs to AAG enhanced luciferase activity significantly without affecting luciferase mRNA expression. The translation efficiencies of T7-5'UTR-Luciferase cassettes determined in vitro were consistent with transfected HepG2 cells and showed that inhibition of translation by the -105 uORF occurred only in the cis configuration and not in the trans configuration and that inhibition of translation by the -105 uORF was independent of the encoded peptide sequence. Characterization of an MRP2 polymorphism, -24C>T, in the MRP2 5'UTR, demonstrated no effect on mRNA expression or downstream ORF translation. These data indicate for the first time that the 5'UTR of MRP2 mRNA transcripts and the uORF at -105 markedly influence MRP2 translation.