Abstracts 210 to 431
Authors: Yuanyuan Zhang, Wei Li, Mary Vore, Irina Bochkis, Nir Rubins, Peter White, Klaus Kaestner, Verena Keitel, Dieter Häussinger, Ralf Kubitz, Marco Marzioni, Gianfranco Alpini, Stefania Saccomanno, Giammarco Fava, Cinzia Candelaresi, Chiara Rychlicki, Heather Francis, Luciano Trozzi, Julie Venter, Antonio Benedetti, Jesus Banales, Pamela Tietz, Seung-Ok Lee, Bing Huang, Tatyana Masyuk, Angela Stroope, Sergio Gradilone, Anatoliy Masyuk, Juan Medina, Nicholas Larusso, Xuefeng Xia, Wenzheng Zhang, Yuhua Xiao, Dongbing Gao, Gene Lesage, Sharon Demor- Row, Shannon Glaser, Shelley Vaculin, Yoshiyuki Ueno, Bradley Vac- Ulin, Sharon Demorrow, Julie Ven- Ter, Eugenio Gaudio, Paolo Onori, Antonio Franchitto, Bradley Vaculin, Kristina Kelley, Luc Guerrier, Stephane Claverol, Laetitia Finzi, Frederic Fortis, Egisto Boschetti, Chantal Housset, Jin Yu, Nina Sheung, Jonathan Dranoff
Journal: Hepatology (2007)

Human multidrug resistance protein 2 (MRP2; ABCC2), an ATPbinding cassette transporter, effluxes organic anions into bile.The absence of functional MRP2 causes Dubin-Johnson syndrome associated with conjugated hyperbilirubinemia.Three transcription initiation sites of the MRP2 gene in liver have been identified at -245, -204 and -99 nucleotide (nt) relative to the MRP2 AUG (numbered +1, +2 and +3 nt).(Hepatology 30:1507).We investigated the distribution of MRP2 transcription initiation sites in human liver and HepG2 cells, and the effect of the untranslated regions (5'UTRs) on in vitro translation.Among the seven upstream start codon AUGs in the MRP2 gene, only the AUG at -105 nt has a perfect Kozak motif and encodes a 22 amino acid peptide from -105 nt to -37 nt.We also studied whether translational regulation is dependent on this putative peptide sequence.Methods and results: 1. Ribonuclease Protection Assay.Human liver and HepG2 cell RNAs were used to detect MRP2 transcription initiation sites.We found the transcription initiation sites at -245, -204, and -99 nt.The distribution patterns of these sites in liver and HepG2 cells were similar, with the -204 nt and -99 nt 5'UTR transcripts were expressed equally and the -245 nt 5'UTR transcript expression was at low level.2. Transient co-transfection assay in HepG2 cells.pGL3 constructs containing the 5'UTRs of -245(Hu-L), -204(Hu-M) and -99(Hu-S) nt upstream of the luciferase reporter gene were cotransfected with pSV40-Ren to determine the effect of the 5'UTRs on firefly luciferase expression.In transfected HepG2 cells, the ratios of Fire/Ren luciferase activities of Hu-L, Hu-M, and Hu-S constructs were 1.2, 1.3, and 3.5, respectively.3.In vitro translation assay.The 5'UTRs (-204(M) and -99(S) nt) were cloned into T7Luc control vector.A mutation at -105ATGÇAAG was introduced into mutM construct to disrupt the upstream open reading frame (uORF).∆hMRP2 construct was prepared to scramble the peptide sequence encoded from -105 nt to -37 nt in hMRP2 construct.Capped reporter messages were prepared from linearized constructs, and in vitro translation rates were determined in Rabbit Reticulocyte Lysate.The translation efficiencies of M, S, and mutM constructs relative to the T7Luc control vector were 0.27, 1.2, and 2.2, respectively.The relative translation efficiencies of hMRP2 and ∆hMRP2 constructs were not different (0.52 and 0.68, respectively).Conclusions: The uORF at -105 nt of the 5'UTR of MRP2 has a significant inhibitory effect on in vitro translation, but this inhibitory effect is not dependent on the sequence of the peptide encoded by this uORF.