Translational regulation of prostaglandin endoperoxide H synthase-1 mRNA in megakaryocytic MEG-01 cells. Specific protein binding to a conserved 20-nucleotide CIS element in the 3'-untranslated region.
Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of PGHS-1 mRNA within a few hours, whereas protein increase occurs after several days of treatment. We now report that the delayed increase in PGHS-1 protein is caused by translational regulation. De novo PGHS-1 synthesis, measured using [(35)S]methionine pulse labeling followed by immunoprecipitation, was detected at day 4 after TPA treatment but not at day 1. To identify a potential element of PGHS-1 mRNA controlling translation, we compared the 3'-untranslated region from different species and identified a 20-nt segment perfectly conserved. The 20-nt segment was used as a probe in RNA gel mobility-shift assays using MEG-01 extracts from control cells or from TPA-treated cells. Four complexes were formed with extracts from control cells or cells treated with TPA for 1 day but were not observed with extracts from cells treated for 4 days. Of the 4 complexes, one was sequence-specific and binding involved uridylate residues and interactions with a 45-kDa protein and a protein doublet of 116 kDa. Binding of this 45/116-kDa complex to the 20-nt conserved cis element most likely regulates negatively PGHS-1 protein accumulation. We have provided evidence that the PGHS-1 gene is regulated at the translational level.
Duquette
Maryse
M
Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa K1H 8M5, Canada.
Laneuville
Odette
O
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D051545:Cyclooxygenase 1; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014542:Uridine Monophosphate / Q000378:metabolism
D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D014542:Uridine Monophosphate; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate