Translational regulation of PGHS-1 mRNA: 5' untranslated region and first two exons conferring negative regulation.
Prostaglandin endoperoxide H synthase-1 gene expression is described as inducible in a few contexts such as differentiation of megakaryoblastic MEG-01 cells into platelet-like structures. In the MEG-01 cells model of PGHS-1 gene induction, we previously reported a delay in protein synthesis and identified the translational step of gene expression as being regulated. In the current study, we mapped PGHS-1 mRNA sequences regulating translational efficiency and identified an RNA binding protein. The 5'UTR and first two exons of the PGHS-1 5' mRNA decreased the synthesis of Luciferase protein by approximately 80% without significant changes in mRNA levels when compared to controls. Both the PGHS-1 5'-UTR and the first two exons were required for activity. Sucrose density gradient fractionations of cytoplasmic extracts from MEG-01 cells infected with reporter constructs, either controls or containing PGHS-1 sequence, presented a similar profile of distribution of reporter transcripts between polysomal and non-polysomal fractions. RNA/protein interaction studies revealed nucleolin binding to the 135 nt PGHS-1 sequence. Mutation of the two NRE elements located in the 5'end of PGHS-1 mRNA sequence partially reduced the negative activity of the 135 nt sequence. Stable secondary structures predicted at the 5' end of the transcript are potentially involved in translational regulation. We propose that the 5'end of PGHS-1 mRNA represses translation and could delay the synthesis of PGHS-1 enzyme.
Bunimov
Natalia
N
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5.
Smith
Jennifer Erin
JE
Gosselin
Dominique
D
Laneuville
Odette
O
D020121:5' Untranslated Regions / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D051545:Cyclooxygenase 1 / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D008533:Megakaryocytes / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D015533:Transcriptional Activation
D020121:5' Untranslated Regions; D008565:Membrane Proteins; D012333:RNA, Messenger; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human