Specific protein binding to a conserved region of the ornithine decarboxylase mRNA 5'-untranslated region.
An RNA gel retardation assay was used to identify one or more cellular protein(s) (ornithine decarboxylase mRNA 5'-UTR binding protein (ODCBP)) that bind specifically to a conserved region of the 5'-untranslated region (5'-UTR) of rat ornithine decarboxylase (ODC) mRNA. Ultraviolet light cross-linking demonstrated that this protein has an apparent Mr = 58,000 in mammalian cells. Treatment with the oxidizing agent diamide prevented binding of the ODCBP to ODC mRNA; addition of beta-mercaptoethanol reversed this inhibition and permitted mRNA.ODCBP complex formation. Cytoplasmic extracts from a variety of animal cells and tissues demonstrated similar binding activities; however, there was marked tissue-specific expression of the protein in the rat, with brain, heart, lung, and testis containing large amounts, and kidney, spleen, and skeletal muscle expressing negligible amounts. Binding was completely prevented by several mutations within a highly conserved heptanucleotide region (CCAU/ACUC) that was within 61 bases of the initiation codon in ODC mRNAs from mammals, Xenopus, and Caenorhabditis elegans; mutations 5' and 3' of the conserved heptanucleotide domain had no effect on binding activity. Binding was not affected by manipulation of cellular polyamine levels or by treatment of cells with agents that stimulate ODC biosynthesis. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 5'-UTR of ODC mRNA from many animal species; functional consequences of this binding remain to be determined.
Manzella
J M
JM
Howard Hughes Medical Institute Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Blackshear
P J
PJ
D000818:Animals; D001483:Base Sequence; D002107:Caenorhabditis / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics* / Q000528:radiation effects; D051381:Rats; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014981:Xenopus
D002352:Carrier Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D009955:Ornithine Decarboxylase