Characterization of the hypoxia-inducible protein binding site within the pyrimidine-rich tract in the 3'-untranslated region of the tyrosine hydroxylase mRNA.
Reduced tension of O2 slows the degradation rate of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. The observed increase in half-life (30 h versus 10 h) correlates with enhanced binding of a 66-kDa protein (hypoxia inducible protein) to the pyrimidine-rich tract located between bases 1552 1578 in the 3 -untranslated region of TH mRNA (hypoxia-inducible protein binding site (HIPBS)). The present study investigates the protein binding site within the 27-base HIPBS, first by using specific cleavages of HIPBS and its flanking sequences with antisense oligodeoxynucleotides and RNase H and then by using mutational analysis of the binding properties. We found that the 27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia-stimulated manner. We further identified the optimal hypoxia-inducible protein binding site that is represented by the motif (U/C)(C/U)CCCU, where the core binding site is indicated by the underlined cytidines. Substitutions of either one of the cytidines with purine or uridine abolished the protein binding. The mutations within HIPBS, which partially reduced binding, did not prevent stimulation of protein binding for extracts from hypoxic cells. The hypoxia-induced increase in complex formation was proportional to the strength of binding using proteins from normoxic cells. The HIPBS element is conserved in TH mRNAs derived from different species.
Czyzyk-Krzeska
M F
MF
Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0576, USA.
Beresh
J E
JE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002395:Catecholamines / Q000096:biosynthesis; D015687:Cell Hypoxia; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016716:PC12 Cells; D014176:Protein Biosynthesis; D011743:Pyrimidines; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D015183:Restriction Mapping; D016914:Ribonuclease H; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D002395:Catecholamines; D016376:Oligonucleotides, Antisense; D011743:Pyrimidines; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014446:Tyrosine 3-Monooxygenase; D016914:Ribonuclease H