Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA.
Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed.
Czyzyk-Krzeska
M F
MF
Department of Physiology, University of North Carolina, Chapel Hill 27599.
Dominski
Z
Z
Kole
R
R
Millhorn
D E
DE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D016716:PC12 Cells; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis*
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014446:Tyrosine 3-Monooxygenase