Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA

M.F Czyzyk-Krzeska,Z Dominski,R Kole,D.E Millhorn

doi:10.1016/s0021-9258(17)36973-9

M.F Czyzyk-Krzeska, Z Dominski + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)36973-9

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Abstract

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.

Highlights

R‘educed oxygen tension induces a 3-foilnd- found that TH gene expression is enhanced by hypoxia in phecrease in stability of mRNA for tyrosine hydroxylase ochromocytoma
The turnover of mRNAs is regulated by interactions among trans-acting factors and specific sequences located mainly within the 3”untranslated regions (3’-UTR)of some mRNAs.In recent yearsa number of unique cis-acting elements, such as the iron-responsive digestion.This fragmentof the 3’-untranslated regionof elements [11] and adenosine/uridine(AU)-rich sequences (12, THmRNA is rich in pyrimidine nucleotides, and the 13), have been shown to bind cytosplamic proteins
We reported recentlythat TH gene expression is stimulated by hypoxiain the 0,sensitive

Summary

RESULTS

RNA gel retardation assays were performed to detertmheine tion, the template DNA was digested with RNase-free DNase RNase T1 Mapping-The binding reaction between the 162-b fragment of the 3'-UTR of TH mRNA and cytoplasmic proteins was performed as described above. This difference was approximately 2.5-fold and was

TH mRNA

There is evidence that these proteins alorecated in thenucleus

DISCUSSION