Post-transcriptional Regulation of Erythropoietin mRNA Stability by Erythropoietin mRNA-binding Protein

We have previously identified a sequence in the 3′-untranslated region (3′-UTR) of erythropoietin (Epo) mRNA which binds a protein(s), erythropoietin mRNA-binding protein (ERBP). A mutant lacking the ERBP binding site (EpoM) was generated. Hep3B cells were stably transfected with a wild-type Epo (Ep...

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Bibliographic Details
Published in:The Journal of biological chemistry 1997-03, Vol.272 (13), p.8628-8634
Main Authors: McGary, Eric C., Rondon, Isaac J., Beckman, Barbara S.
Format: Article
Language:English
Subjects:
Binding Sites
Chloramphenicol O-Acetyltransferase - genetics
Dactinomycin - pharmacology
Erythropoietin - genetics
Genes, Reporter
Half-Life
Humans
Nucleic Acid Synthesis Inhibitors - pharmacology
Oxygen - metabolism
RNA Processing, Post-Transcriptional - drug effects
RNA, Messenger - chemistry
RNA, Messenger - metabolism
RNA-Binding Proteins - pharmacology
Transfection
Tumor Cells, Cultured
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Summary:We have previously identified a sequence in the 3′-untranslated region (3′-UTR) of erythropoietin (Epo) mRNA which binds a protein(s), erythropoietin mRNA-binding protein (ERBP). A mutant lacking the ERBP binding site (EpoM) was generated. Hep3B cells were stably transfected with a wild-type Epo (EpoWT) cDNA or EpoM cDNA construct located downstream of a promoter of cytomegalovirus. Following inhibition of transcription, the half-lives of EpoWT and EpoM mRNAs were 7 h and 2.5 h in normoxia, respectively. The EpoM mRNA half-life remained unchanged in hypoxia. EpoWT mRNA half-life increased ~40% in response to a 6-h hypoxic pre-exposure and an additional ~50% when pre-exposed to 12 h hypoxia. The steady-state level of EpoWT mRNA was 4-fold that of EpoM mRNA reflecting the difference in mRNA decay rates in normoxia. The Epo protein level expressed from exogenous EpoM was unchanged in both normoxia and hypoxia. In contrast, the Epo protein level expressed from exogenous EpoWT increased 50% in hypoxia when compared with normoxia. These observations were further supported by chimeric chloramphenicol acetyltransferase and Epo-3′-UTR constructs. We have demonstrated that Epo mRNA stability was modulated in normoxia and further by hypoxia, therefore, providing evidence that Epo is regulated at the post-transcriptional level through ERBP complex formation.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.272.13.8628