Abstract
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 approximately 40% in response to a 6-h hypoxic pre-exposure and an additional approximately 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.
Highlights
Erythropoietin is a glycosylated protein that plays a central role in erythropoiesis by supporting the proliferation and differentiation of erythroid progenitor cells in the bone marrow [1]
We investigate the effect of erythropoietin mRNA-binding protein (ERBP) complex formation on Epo mRNA stability in normoxia and hypoxia
Deletion of the ERBP Binding Site—We have previously reported that a 120-nucleotide region in the 3ЈUTR of Epo mRNA is necessary for ERBP binding [17]
Summary
Erythropoietin; ERBP, erythropoietin mRNA-binding protein; bp, base pair(s); UTR, untranslated region; CAT, chloramphenicol acetyltransferase; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Bicine, N,N-bis(2-hydroxyethyl)glycine; VEGF, vascular endothelial growth factor; TH, tyrosine hydroxylase. A 3Ј enhancer element flanking the Epo gene as well as a 5Ј minimal sequence required for basal promoter activity have each been shown to confer a 4- to 14-fold induction of Epo gene transcription in response to hypoxia in Hep3B cells [11]. A mutated Epo gene with a deleted ERBP binding region was generated Both wild-type and mutant Epo constructs lack hypoxic responsive elements such that changes in mRNA stability in hypoxia would only reflect hypoxic modulation of ERBP complex formation. The mRNA half-lives, steady-state mRNA levels, and protein levels of Hep3B cells stably transfected with either the exogenous wild-type Epo gene or the mutant Epo gene and exposed to either normoxia or hypoxia were compared. The effect of ERBP complex formation on the mRNA stability of a heterologous gene, chloramphenicol acetyltransferase (CAT), was examined
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