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 (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.