Post-transcriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein

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 (Epo(M)) was generated. Hep3B cells were stably transfected with a wild- type Epo (Epo(WT)) cDNA or Epo(M) cDNA construct located downstream of a promoter of cytomegalovirus. Following inhibition of transcription, the haft- lives of Epo(WT) and Epo(M) mRNAs were 7 h and 2.5 h in normoxia, respectively. The Epo(M) mRNA half-life remained unchanged in hypoxia. Epo(WT) mRNA half-life increased ~40% in response to a 6-h hypoxic pre- exposure and an additional ~50% when preexposed to 12 h hypoxia. The steady- state level of Epo(WT) mRNA was 4-fold that of Epo(M) mRNA reflecting the difference in mRNA decay rates in normoxia. The Epo protein level expressed from exogenous Epo(M) was unchanged in both normoxia and hypoxia. In contrast, the Epo protein level expressed from exogenous Epo(WT) 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.