POSTTRANSCRIPTIONAL REGULATION OF ERYTHROPOIETIN MESSENGER-RNA STABILITY BY ERYTHROPOIETIN MESSENGER-RNA-BINDING PROTEIN

We have previously identified a sequence in the 3'-untranslated region (3'-UTR) of erythropoietin (Epo) mRNA which binds a protein(s), eryth ropoietin mRNA-binding protein (ERBP). A mutant lacking the ERBP bindi ng site (Epo(M)) was generated. Hep3B cells were stably transfected wi th a wild-type Epo (Epo(WT)) cDNA or Epo(M) cDNA construct located dow nstream of a promoter of cytomegalovirus. Following inhibition of tran scription, the half-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 uncha nged in hypoxia, Epo(WT) mRNA half-life increased similar to 40% in re sponse to a 6-h hypoxic pre-exposure and an additional similar to 50% when preexposed to 12 h hypoxia, The steady-state level of Epo(WT) mRN A was 4-fold that of Epo(M) mRNA reflecting the difference in mRNA dec ay rates in normoxia, The Epo protein level expressed from exogenous E po(M) was unchanged in both normoxia and hypoxia, In contrast, the Epo protein level expressed from exogenous Epo(WT) increased 50% in hypox ia when compared with normoxia. These observations were further suppor ted by chimeric chloramphenicol acetyltransferase and Epo-3'-UTR const ructs. We have demonstrated that Epo mRNA stability was modulated in n ormoxia and further by hypoxia, therefore, providing evidence that Epo is regulated at the post-transcriptional level through ERBP complex f ormation.