Translation of the human erythropoietin transcript is regulated by an upstream open reading frame in response to hypoxia

Erythropoietin (EPO) is a key mediator hormone for hypoxic induction of erythropoiesis that also plays important nonhematopoietic functions. It has been shown that EPO gene expression regulation occurs at different levels, including transcription and mRNA stabilization. In this report, we show that expression of EPO is also regulated at the translational level by an upstream open reading frame (uORF) of 14 codons. As judged by comparisons of protein and mRNA levels, the uORF acts as a cis-acting element that represses translation of the main EPO ORF in unstressed HEK293, HepG2, and HeLa cells. However, in response to hypoxia, this repression is significantly released, specifically in HeLa cells, through a mechanism that involves processive scanning of ribosomes from the 5′ end of the EPO transcript and enhanced ribosome bypass of the uORF. In addition, we demonstrate that in HeLa cells, hypoxia induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) concomitantly with a significant increase of EPO protein synthesis. These findings provide a framework for understanding that production of high levels of EPO induced by hypoxia also involves regulation at the translational level.