Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein

• Published in: The Journal of biological chemistry 1991-09, Vol.266 (25), p.16594-16598
• Main Authors: RONDON, I. J, MACMILLAN, L. A, BECKMAN, B. S, GOLDBERG, M. A, SCHNEIDER, T, BUNN, H. F, MALTER, J. S
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Binding, Competitive; Biological and medical sciences; Carcinoma, Hepatocellular; Carrier Proteins - metabolism; Cytosol - metabolism; Erythropoietin - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Humans; Hypoxia; Mice; mRNA-binding protein; Oxygen - metabolism; Proteins; Restriction Mapping; RNA Processing, Post-Transcriptional; RNA, Messenger - metabolism; RNA-Binding Proteins; Tumor Cells, Cultured; Up-Regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)55342-4

The mechanisms which control the production of erythropoietin (Epo) remain enigmatic. Recent data suggest that the half-time of Epo messenger RNA (mRNA) is increased by hypoxia in Hep 3B cells, a human hepatoma line. The post-transcriptional regulation of other rapidly degraded mRNAs is mediated by sequence-specific mRNA binding proteins. In order to determine if Epo mRNA specific binding proteins exist, we probed cytosolic lysates from Hep 3B cells and mouse tissues with radiolabeled Epo RNA. A cytosolic protein that binds specifically to Epo RNA was identified in the Epo-producing, hepatoblastoma Hep 3B cell line by gel mobility shift assay. This protein was identified in both normoxic and hypoxic cells and bound specifically to a 120-base fragment of the 3'-untranslated region (3'-UTR) of Epo mRNA. Binding was completed with unlabeled Epo RNA, but not with granulocyte-macrophage colony-stimulating factor RNA. Ultraviolet light cross-linked Epo RNA-protein complexes migrated as two bands of 70 and 135-140 kD on sodium dodecyl sulfate-polyacrylamide gels. Binding activity was markedly increased in brain and spleen lysates from mice subjected to 24 h of hypoxia. Therefore, the post-transcriptional regulation of Epo expression in response to hypoxia may in part be due to the interaction of Epo RNA with its specific binding protein.