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

Isaac J. Rondon, Lee Ann MacMillan, Barbara S. Beckman, Mark A. Goldberg, Thomas Schneider, H. Franklin Bunn, James S. Malter

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Abstract

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 competed 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.

Original languageEnglish (US)
Pages (from-to)16594-16598
Number of pages5
JournalJournal of Biological Chemistry
Volume266
Issue number25
Publication statusPublished - 1991

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Rondon, I. J., MacMillan, L. A., Beckman, B. S., Goldberg, M. A., Schneider, T., Franklin Bunn, H., & Malter, J. S. (1991). Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein. Journal of Biological Chemistry, 266(25), 16594-16598.