Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein.
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.
Rondon
I J
IJ
Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana 70112.
MacMillan
L A
LA
Beckman
B S
BS
Goldberg
M A
MA
Schneider
T
T
Bunn
H F
HF
Malter
J S
JS
D000818:Animals; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004921:Erythropoietin / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D000860:Hypoxia; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D002352:Carrier Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D004921:Erythropoietin; D010100:Oxygen