Common proteins bind mRNAs encoding erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor.
The hypoxia-inducible genes erythropoietin (Epo), tyrosine hydroxylase (TH), and vascular endothelial growth factor (VEGF) are regulated post-transcriptionally by proteins binding to specific regions located in the 3' untranslated region (UTR) of their mRNAs. To determine whether trans-factors binding to this region in all three of these RNAs are similar, we generated riboprobes containing the 3' UTR of erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor mRNA and assayed them by electrophoretic mobility shift assay (EMSA) and UV cross-linking experiments. Each riboprobe formed similar shifted protein complexes using human hepatoma cell (Hep3B) cytoplasmic lysates in the EMSA. Hep3B proteins bound to each probe could be cross-competed by the specific unlabeled Epo, TH, or VEGF riboprobes. By contrast, a non-specific 3' UTR riboprobe did not compete for binding with the Epo, TH, or VEGF RNA shifted protein complexes. UV cross-linking studies revealed proteins of similar molecular weights for the Epo, TH, and VEGF RNA shifted protein complexes. Taken together, these results suggest a common posttranscriptional regulatory mechanism for hypoxia-inducible genes.
Scandurro
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AB
Department of Pharmacology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA. alibscan@mailhost.tcs.tulane.edu
Beckman
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BS
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000235:genetics; D016228:Endothelial Growth Factors / Q000235:genetics*; D004921:Erythropoietin / Q000235:genetics*; D006801:Humans; D008222:Lymphokines / Q000235:genetics*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D015347:RNA Probes / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*; D014466:Ultraviolet Rays; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D003432:Cross-Linking Reagents; D017931:DNA Primers; D016228:Endothelial Growth Factors; D008222:Lymphokines; D015347:RNA Probes; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D004921:Erythropoietin; D014446:Tyrosine 3-Monooxygenase