The long term objective of this project is to identify the key factors involved in post-transcriptional regulation of oxygen-sensing genes. Although the importance of post-transcriptional mechanisms in oxygen- sensing has been recognized for some time the actual key players have not been clearly defined. Post-transcriptional regulation of the oxygen-sensing genes erythropoietin (Epo), tyrosine hydroxylase (TH) and vascular endothelial growth factor (VEGF) has been shown to be mediated by cis-acting sequences in the 3' untranslated region of their mRNAs and by trans-acting factors binding to these sequences. Our studies suggest that there are common trans-acting factors binding the 3' UTR of Epo, TH and VEGF. These common factors are hypothesized to be responsible for controlled cellular responses to changes in oxygen tension. In this proposal, we plan to identify and functionally characterize these common trans-acting proteins and their 3' UTR binding sites. For this purpose, the erythropoietin gene used effectively as the model gene for oxygen- sensing transcriptional regulation, will be the focus of post- transcriptional studies in this proposal. With the purification and peptide sequencing of specific erythropoietin RNA binding proteins (30 and 70 kDa-ERBPs) considerable progress has been made towards this goal. Establishing that these are the proteins involved in post- transcriptional regulation of oxygen-sensing RNAs will be the focus of the in vitro studies proposed in the first aim.
For specific aim 1 a, these 30 and 70 kDa proteins will be added to an in vitro mRNA decay system to provide evidence for their stabilizing role.
In specific aim 1 b the in vitro mRNA decay assay will be used to map the sequences within the 3' UTR of Epo mRNA important in oxygen-sensing. The corresponding sequences within TH and VEGF mRNAs will be identified in specific aim 1c. In vivo correlations to the findings of specific aim 1 will be the focus of the second aim. First, antibodies to the 30 and 70 kDa proteins will be developed. The antibodies will then be used to learn more about the function of these common oxygen-sensing proteins by analyzing the distribution of these within the cell, in oxygen- sensing and non-sensing cells, in tissues and during development. Lastly, chimeric molecules made up of the 3' UTR sequences identified above an a non-oxygen sensing gene (e.g., GM-CSF) will be tested in vivo to support in vitro findings. Insights derived from these studies will have widespread application beyond oxygen-sensing mechanisms. A better understanding of post-transcriptional mechanisms will provide new targets for tumor therapy, provide improved methodology in problematic gene therapy strategies and in general, provide a more complete picture of cell biology. Further characterization of ERBP will provide a starting point in pursuit of these goals and will provide great insight into a genetic mechanism still largely unexplored.
|Scandurro, A B; Weldon, C W; Figueroa, Y G et al. (2001) Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells. Int J Oncol 19:129-35|