Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation |
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| Authors: | Claudia M. Casanova Peter Sehr Kerstin Putzker Matthias W. Hentze Beate Neumann Kent E. Duncan Christian Thoma |
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| Affiliation: | 1. Department of Medicine II, University Hospital of Freiburg, Freiburg, Germany.; 2. EMBL, Heidelberg, Germany.; 3. Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.; Pohang University of Science and Technology, Republic of Korea, |
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| Abstract: | Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation. |
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