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Molecular & Cellular Analysis Technologies
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A two year plan is proposed to apply a new tagging system to the analysis of specific RNAs in cancer. This new applied technology will enable rapid isolation and quantitative mass spectrometry analysis of RNA/protein complexes. The development of this tagging system will be performed using Chronic Myelogenous Leukemia, a cancerous hematopoietic stem cell disorder driven by the oncogene BcrAbl. The specific focus is on Lymphoid Enhancer Factor-1 mRNA, a transcription factor that mediates Wnt signaling and is an essential factor in CML. We have recently linked BcrAbl action to LEF-1 translation in CML cells, a novel observation since LEF-1 protein is produced by the actions of two internal ribosome entry sites that direct cap-independent initiation of translation. The goal is to analyze LEF-1 mRNA/protein complexes in CML and identify proteins that are sensitive to BcrAbl action. Since a key activity of BcrAbl oncogenic action is misregulated translation, the expectation is that new BcrAbl target proteins will be revealed. The first aim of this proposal is to create a series of LEF-1 mRNAs tagged by stem loops for strong and specific association to epitope-specific matrices and resins. Expression systems in CML cells will be devised such that the tagged RNA can be rapidly isolated either under physiological or denaturing conditions. Negative controls and proof-of-principle experiments will be carried out to make sure that the LEF1 mRNA complexes are authentic. The second aim will refine the expression system for the use of isotope-labeling media (12C6-lysine/12C6-arginine vs. 13C6-lysine/13C6-arginine), such that SILAC-based analyses can be used for quantitative mass spectrometry. Beyond the specific focus of LEF-1 translation in this application, the RNA-tagging method will be developed so that it can be applied to the analysis of different RNP complexes in many contexts: normal cells, cancer, and other aberrant, diseased cell states. The goal is to develop the method to make it reproducible, reliable and able to be established in cell lines, primary cells and whole organisms. Vectors for tagging RNA and proteins will be developed for general use.