LEF-1 translation in chronic myelegenous leukemia

描述（由申请人提供）：提出一项为期两年的计划，将新的标记系统应用于癌症中特定rna的分析。这项新的应用技术将使RNA/蛋白质复合物的快速分离和定量质谱分析成为可能。这种标记系统的开发将使用慢性骨髓性白血病（一种由癌基因BcrAbl驱动的恶性造血干细胞疾病）进行。具体的重点是淋巴细胞增强因子-1 mRNA，这是一种介导Wnt信号的转录因子，是CML的重要因子。我们最近将BcrAbl作用与CML细胞中的LEF-1翻译联系起来，这是一个新的观察结果，因为LEF-1蛋白是由两个内部核糖体进入位点的作用产生的，这些位点直接引导帽独立的翻译起始。目的是分析CML中LEF-1 mRNA/蛋白复合物，并鉴定对BcrAbl作用敏感的蛋白。由于BcrAbl致癌作用的一个关键活性是错误调节的翻译，因此期望新的BcrAbl靶蛋白将被揭示。本提案的第一个目标是创建一系列由茎环标记的LEF-1 mrna，用于与表位特异性基质和树脂的强特异性关联。CML细胞中的表达系统将被设计成这样，标记的RNA可以在生理或变性条件下快速分离。阴性对照和原理验证实验将进行，以确保LEF1 mRNA复合物是真实的。第二个目标将改进使用同位素标记介质的表达系统（12c6 -赖氨酸/ 12c6 -精氨酸vs. 13c6 -赖氨酸/ 13c6 -精氨酸），这样基于silac的分析可以用于定量质谱分析。除了在本应用程序中对LEF-1翻译的特定关注之外，rna标记方法将被开发，以便它可以应用于在许多情况下分析不同的RNP复合物：正常细胞，癌症和其他异常，病变细胞状态。目标是开发一种方法，使其可重复、可靠，并能够在细胞系、原代细胞和整个生物体中建立。用于标记RNA和蛋白质的载体将被开发用于一般用途。

DESCRIPTION (provided by applicant): 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.