LEF-1 translation in chronic myelegenous leukemia

描述（由申请人提供）：提出了一项两年计划，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。该标记系统的开发将使用慢性髓性白血病进行，慢性髓性白血病是一种由癌基因BcrAbl驱动的癌性造血干细胞疾病。特别关注的是类磷脂增强因子-1 mRNA，一种介导Wnt信号传导的转录因子，是CML中的一个重要因子。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，这两个位点直接启动非帽依赖性翻译。目的是分析CML中LEF-1 mRNA/蛋白复合物，并鉴定对BcrAbl作用敏感的蛋白质。由于BcrAbl致癌作用的一个关键活性是错误调节的翻译，因此期望发现新的BcrAbl靶蛋白。该提议的第一个目的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且特异性缔合。将设计CML细胞中的表达系统，使得标记的RNA可以在生理或变性条件下快速分离。将进行阴性对照和原理验证实验，以确保LEF 1 mRNA复合物是真实的。第二个目标是改进使用同位素标记培养基（12 C6-赖氨酸/12 C6-精氨酸vs. 13 C6-赖氨酸/13 C6-精氨酸）的表达系统，使得基于SILAC的分析可用于定量质谱。除了本申请中LEF-1翻译的特定焦点之外，还将开发RNA标记方法，以便其可以应用于许多情况下不同RNP复合物的分析：正常细胞，癌症和其他异常，患病细胞状态。目标是开发方法，使其具有重现性，可靠性，并能够在细胞系，原代细胞和整个生物体中建立。将开发用于标记RNA和蛋白质的载体以供通用。

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.