LEF-1 translation in chronic myelegenous leukemia

描述(由申请人提供)：一项为期两年的计划被提出，将一种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/蛋白质复合体的快速分离和定量质谱分析成为可能。这个标签系统的开发将使用慢性髓系白血病，这是一种由癌基因BcrAbl驱动的癌症造血干细胞疾病。特别关注的是淋巴增强因子-1mRNA，一种介导Wnt信号的转录因子，是慢性粒细胞白血病的关键因子。我们最近将BcrAbl的作用与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，这两个核糖体进入位点直接启动了帽非依赖性的翻译。我们的目标是分析CML中的Lef-1mRNA/蛋白质复合体，并识别对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控的翻译，因此预计新的BcrAbl靶蛋白将被揭示。这项提议的第一个目标是创建一系列由茎环标记的Lef-1 mRNAs，用于与表位特定的基质和树脂进行强烈和特定的关联。CML细胞中的表达系统将被设计成能够在生理或变性条件下快速分离标记的RNA。将进行阴性对照和原则性实验，以确保LEF1 mRNA复合体的真实性。第二个目标是改进用于同位素标记介质(12C6-赖氨酸/12C6-精氨酸与13C6-赖氨酸/13C6-精氨酸)的表达系统，以便基于SILAC的分析可用于定量质谱分析。除了本申请中Lef-1翻译的特定重点之外，RNA标签方法将被开发出来，以便它可以应用于许多背景下的不同RNP复合体的分析：正常细胞、癌症和其他异常、疾病的细胞状态。我们的目标是开发这种方法，使其具有重复性、可靠性，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为通用载体。

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.