LEF-1 translation in chronic myelegenous leukemia

LEF-1 在慢性粒细胞白血病中的翻译

• 批准号: 7692847
• 负责人: Marian L Waterman
• 金额: $ 19.67万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-10 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7692847
• 关键词: Acids; Affinity; Affinity Chromatography; Algorithms; Amino Acids; Arginine; Binding; Binding Proteins; CD34 gene; Cancerous; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Chronic; Chronic Myeloid Leukemia; Complex; Cultured Cells; Cytolysis; Cytoplasm; Databases; Development; Disease; Elements; Epitopes; Family; Gene Expression; Goals; Growth; Health; Hela Cells; Hematopoietic stem cells; Human; Imatinib; Internal Ribosome Entry Site; Isotope Labeling; Joints; K-562; Length; Letters; Light; Link; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methods; Normal Cell; Oncogenes; Oncogenic; Pattern; Peptides; Phenotype; Physiological; Plant Resins; Plasmids; Protein Binding; Proteins; RNA; RNA Polymerase II; RNA analysis; RNA-Binding Proteins; Regulation; Regulatory Element; Relative (related person); Reporter; Research Personnel; Sampling; Series; Signal Transduction; Stable Isotope Labeling; System; TCF Transcription Factor; Techniques; Technology; Translation Initiation; Translations; Trypsin; Untranslated Regions; Validation; Whole Organism; arginyllysine; base; c-Myc Staining Method; cancer therapy; crosslink; discount; expectation; follow-up; in vivo; interest; leukemia; lymphoid enhancer-binding factor 1; mRNA tagging; member; method development; novel; protein complex; research study; retroviral transduction; self-renewal; stem; therapeutic development; transcription factor; vector

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.

描述（由申请人提供）：提出了为期两年的计划，将新标记系统应用于癌症特定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。该标记系统的发展将使用慢性粒细胞性白血病，这是一种由癌基因BCRABL驱动的癌造血干细胞疾病。特定的重点是淋巴增强子1 mRNA，这是一种介导Wnt信号传导的转录因子，是CML的重要因素。我们最近将BCRABL作用与CML细胞中的LEF-1翻译联系起来，这是一种新的观察结果，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，这些内部核糖体入口位点直接无关cap独立于翻译的启动。目的是分析CML中的LEF-1 mRNA/蛋白质复合物，并鉴定对BCRABL作用敏感的蛋白质。由于BCRABL致癌作用的关键活性是不正当调节的翻译，因此期望将揭示新的BCRABL靶蛋白。该提案的第一个目的是创建一系列由STEM循环标记的LEF-1 mRNA，以与表位特异性矩阵和树脂有很强的特定关联。将设计CML细胞中的表达系统，以便可以在生理或变性条件下快速分离标记的RNA。负面对照和原则实验将进行，以确保LEF1 mRNA复合物是真实的。第二个目的将完善用于使用同位素标记介质（12C6-赖氨酸/12C6-精氨酸与13C6-赖氨酸/13C6-精氨酸）的表达系统，因此可以将基于SILAC的分析用于定量质谱法。除了在本应用中LEF-1翻译的特定焦点之外，还将开发RNA标记方法，以便在许多情况下将其应用于对不同RNP复合物的分析：正常细胞，癌症和其他异常，患病的细胞状态。目的是开发使其可重现，可靠并能够在细胞系，原代细胞和整个生物体中建立的方法。将开发用于标记RNA和蛋白质的向量供一般使用。