RNA Dynamics of Transient Macromolecular Complexes in Cancer Cells

癌细胞中瞬时大分子复合物的 RNA 动力学

基本信息

批准号：
8701882
负责人：
JACK D KEENE
金额：
$ 31.6万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8701882
关键词：
Address Affect Animal Cancer Model Apoptosis Inhibitor Binding Binding Sites Biochemical Biology Cancerous Cell Cycle Proteins Cells ChIP-seq Complex Cytoplasm Cytoplasmic Granules Data Development Disease ERG gene Embryo Epithelial Cells Event Gene Expression Genes Genome Goals Growth Heat shock proteins Hereditary Breast Carcinoma Human Hypoxia Imagery Immune Immunoprecipitation Intervention Laboratories Link Macromolecular Complexes Malignant - descriptor Malignant Epithelial Cell Malignant Neoplasms Mammalian Cell Maps Messenger RNA Methods MicroRNAs Microscopic Mitogens Modeling Molecular Oncogenic Oxidative Stress Oxidative Stress Induction Pathway interactions Pharmaceutical Preparations Phenotype Phorbol Esters Phosphorylation Population Population Dynamics Precancerous Conditions Precipitation Procedures Process Prognostic Factor Property Protein Binding Protein Isoforms Proteins Proteomics Proto-Oncogenes Publications RNA RNA Binding RNA-Binding Proteins Regulation Research Ribonucleoproteins Ribonucleosides Role Sampling Site Specificity Staging Statistical Models Stress System T-Lymphocyte Target Populations Technology Telomerase Testing Tretinoin Ultraviolet Rays Work cancer cell carcinogenesis cell immortalization cell transformation crosslink drug candidate functional outcomes genome-wide immortalized cell innovation leukemia novel prevent research study response small molecule stress protein tumor progression

项目摘要

DESCRIPTION (provided by applicant): In this proposal, we seek to understand RNA dynamics in transient cellular complexes and to use that information to identify candidate drugs that may modulate the pathways to malignancy. While protein components of RNP granules have been studied in mammalian cells, quantitative RNA population dynamics have not been analyzed for lack of suitable technologies. Our laboratory focuses on multi-targeting of populations of mRNAs regulated by RNA-binding proteins (RBPs) and microRNAs. We have demonstrated that sequence specific RBPs coordinately regulate groups of functionally related mRNAs and these RNPs are remodeled during activation of cells with small molecule drugs. For example, the mRNAs associated with several RBPs have been shown to change coordinately following treatment of embryonic carcinoma cells with retinoic acid or leukemia-derived immune T cells with phorbol esters plus mitogens. We hypothesize that dynamic changes in RNA populations within these transient RNP complexes coordinate functionally-related subsets of mRNAs that encode proteins whose synchronized expression is required for oncogenic transformation. Here, we will use a probabilistic approach that quantifies dynamic RNA changes en masse to analyze mRNAs associated with RBPs during progression to malignancy. We will examine the transition from a precancerous state to a cancerous state in primary epithelial cells using methods pioneered in the laboratory of Robert Weinberg with RAS, telomerase and four other transforming proteins quantify dynamic changes in RNAs associated with HuR (early response gene mRNAs), TIAR (stress granule RNAs) and AGO2/RISC (processing "P" body RNAs). HuR shift to the cytoplasm alters its mRNA targeting and has been claimed as a prognostic factor in hereditary breast cancer. We will quantify the levels of mRNAs and microRNAs in these complexes and determine how they change in response to progressive development of a transformed phenotype, as well as after inducing oxidative stress and hypoxia. The RNP-Immunoprecipitation microarray (RIP-Chip) procedure will be used to identify and quantify mRNAs associated with specific RBPs; an ultraviolet light crosslinking procedure with high specificity and efficiency termed PAR-CLIP recently developed in the laboratory of our collaborator, Thomas Tuschl, will be used to identify the precise binding sites of these RBPs and microRNAs. Dynamic changes in sites of microRNA binding to mRNAs will be globally integrated with RBP binding sites. We will construct a quantitative dynamic model of these events and use these data to query the drug-genome Connectivity Map for drugs that affect these processes as demonstrated in our recent publications. These compounds will be used to further investigate the underlying biology of carcinogenesis in this system. Microscopic visualization at each stage of progression will be used to confirm the RNA/RBP localization and phenotypic effects of drug treatments. It is our long-term plan to use this quantitative probabilistic approach of RNA targeting to investigate these and other transient macromolecular RNP complexes involved in posttranscriptional gene expression using animal models of cancer.

描述（由申请人提供）：在此提案中，我们试图了解瞬时细胞复合物中的RNA动力学，并使用该信息来识别可能调节恶性途径的候选药物。虽然已经在哺乳动物细胞中研究了RNP颗粒的蛋白质成分，但由于缺乏合适的技术，尚未分析定量RNA种群动力学。我们的实验室着重于由RNA结合蛋白（RBP）和microRNA调节的mRNA种群的多目标。我们已经证明了序列特定的RBP协同调节功能相关的mRNA组，并且在用小分子药物激活细胞期间，对这些RNP进行了重塑。例如，在用视黄酸或白血病衍生的免疫T细胞和凤凰酯加有丝分裂剂治疗胚胎癌细胞后，已证明与几个RBP相关的mRNA被证明会改变协调一致。我们假设这些瞬时RNP复合物中RNA群体的动态变化坐标了与蛋白质相关的MRNA的子集，该子集编码了蛋白质的蛋白质，其肿瘤转化需要其同步表达。在这里，我们将使用一种概率方法来量化动态RNA的变化，以分析与恶性肿瘤发展过程中与RBP相关的mRNA。 We will examine the transition from a precancerous state to a cancerous state in primary epithelial cells using methods pioneered in the laboratory of Robert Weinberg with RAS, telomerase and four other transforming proteins quantify dynamic changes in RNAs associated with HuR (early response gene mRNAs), TIAR (stress granule RNAs) and AGO2/RISC (processing "P" body RNAs). HUR向细胞质的转移会改变其mRNA靶向，并被称为遗传性乳腺癌的预后因素。我们将量化这些复合物中的mRNA和microRNA的水平，并确定它们如何在转化表型的逐步发展以及诱导氧化应激和缺氧后的响应中变化。 RNP免疫沉淀微阵列（RIP-CHIP）程序将用于识别和量化与特定RBP相关的mRNA；最近在我们的合作者Thomas Tuschl实验室开发的具有高特异性和效率的紫外线交联过程将用于识别这些RBP和MicroRNA的精确结合位点。 MicroRNA结合与mRNA的位点的动态变化将与RBP结合位点全局整合。我们将构建这些事件的定量动态模型，并使用这些数据来查询药物基因组连通性图，以影响我们最近出版物所证明的影响这些过程的药物。这些化合物将用于进一步研究该系统中癌变的潜在生物学。每个进展阶段的微观可视化将用于确认药物处理的RNA/RBP定位和表型效应。我们的长期计划是使用RNA靶向的这种定量概率方法研究使用癌症动物模型参与转录后基因表达的这些和其他瞬时大分子RNP复合物。