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

• 批准号: 8504813
• 负责人: JACK D KEENE
• 金额: $ 30.62万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 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/8504813
• 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

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）：在本提案中，我们寻求了解瞬时细胞复合物中的 RNA 动态，并利用该信息来识别可能调节恶性肿瘤途径的候选药物。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，尚未对定量 RNA 群体动态进行分析。我们的实验室专注于由 RNA 结合蛋白 (RBP) 和 microRNA 调控的 mRNA 群体的多靶点。我们已经证明，序列特异性 RBP 协调调节功能相关 mRNA 组，并且这些 RNP 在小分子药物激活细胞过程中被重塑。例如，用视黄酸处理胚胎癌细胞或用佛波酯加促细胞分裂剂处理白血病衍生的免疫 T 细胞后，与几种 RBP 相关的 mRNA 已被证明会发生协调变化。我们假设这些瞬时 RNP 复合物内 RNA 群体的动态变化协调功能相关的 mRNA 子集，这些子集编码致癌转化所需同步表达的蛋白质。在这里，我们将使用一种概率方法来量化动态 RNA 的整体变化，以分析在恶性肿瘤进展过程中与 RBP 相关的 mRNA。我们将使用 Robert Weinberg 实验室首创的方法，利用 RAS、端粒酶和其他四种转化蛋白来检测原代上皮细胞从癌前状态到癌性状态的转变，量化与 HuR（早期反应基因 mRNA）、TIAR（应激颗粒 RNA）和 AGO2/RISC（处理“P”体 RNA）相关的 RNA 的动态变化。 HuR 向细胞质的转移改变了其 mRNA 靶向，并被认为是遗传性乳腺癌的预后因素。我们将量化这些复合物中 mRNA 和 microRNA 的水平，并确定它们如何响应转化表型的逐渐发展以及诱导氧化应激和缺氧后发生变化。 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；我们的合作者 Thomas Tuschl 的实验室最近开发了一种具有高特异性和效率的紫外光交联程序，称为 PAR-CLIP，将用于识别这些 RBP 和 microRNA 的精确结合位点。 microRNA 与 mRNA 结合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接图，以查找影响这些过程的药物，如我们最近的出版物所示。这些化合物将用于进一步研究该系统中致癌作用的基础生物学。进展每个阶段的显微可视化将用于确认 RNA/RBP 定位和药物治疗的表型效应。我们的长期计划是利用这种 RNA 靶向的定量概率方法，利用癌症动物模型来研究这些和其他参与转录后基因表达的瞬时大分子 RNP 复合物。