Mechanisms directing oncoprotein and cytokine mRNA decay

指导癌蛋白和细胞因子 mRNA 衰减的机制

基本信息

批准号：
7622813
负责人：
Gerald M. Wilson
金额：
$ 36.28万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-07-01 至 2009-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7622813
关键词：
Adopted Affinity Binding Binding Proteins Binding Sites Biochemical Biological Biological Assay Biological Models C-terminal Cell physiology Cells Characteristics Chronic Complex Coupled Data Dimerization Disease Disruption Drug Design Elements Equilibrium Event Family Fibrinogen Fluorescence Funding Gene Chips Gene Expression Genes Human In Vitro Individual Inflammatory Kinetics Lead Learning Link Localized Malignant Neoplasms Mammals Mechanics Mediator of activation protein Messenger RNA Metabolic Metabolism MicroRNAs Modeling Molecular Monitor Neoplasms Numbers Oncogene Proteins Peptides Phosphorylation Population Positioning Attribute Post-Transcriptional Regulation Production Protein Binding Protein Isoforms Proteins RNA RNA Conformation RNA Folding RNA Recognition Motif RNA Sequences RNA Splicing RNA-Binding Proteins Range Rate Regulation Regulator Genes Relative (related person)Reporting Ribonucleoproteins Role Series Site Structure Surveys Syndrome System TIS11 protein Time Transcript Translations Tumor Necrosis Factor-alpha Tumor Necrosis Factors Untranslated Regions base cytokine high throughput screening human TNF protein insight mRNA Decay mutant novel size tumorigenesis

项目摘要

The stability and translation of many mRNAs encoding oncoproteins and cytokines are regulated by AU-rich elements (AREs), a diverse but evolutionarily conserved family of RNA sequences localized to their 3¿ untranslated regions. Disruption of ARE-directed regulatory mechanisms can contribute to oncogenesis and severe inflammatory syndromes. Our long-term objectives are to determine how the size and sequence diversity of AREs directs post-transcriptional regulation at the gene-specific level, and how gene-specific characteristics of AREs might ultimately be exploited as targets for novel therapies to treat some cancers and chronic inflammatory diseases. Our central hypothesis is that the metabolic fate of any ARE-containing mRNA is directed by the population of cellular trans-factors targeting each transcript; however, the biochemical basis for selecting one factor over another remains poorly defined. Recent findings indicate that some ARE-binding factors target distinct but overlapping mRNA subpopulations, and that local RNA secondary structure can influence trans-factor selectivity. Also, some factors can remodel local RNA structure or form oligomeric complexes on AREs. This project uses a series of biochemical and molecular biological strategies to define the roles of specific molecular determinants in the formation of stable, functional ribonucleoprotein (RNP) complexes on AREs. Using the ubiquitously expressed ARE-binding proteins AUF1 and HuR as model systems, we will first characterize specific protein subdomains contributing to ARE binding affinity and RNA-dependent protein oligomerization (Aim 1). Second, we will identify specific and non-specific RNA primary structural requirements for AUF1 and HuR binding, and assess the use of these sequences among the cellular mRNA subpopulation(s) interacting with these factors (Aim 2). Finally, we will determine how the energetics of local ARE structure direct the recruitment and positioning of AUF1 and HuR on RNA substrates (Aim 3). We anticipate that our approach will permit the mechanics of protein selectivity and binding to be evaluated in much greater detail than previously reported, largely through the use of steadystate and time-resolved fluorescence-based assay systems that we have adapted to study RNA-protein binding equilibria and RNA conformational events. Together, these studies will further our understanding of the relationships between ARE structure, trans-factor recognition, and the cellular functions of resulting RNP complexes.

许多编码癌蛋白和细胞因子的 mRNA 的稳定性和翻译受到以下因素的调节：富含 AU 的元件 (ARE)，一个多样化但进化上保守的 RNA 序列家族，定位于其 3¿ 非翻译区域的破坏可能导致肿瘤发生和我们的长期目标是确定严重炎症综合征的大小和顺序。 ARE 的多样性在基因特异性水平上指导转录后调控，以及基因特异性如何 ARE 的特性最终可能会被用作治疗某些癌症的新疗法的靶点我们的中心假设是任何含有 ARE 的物质的代谢命运。 mRNA 受针对每个转录物的细胞反式因子群的指导；最近的研究结果表明，选择一种因素而不是另一种因素的生化基础仍然不明确。一些 ARE 结合因子针对不同但重叠的 mRNA 亚群，并且局部 RNA 二级结构可以影响反式因子选择性，而且一些因素可以重塑局部 RNA 结构。或在 ARE 上形成寡聚复合物该项目使用了一系列生物化学和分子生物学。定义特定分子决定因素在稳定、功能性物质形成中的作用的策略使用普遍表达的 ARE 结合蛋白 AUF1 上的核糖核蛋白 (RNP) 复合物。和 HuR 作为模型系统，我们将首先表征有助于 ARE 结合的特定蛋白质子结构域其次，我们将识别特异性和非特异性。 AUF1 和 HuR 结合的 RNA 一级结构要求，并评估这些序列的使用最后，我们将确定与这些因素相互作用的细胞 mRNA 亚群。局部 ARE 结构的能量如何指导 AUF1 和 HuR 在 RNA 上的招募和定位我们预计我们的方法将允许蛋白质选择性和机制。结合力的评估比以前报道的要详细得多，主要是通过使用稳态以及基于时间分辨荧光的检测系统，我们已将其用于研究 RNA 蛋白质这些研究将进一步加深我们对结合平衡和 RNA 构象事件的理解。 ARE 结构、反式因子识别和所得 RNP 的细胞功能之间的关系复合物。