Mechanisms directing oncoprotein and cytokine mRNA decay

指导癌蛋白和细胞因子 mRNA 衰减的机制

• 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
• 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

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（战神），一个多样化但进化上保守的RNA序列家族，定位于其3 ′端。 未翻译的区域ARE指导的调节机制的破坏可有助于肿瘤发生， 严重的炎症综合征。我们的长期目标是确定 战神的多样性在基因特异性水平指导转录后调节，以及基因特异性如何影响转录后调节。 战神的特性可能最终被开发为治疗某些癌症的新疗法的靶点， 慢性炎症性疾病。我们的中心假设是，任何含有ARE的代谢命运 mRNA由靶向每个转录物的细胞反式因子群体指导;然而， 选择一个因素而不是另一个因素的生物化学基础仍然不清楚。最近的研究结果表明， 一些ARE结合因子靶向不同但重叠mRNA亚群，且局部RNA 二级结构可影响反式因子选择性。此外，一些因素可以重塑局部RNA结构 或在战神上形成低聚复合物。该项目采用一系列生物化学和分子生物学方法， 战略，以确定在形成稳定的，功能性的特定分子决定因素的作用， 核糖核蛋白（RNP）复合物。利用广泛表达的ARE结合蛋白AUF 1 和HuR作为模型系统，我们将首先表征有助于ARE结合的特定蛋白质亚结构域 亲和力和RNA依赖性蛋白质寡聚化（Aim 1）。其次，我们将确定具体和非具体 AUF 1和HuR结合的RNA一级结构要求，并评估这些序列的使用 在与这些因子相互作用的细胞mRNA亚群中（目的2）。最后，我们将确定 局部ARE结构的能量学如何指导AUF 1和HuR在RNA上的募集和定位 底物（目标3）。我们预计，我们的方法将允许蛋白质选择性的机制， 结合进行评估，在更大程度上比以前报告的细节，主要是通过使用稳态 和基于时间分辨荧光的分析系统，我们已经适应了研究RNA-蛋白质 结合平衡和RNA构象事件。总之，这些研究将进一步加深我们对 ARE结构、反式因子识别与RNP细胞功能之间的关系 配合物