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 (ARE)，一个多样化但进化上保守的 RNA 序列家族，定位于其 3¿ 未翻译的区域。 ARE 调控机制的破坏可能导致肿瘤发生和 严重的炎症综合征。我们的长期目标是确定大小和顺序 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 慢性炎症性疾病。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；然而， 选择一种因素而不是另一种因素的生化基础仍然不明确。最近的研究结果表明 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。此外，一些因素可以重塑局部 RNA 结构 或在 ARE 上形成寡聚复合物。该项目采用了一系列生化和分子生物学技术 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 ARE 上的核糖核蛋白 (RNP) 复合物。使用普遍表达的 ARE 结合蛋白 AUF1 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 亲和力和 RNA 依赖性蛋白质寡聚化（目标 1）。其次，我们要区分具体的和非具体的 AUF1 和 HuR 结合的 RNA 一级结构要求，并评估这些序列的使用 与这些因子相互作用的细胞 mRNA 亚群（目标 2）。最后，我们将确定 局部 ARE 结构的能量如何指导 AUF1 和 HuR 在 RNA 上的招募和定位 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和 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 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、反式因子识别和所得 RNP 的细胞功能之间的关系 复合物。