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: 8063923
• 负责人: Gerald M. Wilson
• 金额: $ 25.8万
• 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 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063923
• 关键词: 3' Untranslated Regions; Adopted; Affinity; Beryllium; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; C-terminal; Cell Differentiation process; Cell Proliferation; Cell physiology; Cells; Characteristics; Chronic; Clinical; Competitive Binding; Complex; Coupled; Data; Dimerization; Disease; Drug Design; Elements; Equilibrium; Event; Family; Fluorescence; Funding; Gene Chips; Gene Expression; Genes; Genetic Translation; Health; Human; In Vitro; Individual; Inflammation Mediators; Inflammatory; Kinetics; Lead; Learning; Link; Malignant Neoplasms; Mammals; Mechanics; Messenger RNA; Metabolic; Metabolism; MicroRNAs; Modeling; Molecular; Monitor; Neoplasms; Oncogene Proteins; Peptides; Phosphorylation; Population; Positioning Attribute; Post-Transcriptional Regulation; Production; Protein Binding; Protein Isoforms; Proteins; RNA; RNA Conformation; RNA Decay; RNA Folding; RNA Recognition Motif; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Regulation; Regulator Genes; Relative (related person); Reporting; Ribonucleoproteins; Role; Series; Site; Structure; Surveys; Syndrome; System; TIS11 protein; Testing; Time; Transcript; Translations; Tumor Necrosis Factor-alpha; base; combinatorial; cytokine; high throughput screening; insight; mutant; novel; protein expression; tumorigenesis

DESCRIPTION (provided by applicant): 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, and test the functional significance of these domains in cells (Aim 1). Second, we will identify specific and non-specific RNA primary structural requirements for protein 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 local RNA structure directs the recruitment and positioning of AUF1 and HuR on ARE substrates and influences the cellular consequences of these interactions (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 steady-state 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. PUBLIC HEALTH RELEVANCE: Cancer and other serious clinical syndromes result when the expression of proteins regulating cell proliferation and differentiation becomes uncontrolled. Cells use many mechanisms to limit the production of these proteins, including rapid destruction or silencing of the messenger RNAs (mRNAs) encoding them through a family of RNA sequences called AU-rich elements (AREs). The proposed studies will determine how different cellular factors recognize unique ARE sequences and target their associated mRNAs for destruction, opening the possibility that these gene-specific interactions might be exploited as targets for novel therapies to treat some cancers and chronic inflammatory diseases.

描述（由申请人提供）：编码癌蛋白和细胞因子的许多mRNA的稳定性和翻译受富含AU的元件（战神）调节，ARE是位于其3'非翻译区的RNA序列的多样但进化上保守的家族。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战神的大小和序列多样性如何在基因特异性水平指导转录后调控，以及战神的基因特异性特征最终如何被利用作为治疗某些癌症和慢性炎症性疾病的新疗法的靶点。我们的中心假设是，任何ARE的mRNA的代谢命运是由细胞的反式因子的人口针对每个转录本，然而，选择一个因素在另一个的生化基础仍然定义不清。最近的研究结果表明，一些ARE结合因子的目标是不同的，但重叠的mRNA亚群，和当地的RNA二级结构可以影响反式因子的选择性。此外，一些因子可以重塑局部RNA结构或在战神上形成寡聚复合物。本项目采用一系列生物化学和分子生物学策略来确定特定分子决定簇在战神上形成稳定的功能性核糖核蛋白（RNP）复合物中的作用。使用广泛表达的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结合亲和力和RNA依赖性蛋白寡聚化的特定蛋白质亚结构域，并测试这些结构域在细胞中的功能意义（目的1）。其次，我们将确定蛋白质结合的特异性和非特异性RNA一级结构要求，并评估这些序列在与这些因子相互作用的细胞mRNA亚群中的应用（目的2）。最后，我们将确定局部RNA结构如何指导AUF 1和HuR在ARE底物上的募集和定位，并影响这些相互作用的细胞后果（目的3）。我们预计，我们的方法将允许力学的蛋白质的选择性和结合进行评估，在更详细的比以前报道的，主要是通过使用稳态和时间分辨荧光为基础的测定系统，我们已经适应研究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复合物的细胞功能之间关系的理解。 公共卫生相关性：当调节细胞增殖和分化的蛋白质的表达变得不受控制时，会导致癌症和其他严重的临床综合征。细胞使用许多机制来限制这些蛋白质的产生，包括通过称为富含AU元件（战神）的RNA序列家族快速破坏或沉默编码这些蛋白质的信使RNA（mRNA）。拟议的研究将确定不同的细胞因子如何识别独特的ARE序列并靶向其相关的mRNA进行破坏，从而打开了这些基因特异性相互作用可能被用作治疗某些癌症和慢性炎症性疾病的新疗法的靶点的可能性。