Mechanisms directing oncoprotein and cytokine mRNA decay

描述（由申请人提供）：许多编码癌蛋白和细胞因子的mrna的稳定性和翻译受富au元件（AREs）的调控，AREs是一个多样化但进化上保守的RNA序列家族，定位于它们的3'非翻译区。破坏定向调控机制可导致肿瘤发生和严重炎症综合征。我们的长期目标是确定AREs的大小和序列多样性如何在基因特异性水平上指导转录后调控，以及AREs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，任何含有are的mRNA的代谢命运都是由针对每个转录物的细胞反式因子群体指导的；然而，选择一个因素而不是另一个因素的生化基础仍然不明确。最近的研究结果表明，一些ARE-binding因子靶向不同但重叠的mRNA亚群，并且局部RNA二级结构可以影响反式因子的选择性。此外，一些因子可以重塑局部RNA结构或在AREs上形成寡聚复合物。该项目使用一系列生化和分子生物学策略来确定特定分子决定因素在AREs上形成稳定、功能性核糖核蛋白（RNP）复合物中的作用。使用普遍表达的ARE结合蛋白AUF1和HuR作为模型系统，我们将首先表征对ARE结合亲和力和rna依赖性蛋白寡聚化有贡献的特定蛋白质亚域，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初级结构要求，并评估这些序列在与这些因素相互作用的细胞mRNA亚群中的使用（目的2）。最后，我们将确定局部RNA结构如何指导AUF1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，我们的方法将允许蛋白质选择性和结合的机制得到比以前报道的更详细的评估，主要是通过使用稳态和时间分辨荧光分析系统，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、反式因子识别和由此产生的RNP复合物的细胞功能之间关系的理解。

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.