Mechanisms directing oncoprotein and cytokine mRNA decay

描述(申请人提供)：许多编码癌蛋白和细胞因子的mRNAs的稳定性和翻译由富含AU的元件(ARES)调节，ARES是一个多样化但进化保守的RNA序列家族，定位于其3‘非翻译区。ARE导向的调节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的基因特异性最终如何被用作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本的细胞反式因子的群体决定的；然而，选择一个因子而不是另一个因子的生化基础仍然没有明确的定义。最近的发现表明，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的选择性。此外，一些因素可以重塑局部RNA结构或在Ares上形成寡聚复合体。该项目使用一系列生化和分子生物学策略来确定特定的分子决定因素在ARES上形成稳定的、功能性的核糖核蛋白(RNP)复合体中的作用。使用普遍表达的ARE结合蛋白AUF1和HUR作为模型系统，我们将首先表征参与ARE结合亲和力和RNA依赖的蛋白质齐聚的特定蛋白质亚域，并测试这些结构域在细胞中的功能意义(目标1)。其次，我们将确定蛋白质结合的特定和非特定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信使核糖核酸亚群(S)中的使用(目标2)。最后，我们将确定局部RNA结构如何指导AUF1和HUR在ARE底物上的招募和定位，并影响这些相互作用的细胞后果(目标3)。我们预计，我们的方法将允许比以前报道的更详细地评估蛋白质选择性和结合的机制，主要是通过使用稳态和时间分辨的荧光分析系统，我们已经采用这些系统来研究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、跨因子识别和所产生的RNP复合体的细胞功能之间的关系的理解。与公共卫生相关：当调节细胞增殖和分化的蛋白质表达失控时，就会导致癌症和其他严重的临床症状。细胞使用许多机制来限制这些蛋白质的产生，包括通过一系列称为富含AU的元素(ARES)的RNA序列来快速破坏或沉默编码这些蛋白质的信使RNA(MRNAs)。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，并针对其相关的mRNAs进行破坏，从而打开了利用这些基因特异性相互作用作为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。

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.