Post-Transcriptional controls in Mammalian Erythroid Differentiation

描述(由申请者提供)：这项资助的长期目标是建立对哺乳动物红系分化至关重要的转录后调控的全面了解。红系分化的标志是一系列戏剧性的形态和功能变化，其中大部分过程发生在转录沉默的环境中。因此，红系分化在很大程度上依赖于转录后调控。核糖核酸结合蛋白(RNAbindingProteins，RBPs)是转录后调控的主要调控因子，对转录产物在细胞核中的加工以及细胞质中mRNA的稳定性和功能具有重要影响。限制性商业惯例在其结构、结合特异性和细胞区划方面有很大的不同，可以通过直接作用和/或通过招募效应复合体来调节RNA功能。识别限制性商业惯例、它们的信使核糖核酸靶标和相应的转录后调控促进了我们对许多发育过程的理解，并揭示了意想不到的病理生理途径。通过定义红系分化中的转录后调控，我们将扩大我们对遗传性和获得性红细胞生成障碍的理解，并为其他系统中的类似研究建立一个模板。为了实现这一目标，我们将进行一种新颖的、最先进的转录组分析，以揭示伴随着红系末端分化动态过程的全方位的mRNA-蛋白质(MRNP)相互作用。这种方法将生化方法与创新的信息管道相结合，专门为全面描述复杂的RNA-蛋白质相互作用而设计。这一无偏见的分析将与一系列有针对性的深入机制研究相结合，这些研究侧重于我们已确定在红系分化中发挥核心作用的两组mRNA/蛋白质相互作用。这些互补方法的组合输出将在这个发育健壮和临床相关的模型系统中建立独特的转录后控制网络。这项提议包括三个具体目标。目的1.研究多聚C结合蛋白�1和�2作为转录后整合因子在红系分化中的作用。目的2.确定Polya结合蛋白在定义和维持红系转录组中的关键作用(S)。目的3.绘制红系细胞中mRNA/蛋白质相互作用的整体结构图，并确定这些相互作用在分化过程中的动态性质。结合转录组范围的分析和有针对性的机制研究，将产生一个强大的途径，以获得构成红系分化的关键决定因素的复杂的RNA/蛋白质相互作用阵列。这些研究的成功将从根本上改变我们对这一深入研究的途径的理解；作为其他系统转录后调控研究的原型，并为未来的诊断和治疗创新提供新的靶点。

DESCRIPTION (provided by applicant): The long-term goal of this grant is to establish a comprehensive understanding of post-transcriptional controls critical to mammalian erythroid differentiation. Erythroid differentiation is marked by a set of dramatic morphologic and functional changes, much of this process occurring in a transcriptionally-silent environment. For this reason erythroid differentiation is heavily dependent on post-transcriptional controls. RNA binding proteins (RBPs), the major regulators of post-transcriptional controls, impact on transcript processing in the nucleus as well as the stability and function of the mRNA in the cytoplasm. RBPs vary widely in their structures, binding specificities, and cellular compartmentalization and can modulate RNA functions by direct actions and/or via the recruitment of effector complexes. Identification of RBPs, their mRNA targets, and corresponding post-transcriptional controls have advanced our understanding of numerous developmental processes and have revealed unanticipated pathophysiologic pathways. By defining post-transcriptional controls in erythroid differentiation, we will expand our understanding of inherited and acquired disorders of erythropoiesis and establish a template for similar investigations in other systems. To achieve this goal, we will carry out a novel and state-of-the-art transcriptome-wide analyses to reveal the full range of mRNA-protein (mRNP) interactions that accompany the dynamic process of erythroid terminal differentiation. This approach combines biochemical methodologies with innovative informatic pipelines specifically designed for comprehensive descriptions of complex RNA-protein interactions. This unbiased analysis will be combined with a series of targeted in-depth mechanistic studies that focus on two sets of mRNA/protein interactions that we have identified to play central roles in erythroid differentiation. The combined output of these complementing approaches will establish the unique network of post-transcriptional controls in this developmentally robust and clinically relevant model system. This proposal encompasses three Specific Aims. Aim 1. Characterize the roles of the polyC-binding proteins, �1 and �2, as post- transcriptional integrators of erythroid differentiation. Aim 2. Identify the critical role(s) of the polyA binding protein, PABPC, in defining and sustaining the erythroid transcriptome. Aim 3. Map the global structure of mRNA/protein interactions in erythroid cells and define the dynamic nature of these interactions in the differentiation process. Combining transcriptome-wide analyses with targeted mechanistic studies will generate a powerful access to the complex array of RNA/protein interactions that constitute critical determinants of erythroid differentiation. Success in these studies will fundamentally alter our understanding of this intensively studied pathway; serve as a prototype for investigations of post- transcriptional controls in other systems, and present novel targets fo future diagnostic and therapeutic innovations.