Mechanism of RNA Localization in Drosophila Development

描述(由申请人提供)：发育过程中不对称性的产生和分化细胞的极化需要细胞质蛋白质的不对称分布。信使RNA定位通过将蛋白质的合成定位于特定的亚细胞结构域，为产生蛋白质不对称性提供了一个重要的机制。两极化的细胞功能，如成纤维细胞的运动性和树突的突触可塑性，发芽酵母的不对称分裂，以及非洲爪哇和果蝇的胚轴形成，都需要由定位的mRNAs合成的蛋白质。这项拟议的研究旨在阐明在发育过程中用于产生极性的mRNA本地化机制。这些研究集中在Nanos mRNA上，其定位于果蝇胚胎的后极，对于产生Nanos蛋白梯度以形成前后体轴是必不可少的。前一批赠款期间的工作表明，对于生殖细胞的功能和外周神经元的正常发育，Nanos的本地化也是必需的。拟议的研究结合了分子、生化和遗传学的方法，以确定像Nanos这样的mRNAs是如何被定位因子特异性识别的，以及这些RNA-蛋白质相互作用如何导致mRNAs运输到它们的目标目的地并保持它们在那里。现在，实时成像可以研究Nanos信使核糖核酸在不同细胞环境中定位的机制。目的1重点是对前一资助时期分离的Nanos定位因子进行生化分析，以及使用亲和纯化策略纯化额外的因子。目的2采用一种互补的方法，通过敏化的遗传筛选来鉴定与Nanos mRNA定位有关的蛋白质。AIMS 3和4利用体内mRNAs荧光标记系统来跟踪活胚胎和神经元中Nanos定位的动态。此外，一个允许同时显示两个mRNAs的新系统将扩大我们对多个mRNAs定位途径如何相交的理解。 公共卫生相关性：Messenger RNA定位产生胚胎轴形成和发育过程中细胞不对称分裂所需的局部蛋白质分布，以及成纤维细胞的运动性和树突的突触可塑性等极化细胞功能所需的蛋白质分布。与信使RNA结合并指导其定位的蛋白质水平的改变与多种癌症有关，这些蛋白质的致瘤性可能反映了它们在细胞命运指定、细胞极性和迁移的mRNAs定位中的作用。拟议的研究将阐明RNA-蛋白质相互作用提供对发育、生长和分化所需的基本细胞过程的高度选择性控制的机制，以及这些过程的中断可能如何导致癌症或神经功能障碍等疾病。

DESCRIPTION (provided by applicant): The generation of asymmetry during development and the polarization of differentiated cells require asymmetric distributions of cytoplasmic proteins. Messenger RNA localization provides an important mechanism for generating protein asymmetries by targeting the synthesis of such proteins to specific subcellular domains. Polarized cellular functions like motility in fibroblasts and synaptic plasticity in dendrites, asymmetric division of budding yeast, and embryonic axis formation in Xenopus and Drosophila all require proteins that are synthesized from localized mRNAs. The proposed research aims to elucidate mechanisms of mRNA localization used to generate polarity during development. These studies focus on nanos mRNA, whose localization to the posterior pole of the Drosophila embryo is essential for production of a Nanos protein gradient that patterns the anterior-posterior body axis. Work during the previous grant period has shown that localization of nanos is also required for germ cell function and for the proper development of peripheral neurons. The proposed studies combine molecular, biochemical, and genetic approaches to determine how mRNAs like nanos are specifically recognized by localization factors and how these RNA-protein interactions result in transport of mRNAs to their target destinations and maintain them there. Live imaging now permits investigation of mechanisms used by nanos mRNA for localization in different cellular contexts. Aim 1 focuses on biochemical analysis of nanos localization factors isolated during the previous funding period as well as purification of additional factors using an affinity purification strategy. Aim 2 takes a complementary approach toward identification of proteins involved in nanos mRNA localization through a sensitized genetic screen. Aims 3 and 4 take advantage of a system for in vivo fluorescent labeling of mRNAs to follow the dynamics of nanos localization in live embryos and in neurons. In addition, a new system that permits visualization of two mRNAs simultaneously will expand our understanding of how multiple mRNA localization pathways intersect. Public Health Relevance: Messenger RNA localization produces localized protein distributions required for embryonic axis formation and asymmetric cell division during development and for polarized cellular functions like motility in fibroblasts and synaptic plasticity in dendrites. Altered levels of proteins that bind to messenger RNAs and direct their localization have been associated with a variety of cancers and the tumorigenicity of these proteins may reflect their roles in localizing mRNAs for cell fate specification, cell polarity, and migration. The proposed studies will shed light on mechanisms by which RNA-protein interactions provide the highly selective control of basic cellular processes needed for development, growth, and differentiation and how the disruption of these processes may lead to diseases like cancer or neurological dysfunction.