Mechanism of RNA Localization in Drosophila Development

描述（由申请人提供）：发育过程中不对称的产生和分化细胞的极化需要细胞质蛋白的不对称分布。信使RNA定位通过将蛋白质的合成靶向到特定的亚细胞结构域，为产生蛋白质不对称提供了重要的机制。分化的细胞功能，如成纤维细胞的运动性和树突的突触可塑性，出芽酵母的不对称分裂，爪蟾和果蝇的胚胎轴形成，都需要由定位mrna合成的蛋白质。本研究旨在阐明发育过程中产生极性的mRNA定位机制。这些研究集中在纳米mRNA上，其定位于果蝇胚胎的后极，对于产生纳米蛋白梯度是必不可少的，这种梯度是前后体轴的模式。在之前的资助期间的工作表明，纳米定位也是生殖细胞功能和周围神经元正常发育所必需的。拟议的研究结合了分子、生化和遗传方法，以确定像纳米这样的mrna是如何被定位因子特异性识别的，以及这些rna -蛋白质相互作用是如何导致mrna转运到它们的目标目的地并维持它们在那里的。实时成像现在允许研究纳米mRNA在不同细胞环境中定位的机制。目标1侧重于对先前资助期间分离的纳米定位因子的生化分析，以及使用亲和纯化策略纯化其他因子。Aim 2采用一种互补的方法，通过增敏遗传筛选，鉴定参与纳米mRNA定位的蛋白质。目的3和4利用mrna的体内荧光标记系统来跟踪活胚胎和神经元中纳米定位的动态。此外，一种允许同时可视化两个mRNA的新系统将扩展我们对多个mRNA定位途径如何相交的理解。

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.