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的不对称分布。信使RNA定位提供了一个重要的机制，产生蛋白质的不对称性，通过靶向特定的亚细胞结构域的蛋白质的合成。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，芽殖酵母的不对称分裂，以及非洲爪蟾和果蝇的胚胎轴形成，都需要从局部mRNA合成的蛋白质。拟议的研究旨在阐明在发育过程中用于产生极性的mRNA定位机制。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图案化前后体轴的Nanos蛋白梯度是必不可少的。在前一个资助期的工作表明，nanos的定位也是生殖细胞功能和外周神经元正常发育所必需的。拟议的研究结合了联合收割机分子、生物化学和遗传学方法，以确定定位因子如何特异性识别像纳米这样的mRNA，以及这些RNA-蛋白质相互作用如何导致mRNA运输到其目标目的地并将其维持在那里。实时成像现在允许研究nanos mRNA在不同细胞环境中定位的机制。目标1侧重于生化分析的纳米定位因子分离在以前的资助期间，以及使用亲和纯化策略的其他因素的纯化。目的2采取了一种互补的方法，通过敏化遗传筛选来鉴定参与纳米mRNA定位的蛋白质。目的3和4利用用于mRNA的体内荧光标记的系统来跟踪在活胚胎和神经元中纳米定位的动力学。此外，一个新的系统，允许可视化的两个mRNA同时将扩大我们的理解如何多个mRNA定位途径相交。 公共卫生相关性：信使RNA定位产生的蛋白质分布所需的胚胎轴的形成和不对称的细胞分裂过程中的发展和极化的细胞功能，如运动成纤维细胞和突触可塑性树突。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相关，这些蛋白质的致瘤性可能反映了它们在定位mRNA以确定细胞命运、细胞极性和迁移中的作用。拟议的研究将阐明RNA-蛋白质相互作用提供高度选择性控制发育，生长和分化所需的基本细胞过程的机制，以及这些过程的破坏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.