Mechanism of RNA Localizatiion in Drosophila Development

描述（由申请人提供）：细胞内mRNA定位是动物发育所必需的基因表达空间调控的重要机制。通过限制发育调节蛋白的分布到卵母细胞和胚胎的特定区域，mRNA定位在多种生物体胚胎发育期间的体轴图案化和细胞命运的规范中起着关键作用。此外，RNA定位产生蛋白质不对称性，这是许多特化细胞类型随后分化和功能所必需的。现在已经在许多定位的mRNA中鉴定了介导定位的顺式作用信号。然而，关于这些信号被细胞定位机制特异性识别的机制以及它们如何将其RNA靶向到独特的细胞内位置，我们知之甚少。 将Nanos蛋白限制在果蝇胚胎的后部对于前后体轴的正确模式至关重要。通过RNA定位和翻译控制的组合，Nanos合成仅限于胚胎的后极。nanos mRNA定位于胚胎的后极，在腹部发育的后部产生临界浓度的Nanos蛋白，并且对于激活nanos翻译至关重要。 nanos的定位由其3'非翻译区（3' UTR）内的顺式作用定位信号复合物介导。从以前的和初步的研究结果表明，细胞质定位因子识别不同的序列或结构基序内的定位信号。使用nanos作为模型，拟议的工作将深入了解复杂的RNA定位信号如何被细胞定位机制识别，以及这些RNA-蛋白质相互作用如何通过定位途径介导运输和锚定。更一般地说，这些研究将揭示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。具体目标1包括纳米定位信号的突变分析，以确定定位信号识别和功能的序列和结构要求。这项工作将有助于系统发育分析nanos 3 'UTR从十个不同的果蝇物种。初步的工作已经导致一个候选人nanos定位因子的纯化和第二个的生化鉴定。目的2集中于这些因子的生化和遗传特性，以确定它们在纳米定位中的功能。此外，提出了一种新的生化分离定位复合物的策略。在目标3中，纳米定位因子的遗传筛选将补充目标2的生物化学方法。目的4利用绿色荧光蛋白（GFP）标记nanos RNA的新系统，研究卵子发生过程中nanos定位的动态途径。

DESCRIPTION (provided by applicant): Intracellular mRNA localization is an important mechanism for the spatial regulation of gene expression necessary for animal development. By restricting the distributions of developmental regulatory proteins to particular regions of oocytes and embryos, mRNA localization plays a critical role in patterning of body axes and specification of cell fates during embryonic development in a variety of organisms. In addition, RNA localization generates protein asymmetries necessary for the subsequent differentiation and function of many specialized cell types. Cis-acting signals that mediate localization have now been identified in a number of localized mRNAs. Little is known, however, about the mechanisms by which these signals are recognized specifically by cellular localization machinery and how they target their RNAs to unique intracellular locations. Restriction of Nanos protein to the posterior of the Drosophila embryo is essential for proper patterning of the anterior-posterior body axis. Nanos synthesis is limited to the posterior pole of the embryo by a combination of RNA localization and translational control. Localization of nanos mRNA to the posterior pole of the embryo generates the critical concentration Nanos protein in the posterior for abdominal development and is essential to activate nanos translation. Localization of nanos is mediated by a complex a cis-acting localization signal within its 3' untranslated region (3'UTR). Results from previous and preliminary studies indicate that cytoplasmic localization factors recognize different sequence or structural motifs within this localization signal. Using nanos as a model, the proposed work will provide insight into how complex RNA localization signals are recognized by the cellular localization machinery and how these RNA-protein interactions mediate transport and anchoring by localization pathways. More generally, these 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. Specific Aim 1 encompasses mutational analysis of the nanos localization signal to determine sequence and structural requirements for localization signal recognition and function. This work will be facilitated by phylogenetic analysis ofnanos 3'UTRs from ten different drosophilid species. Preliminary work has led to purification of one candidate nanos localization factor and biochemical identification of a second. Aim 2 focuses on biochemical and genetic characterization of these factors to determine their function in nanos localization. In addition, a new strategy for biochemical isolation of localization complexes is proposed. In Aim 3, a genetic screen for nanos localization factors will complement the biochemical approaches of Aim 2. Aim 4 takes advantage of a new system for GFP labeling of nanos RNA in vivo to investigate the dynamic pathway of nanos localization during oogenesis.