Mechanism of RNA Localization in Drosophila Development

描述(申请人提供)：细胞内信使核糖核酸定位是动物发育所必需的基因表达空间调控的重要机制。通过将发育调节蛋白的分布限制在卵母细胞和胚胎的特定区域，在多种生物的胚胎发育过程中，mRNA的定位在体轴的形成和细胞命运的指定中起着关键作用。此外，RNA定位产生了许多特定细胞类型随后分化和功能所必需的蛋白质不对称。介导定位的顺式作用信号现在已经在一些定位的mRNAs中被识别。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靶向独特的细胞内位置，人们知之甚少。 将Nanos蛋白限制在果蝇胚胎的后部是形成适当的前后体轴模式所必需的。通过RNA定位和翻译控制的组合，Nanos的合成被限制在胚胎的后极。Nanos mRNA定位于胚胎的后极，在腹部发育的后极产生临界浓度的Nanos蛋白，是激活Nanos翻译所必需的。 Nanos的定位是由其3‘非翻译区(3’UTR)内的顺式作用定位信号介导的。先前和初步的研究结果表明，细胞质定位因子识别这种定位信号中不同的序列或结构基序。以Nanos为模型，这项拟议的工作将深入了解复杂的RNA定位信号是如何被细胞定位机制识别的，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。更广泛地说，这些研究将阐明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。具体目标1包括对Nanos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。这项工作将通过对来自10个不同果蝇物种的Nanos 3‘UTRs的系统发育分析来促进。初步工作已经导致了一个候选Nanos定位因子的纯化和第二个候选Nanos定位因子的生化鉴定。目的2重点研究这些因子的生物化学和遗传学特征，以确定它们在纳米定位中的作用。此外，还提出了一种新的生化分离定位络合物的策略。在Aim 3中，对Nanos定位因子的遗传筛选将补充Aim 2的生化方法。Aim 4利用一种新的体内纳米RNA GFP标记系统来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.