Mechanism of RNA Localization in Drosophila Development

描述(由申请人提供)：信使RNA定位在创造细胞和发育极性所必需的蛋白质的不对称分布方面起着关键作用。将特定的mRNAs分选到不同的亚细胞结构域是一个复杂的过程，涉及到大的核糖核蛋白(RNP)复合体的组装和运输。这一过程的特异性是如何被赋予的，特别是在几个mRNAs定位通路同时运行的细胞中，人们还知之甚少。该方案集成了基于生化、遗传和成像的方法来研究mRNAs是如何被特异性识别和包装成具有定位能力的颗粒，这些RNP颗粒如何适应不同的定位机制，定位途径相互连接的程度，以及多条定位途径如何在单个细胞内协调。果蝇卵母细胞和早期胚胎为这些研究提供了理想的模型系统，因为它们配备了几条机械上不同的运输途径，指导着 对胚轴形成和生殖系发育至关重要的mRNA。在目标1中，我们将使用包括串联RNA亲和纯化在内的生化方法来研究RNP颗粒组装的机制，以分离和表征纳米RNP复合体的组成。在目标2中，这些研究将得到两种基于成像的方法的补充，这两种方法调查并发贩运路径是否通过不同mRNA共享RNP颗粒而得到协调。目的3通过一种新的全基因组筛选神经元中亚细胞分布不对称的转录本，扩大我们的研究范围，以确定mRNA定位在极化细胞的发育和功能中的更广泛意义。我们的合作者在这个屏幕和其他细胞类型的屏幕上发现了新的定位的mRNAs，这将有助于揭示mRNA靶向特异性的决定因素，并可能揭示定位mRNAs在极化细胞的发育和功能中的新角色。

DESCRIPTION (provided by applicant): Messenger RNA localization plays a key role in creating the asymmetric distributions of proteins necessary for cellular and developmental polarity. The sorting of specific mRNAs to different subcellular domains is a complex process involving the assembly and trafficking of large ribonucleoprotein (RNP) complexes. How specificity is conferred on this process, particularly in cells where several mRNAs localization pathways operate concurrently, is poorly understood. This proposal integrates biochemical, genetic, and imaging-based approaches to investigate how mRNAs are specifically recognized and packaged into localization competent particles, how these RNP particles are adapted to different localization mechanisms, the extent to which localization pathways are interconnected, and how multiple localization pathways are coordinated within a single cell. The Drosophila oocyte and early embryo provide ideal model systems for these studies because they are equipped with several mechanistically distinct trafficking pathways that direct the localization of mRNAs essential for axis formation and germline development. In Aim 1, we will investigate mechanisms of RNP particle assembly using biochemical approaches including tandem RNA affinity purification to isolate and characterize the components of Nanos RNP complexes. These studies will be complemented in Aim 2 by two imaging-based approaches that investigate whether concurrent trafficking pathways are coordinated through the sharing of RNP particles by different mRNAs. Aim 3 expands our studies to determine the broader significance of mRNA localization in the development and function of polarized cells through a novel genome- wide screen for transcripts with asymmetric subcellular distributions in neurons. The identification of new localized mRNAs in this screen and screens in other cell types performed by our collaborators will shed light on determinants of mRNA targeting specificity and may uncover novel roles for localized mRNAs in development and function of polarized cells.