Mechanism of RNA Localization in Drosophila Development

描述（由申请人提供）：信使RNA定位在产生细胞和发育极性所需的蛋白质的不对称分布中起关键作用。将特定的mRNA分选到不同的亚细胞结构域是一个复杂的过程，涉及大的核糖核蛋白（RNP）复合物的组装和运输。如何赋予这个过程的特异性，特别是在几个mRNA定位途径同时运作的细胞中，是知之甚少。该提案整合了生物化学，遗传学和基于成像的方法来研究mRNA如何被特异性识别并包装成定位活性颗粒，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个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，因为它们配备了几种机制上不同的运输途径，这些途径指导了 轴形成和生殖系发育所必需的mRNA。在目标1中，我们将使用生物化学方法（包括串联RNA亲和纯化）研究RNP颗粒组装的机制，以分离和表征Nanos RNP复合物的组分。这些研究将在目标2中通过两种基于成像的方法进行补充，这些方法研究是否通过不同mRNA共享RNP颗粒来协调并行运输途径。目的3扩展了我们的研究，通过一种新的全基因组筛选神经元中具有不对称亚细胞分布的转录本，确定mRNA定位在极化细胞发育和功能中的更广泛意义。我们的合作者在该筛选和其他细胞类型中进行的新定位mRNA的鉴定将揭示mRNA靶向特异性的决定因素，并可能揭示定位mRNA在极化细胞发育和功能中的新作用。

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.