Deciphering the Composition of oskar mRNP via in vivo Fluorescence Imaging

描述（申请人提供）：信使RNA的亚细胞定位是基因转录后调控基因表达的重要机制，与翻译的暂时调控相结合。不正确的mRNA定位会破坏不对称的细胞分裂、长期记忆的形成以及基本体轴的建立。因此，不能正确定位会对学习和记忆基础上的突触可塑性造成灾难性影响，并可能导致脆性X综合征和脊髓性肌萎缩等疾病。核转录后，mRNA以mRNA：蛋白质（mRNP）复合物的形式运输到细胞质中的特定目的地。mrna的有效运输需要细胞核和细胞质蛋白与转录物之间高度协调的相互作用。破译这些动态的、有时转瞬即逝的相互作用的空间和时间组织需要在体内直接观察。利用高分辨率荧光成像，我们的长期目标是描述体内重要的大型mRNP复合物的组成和功能作用。为此，本应用程序的目的是研究黑腹果蝇卵母细胞mRNA运输过程中反式作用因子的时空要求。该应用程序的中心假设是后极决定因素oskar mRNA在mRNA转运的动态过程中与几种蛋白质相互作用，这是一个多步骤机制。本研究的基本原理是通过结合先进的荧光探针和显微技术，我们将为研究活细胞中的动态分子相互作用提供新的途径。

DESCRIPTION (provided by applicant): Coupled with temporally regulated translation, subcellular localization of messenger RNA is an important mechanism of post-transcriptional regulation of gene expression. Incorrect mRNA localization disrupts asymmetric cell division, long-term memory formation, as well as the establishment of basic body axes. Consequently, failure to localize correctly can have catastrophic effects on synaptic plasticity underlying learning and memory, and may lead to diseases such as the Fragile X syndrome and spinal muscular atrophy. After nuclear transcription, mRNA is trafficked to specific destinations in the cytoplasm as mRNA: protein (mRNP) complexes. Efficient transport of mRNAs requires highly orchestrated interactions between nuclear and cytoplasmic proteins and the transcript. Deciphering the spatial and temporal organization of these dynamic and sometimes fleeting interactions requires direct observation in vivo. Using high resolution fluorescence imaging, our long-term goal is to describe the composition and functional role of important large mRNP complexes in vivo. To that end, the objective of this application is to examine the spatio-temporal requirements of trans-acting factors during mRNA transport in Drosophila melanogaster oocytes. The central hypothesis of the application is that the posterior pole determinant, oskar mRNA, interacts with several proteins during the dynamic process of mRNA transport in a multi-step mechanism. The rationale for the proposed research is that by combining advanced fluorescent probes and microscopic techniques, we will provide a new avenue to study dynamic molecular interactions in living cells. In order to test how oskar mRNA is processed during Drosophila oogenesis, the following two specific aims will be pursued: 1. Characterize the dynamic steps of oskar mRNA transport during mid-oogenesis. 2. Identify the spatio-temporal relationship of the trans-acting proteins and oskar mRNA during its transport at mid-oogenesis. The proposed work is innovative because it will allow for the first time a real time view of the molecular dynamics of endogenous mRNAs and associating proteins by taking advantage of advanced fluorescence imaging techniques. The complex composition of mRNPs will be resolved in space and time. These studies will improve the models of RNA transport and localization and contribute to a better understanding of the pathogenesis of diseases caused by genetic errors that affect these processes.