Deciphering the Composition of oskar mRNP via in vivo Fluorescence Imaging

描述（由申请人提供）：与时间调节的翻译相结合，信使RNA的亚细胞定位是基因表达的转录后调节的重要机制。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，以及基本身体轴的建立。因此，未能正确定位可能对学习和记忆基础的突触可塑性产生灾难性影响，并可能导致脆性X综合征和脊髓性肌萎缩症等疾病。在核转录后，mRNA作为mRNA：蛋白质（mRNP）复合物被运输到细胞质中的特定目的地。mRNA的有效转运需要核蛋白和细胞质蛋白与转录本之间高度协调的相互作用。解读这些动态的、有时是短暂的相互作用的空间和时间组织需要在体内进行直接观察。使用高分辨率荧光成像，我们的长期目标是描述重要的大型mRNP复合物在体内的组成和功能作用。为此，本申请的目的是研究果蝇卵母细胞mRNA运输过程中反式作用因子的时空要求。该应用的中心假设是后极决定子oskar mRNA在mRNA转运的动态过程中以多步机制与几种蛋白质相互作用。这项研究的基本原理是，通过结合先进的荧光探针和显微技术，我们将提供一种新的途径来研究活细胞中的动态分子相互作用。 为了测试oskar mRNA在果蝇卵子发生期间如何加工，将追求以下两个具体目标： 1.描述卵子发生中期oskar mRNA运输的动态步骤。 2.确定反式作用蛋白和oskar mRNA在卵子发生中期转运过程中的时空关系。 拟议的工作是创新的，因为它将允许第一次真实的时间视图的内源性mRNA和相关蛋白质的分子动力学，利用先进的荧光成像技术。mRNP的复杂组成将在空间和时间上得到解决。这些研究将改进RNA转运和定位的模型，并有助于更好地理解由影响这些过程的遗传错误引起的疾病的发病机制。

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.