Dynamic regulation of mRNA transport and translation in the Drosophila germline

mRNA定位是一个强大的和进化上保守的策略，耦合到翻译控制允许精确的空间和时间控制细胞内的基因表达。oskar mRNA是mRNA定位和翻译控制的范例，其蛋白产物诱导果蝇胚胎中的生殖系形成和后部图案化。调节奥斯卡核糖核蛋白颗粒（RNP）行为的关键成分包括外显子连接复合物（EJC）和相关的剪接奥斯卡定位元件（SOLE），双链RNA结合蛋白平等主义和Staufen，以及重要的动力蛋白和驱动蛋白-1，相反极性的微管马达蛋白，将奥斯卡mRNA从其在保育细胞中的合成位点顺序运输到卵母细胞后极。为了更深入地了解RNP的组装和调控，在第一个资助期，我们开发了一种严格的生化测定，ipaRT，我们阐明了果蝇EJC结合景观和影响EJC定位转录组范围内的成年苍蝇，包括oskar mRNA的因素。我们还开发了一种转录特异性RNP纯化方法，并将其应用于奥斯卡条件下，mRNA被运输或隔离在处理机构，从而确定了以前已知的和新的候选奥斯卡mRNP组件，包括翻译调节。在第二个资助期内，我们将（1）评估这些候选蛋白中的几种在体内RNA调控中的作用，（2）解决EJC，平等主义者和Staufen在mRNP组装中的作用，以及在oskar mRNA转运过程中从动力蛋白到驱动蛋白的转换。

mRNA localization is a powerful and evolutionarily conserved strategy that coupled to translational control allows precise spatial and temporal control of gene expression within cells. oskar mRNA, whose protein product induces germline formation and posterior patterning in the Drosophila embryo, is a paradigm for mRNA localization and translational control. Key components regulating oskar ribonucleoprotein particle (RNP) behavior include the Exon Junction Complex (EJC) and the associated spliced oskar localization element (SOLE), the double-stranded RNA-binding proteins Egalitarian and Staufen, and importantly dynein and kinesin-1, microtubule motor proteins of opposite polarity that transport oskar mRNA sequentially from its site of synthesis in the nurse cells to the oocyte posterior pole. To gain deeper insight into RNP assembly and regulation, in the first funding period, we developed a stringent biochemical assay, ipaRT, with which we elucidated the Drosophila EJC binding landscape and the factors affecting EJC positioning transcriptome-wide in adult flies, including on oskar mRNA. We also developed a transcript-specific RNP purification method and applied it to oskar under conditions where the mRNA is either transported or sequestered in processing bodies, and thus identified previously known and new candidate oskar mRNP components, including translation regulators. In the second funding period, we will (1) assess the roles of several of these candidate proteins in RNA regulation in vivo and (2) address the roles of the EJC, Egalitarian and Staufen in mRNP assembly and the switch from dynein to kinesin during oskar mRNA transport.