Dynamic regulation of mRNA transport and translation in the Drosophila germline

MRNA定位是一种强大的进化保守策略，与翻译控制相结合，可以对细胞内的基因表达进行精确的时空控制。Oskar mRNA是一种基因定位和翻译调控的范例，其蛋白产物可诱导果蝇胚胎中生殖系的形成和后部模式的形成。调控Oskar核糖核蛋白颗粒(RNP)行为的关键成分包括外显子连接复合体(EJC)及其相关的剪接Oskar定位元件(SOLE)、双链RNA结合蛋白EQUALITAL和Staufen，以及重要的Dynein和Kinesin-1，它们是极性相反的微管马达蛋白，将Oskar mRNA从其在哺育细胞中的合成部位顺序输送到卵母细胞的后极。为了更深入地了解RNP的组装和调控，在第一个资助期，我们建立了一种严格的生化分析方法iPart，用它我们阐明了果蝇EJC的结合情况和影响成年果蝇EJC转录定位的因素，包括Oskar mRNA。我们还开发了一种转录特异的RNP纯化方法，并将其应用于Oskar，在mRNA在加工体中运输或隔离的条件下，从而确定了先前已知的和新的候选Oskar mRNP组分，包括翻译调节因子。在第二个资助期，我们将(1)评估这些候选蛋白中的几个在体内RNA调节中的作用，(2)解决EJC、EQUALITIVE和Staufen在mRNP组装中的作用以及在Oskar基因mRNA运输过程中从动力蛋白到动蛋白的转换。

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.