mRNP assembly and remodeling for transport and translational control in Drosophila

胞质mRNA定位是一种强大的和保守的机制，实现本地化的蛋白质表达在真核细胞中，它是普遍存在于果蝇，其卵母细胞定位的转录本编码胚胎轴决定簇已成为模型的mRNA运输，本地翻译和锚定的研究。由于其高度发达的实验分析工具，果蝇是生物化学，遗传学和细胞生物学研究mRNA定位机制的理想模型系统，因为它们发生在体内。一个范例是果蝇oskar mRNA，其定位是一个多步骤的过程，包括：（1）在oskar mRNA编码区内的二分定位信号的核剪接依赖性产生和（2）伴随的外显子连接复合物（EJC）在mRNA上的剪接依赖性沉积;（3）细胞质中蛋白质的募集（4）微管马达依赖的oskar信使核糖核蛋白（mRNP）转运通过微管马达、动力蛋白从滋养细胞进入卵母细胞，并通过驱动蛋白-1到达后极;（5）转运中mRNA的翻译抑制和（6）卵母细胞后部Oskar mRNA的局部翻译激活，这是（7）Oskar蛋白依赖性mRNA锚定在后极所必需的。虽然（主要是）遗传学已经鉴定出参与这种复杂调控的一些蛋白质，但既不知道蛋白质的完整补充，也不知道它们如何以及何时在这个过程中起作用。作为我们项目的第一个目标，我们计划解决果蝇EJC在果蝇mRNA定位中的更广泛的作用，并了解决定其稳定和选择性结合mRNA上特定位点的细胞事件和分子特征。该项目的第二个目的是开发和应用一种用于果蝇细胞内转运mRNP的转录和翻译状态特异性纯化的方法。作为一个原则的证明，我们将使用oskar mRNA，然后扩展到其他果蝇本地化的mRNA对定位mRNP组成和其功能分析在体内的苍蝇的系统视图。

Cytoplasmic mRNA localization is a powerful and conserved mechanism for achieving localized protein expression in eukaryotic cells; it is prevalent in Drosophila, whose oocyte-localized transcripts encoding embryonic axis determinants have become models for the study of mRNA transport, local translation and anchoring. With its highly developed tools for experimental analysis, Drosophila is an ideal model system for biochemical, genetic and cell biological investigation of mRNA localization mechanisms as they occur in vivo. A paradigm is Drosophila oskar mRNA, whose localization is a multi-step process that includes: (1) nuclear splicing-dependent generation of a bipartite localization signal within the oskar mRNA coding region and (2) concomitant, splicing dependent deposition of the Exon Junction Complex (EJC) on the mRNA; (3) cytoplasmic recruitment of proteins required for (4) microtubule motor-dependent transport of oskar messenger ribonucleoproteins (mRNPs) from the nurse cells into the oocyte by the microtubule motor, dynein and to the posterior pole by kinesin-1; (5) translational repression of the mRNA in transport and (6) localized translational activation of oskar mRNA at the oocyte posterior, which is required for the (7) Oskar protein dependent mRNA anchoring at the posterior pole. While (mainly) genetics has led to the identification of a few of the proteins involved in this complex regulation, neither is the full complement of proteins known, nor are how and when they (inter)act in the process understood. As a first aim of our project, we plan to address the broader role of the Drosophila EJC in mRNA localization in the fly and to understand cellular events and molecular features that determine its stable and selective binding to specific sites on mRNAs. The second aim of the project is to develop and apply a method for transcript- and translational status-specific purification of intracellular transport mRNPs in Drosophila. As a proof of principle, we will use oskar mRNA, then extend to other Drosophila localized mRNAs towards a systemic view of localization mRNP composition and its functional analysis in vivo in the fly.