mRNP assembly and remodeling for transport and translational control in Drosophila

细胞质mRNA定位是真核细胞中实现蛋白本地化表达的一种强大而保守的机制；它在果蝇中普遍存在，其卵母细胞定位转录物编码胚胎轴决定因子已成为mRNA转运、局部翻译和锚定研究的模型。果蝇具有高度发达的实验分析工具，是生物化学、遗传和细胞生物学研究mRNA在体内定位机制的理想模型系统。一个典型的例子是果蝇oskar mRNA，其定位是一个多步骤的过程，包括：(1)在oskar mRNA编码区内核剪接依赖的双部定位信号的产生；(2)伴随剪接依赖的外显子连接复合体（EJC）在mRNA上的沉积；(4) oskar信使核糖核蛋白（mRNPs）的微管运动依赖运输通过微管运动、动力蛋白进入卵母细胞，并通过动力蛋白-1到达后极；(5)转运过程中mRNA的翻译抑制；(6)oskar mRNA在卵母细胞后部的本地化翻译激活，这是oskar蛋白依赖的mRNA锚定在后极所必需的。虽然（主要）遗传学已经确定了参与这种复杂调节的一些蛋白质，但既不知道蛋白质的全部补充，也不知道它们在这个过程中如何以及何时（相互作用）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定位中的更广泛作用，并了解决定其稳定和选择性结合mRNA特定位点的细胞事件和分子特征。该项目的第二个目标是开发和应用果蝇细胞内转运mrna的转录和翻译状态特异性纯化方法。作为原理证明，我们将使用oskar mRNA，然后扩展到其他果蝇定位mRNA，以系统地观察定位mRNP组成及其在果蝇体内的功能分析。

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.