mRNP assembly and remodeling for transport and translational control in Drosophila

细胞质mRNA定位是在真核细胞中实现蛋白质定位表达的一种强大而保守的机制，广泛存在于果蝇体内，其编码胚轴决定因素的卵母细胞定位转录产物已成为研究mRNA转运、局部翻译和锚定的模型。果蝇凭借其高度发达的实验分析工具，是研究体内发生的mRNA定位机制的生化、遗传学和细胞生物学研究的理想模型系统。一个范例是果蝇Oskar mRNA，它的定位是一个多步骤的过程，包括：(1)依赖于核剪接在Oskar mRNA编码区内产生一个两部分定位信号，(2)伴随着依赖剪接的外显子连接复合体(EJC)在mRNA上的沉积；(3)细胞质中募集到(4)微管运动依赖的Oskar信使核糖核蛋白(MRNPs)通过微管运动和动力蛋白(Dynein)从哺育细胞运输到卵母细胞，并通过kinesin-1到达后极；(5)运输中的翻译抑制和(6)Oskar mRNA在卵母细胞后部的局部翻译激活，这是(7)Oskar蛋白依赖的mRNA锚定在卵母细胞后极所必需的。虽然(主要)遗传学导致了参与这一复杂调控的少数蛋白质的鉴定，但既不知道蛋白质的全部组成，也不知道它们(相互)如何以及何时在这个过程中发挥作用。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与mRNAs上特定位点结合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一种针对转录和翻译状态特异性纯化果蝇细胞内转运mRNPs的方法。作为原理的证明，我们将使用Oskar mRNA，然后扩展到其他果蝇定位的mRNAs，以系统地了解mRNP的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.