Shedding light on differential mRNA localisation and RNP dynamics in vitro and in vivo

为了让细胞发挥其复杂的功能，必须在正确的时间将各种组件运送到正确的地点。马达蛋白是这项任务的核心。这些分子机器对接到细胞组件上，并通过沿着细胞内的轨道网络行走将它们运送到目的地。分子马达的关键“货物”之一是信使RNA(信使RNA)，信使RNA是生产蛋白质的模板分子。将信使核糖核酸定位到细胞内的特定位置是控制其蛋白质产物在哪里运作的有效方法。因此，这一过程被用于许多需要区分细胞活动的过程中，包括学习和记忆、胚胎发育和细胞运动。尽管它广泛发生，但我们对mRNAs如何通过马达蛋白传递到细胞内的离散位置知之甚少。解决这个问题的一个容易处理的系统是果蝇的发育中的卵(卵母细胞)。在这里，将mRNA贩运到不同的位置指定了动物未来的身体轴线：从头到尾和从前到后。值得注意的是，将mRNAs传递到卵母细胞中的每个位置都涉及到相同的运动，动力蛋白，它走向极化微管轨迹的‘负端’。几十年来的遗传学研究已经确定了将动力蛋白相关的mRNAs定向到卵母细胞中离散位置所需的蛋白质，但它们是如何做到这一点的尚不清楚。我们最近成功地利用纯化的成分重建了基于动力蛋白的核心信使核糖核酸在细胞外的运输机制；这是一个重大的进步，因为它允许详细地剖析运输过程，包括可视化信使核糖核酸和蛋白质的单分子的行为。我们将在这个系统的基础上理解卵母细胞内差异mRNA定位的机制基础。我们将基于我们最近未发表的结果来检验这一假设，即颗粒中RNA和马达分子的聚集是mRNA在卵母细胞中目的地的关键决定因素。这将通过使用人工构建的RNA-蛋白质组件或与已知对特定mRNAs在卵母细胞中定位重要的蛋白质组装来实现。我们还将通过在玻璃表面构建定义的微管图案，测试微管细胞骨架的结构对不同RNP物种的运输和锚定的影响。在一个互补的方法中，我们将破坏一个关键的mRNA运输蛋白的“低复杂性”片段，这些片段被认为是在其他环境中控制颗粒组装的，并监测卵母细胞内外的影响。这些实验将通过我们小组最近开发的针对苍蝇的高效基因组编辑技术来促进。总而言之，这项工作将为mRNAs在同一细胞中如何不同地分类，以及将RNA和蛋白质组装成颗粒如何影响它们的功能提供独特的见解。通过揭示可用于调节基于动力蛋白的运输的策略，我们的发现也将有助于理解发动机如何运输其他货物，包括膜结合的囊泡和病毒。

In order for cells to perform their elaborate functions, various components must be delivered to the right place at the right time. Motor proteins are central to this task. These are molecular machines that dock onto cellular components and transport them to their destination by walking along a network of tracks in the cell. One of the key 'cargoes' for molecular motors is messenger RNA (mRNA), molecules that are the templates for the production of proteins. Localising an mRNA to a specific site within the cell is an effective way of controlling where its protein product operates. This process is therefore used in many processes that require compartmentalised cell activities, including learning and memory, embryonic development and cell movement. Despite its widespread occurrence we have a poor understanding of how mRNAs are delivered to discrete sites within cells by motor proteins. A tractable system for addressing this issue is the developing egg (the oocyte) of the fruit fly, Drosophila. Here, trafficking of mRNAs to different locations specifies the future body axes of the animal: head-to-tail and front-to-back. Remarkably, delivery of mRNAs to each site in the oocyte involves the same motor, dynein, which walks towards the 'minus-end' of polarised microtubule tracks. Genetic research over several decades has identified proteins that are required to direct dynein-associated mRNAs to discrete sites in the oocyte but how they do this is not clear. We have recently succeeded in reconstituting the core dynein-based mRNA transport machinery outside the cell using purified components; this is a significant advance as it allows the trafficking process to be dissected in detail, including the visualisation of the behaviour of single molecules of mRNA and protein. We will build on this system to understand the mechanistic basis of differential mRNA localisation within the oocyte. We will test the hypothesis, based on our recent unpublished results, that clustering of RNA and motor molecules in granules is a key determinant of an mRNA's destination in the oocyte. This will be achieved using RNA-protein assemblies that are built artificially or with proteins that are known to be important for localisation of specific mRNAs in the oocyte. We will also test the influence of the architecture of the microtubule cytoskeleton on transport and anchorage of different RNP species by constructing defined patterns of microtubules on a glass surface. In a complementary approach we will disrupt a key mRNA trafficking protein's 'low complexity' segments, which have been implicated in controlling granule assembly in other contexts, and monitor the effects inside and outside the oocyte. These experiments will be facilitated by efficient genome editing techniques for the fly that were recently developed in our group. Collectively, this work will provide unique insights into how mRNAs are sorted differentially in the same cell and how the assembly of RNAs and proteins into granules affects their function. By revealing strategies that can be used to regulate dynein-based transport, our findings will also inform efforts to understand how the motor traffics other cargoes, including membrane-bound vesicles and viruses.