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

阐明体外和体内差异 mRNA 定位和 RNP 动态

• 批准号: BB/T00696X/1
• 负责人: Simon Bullock
• 金额: $ 31.63万
• 依托单位: MRC Laboratory of Molecular Biology
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT00696X%2F1
• 关键词: Shedding; light; differential; mRNA; localisation

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 We will test the hypothesis, based on our recent unpublished results, that clustering of RNA and motor molecules in granules is a key determinant of an mRNA's destination in the oocyte.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.

为了让细胞发挥其复杂的功能，各种成分必须在正确的时间被送到正确的地方。运动蛋白是这项任务的核心。这是一种分子机器，它停靠在细胞成分上，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 (mRNA)，这些分子是生产蛋白质的模板。将mRNA定位到细胞内的特定位置是控制其蛋白质产物运作的有效方法。因此，这一过程被用于许多需要分隔细胞活动的过程，包括学习和记忆、胚胎发育和细胞运动。尽管其广泛发生，但我们对mrna如何通过运动蛋白递送到细胞内的离散位点知之甚少。解决这个问题的一个容易处理的系统是果蝇发育中的卵（卵母细胞）。在这里，将mrna运送到不同的位置指定了动物未来的身体轴：从头到尾和从前面到后面。值得注意的是，将mrna传递到卵母细胞的每个位置涉及到相同的马达，动力蛋白，它走向极化微管轨道的“负端”。几十年来的遗传研究已经确定了将动力蛋白相关mrna引导到卵母细胞中离散位点所需的蛋白质，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在细胞外重建了核心动力蛋白为基础的mRNA转运机制；这是一个重大的进步，因为它允许详细剖析贩运过程，包括mRNA和蛋白质单分子行为的可视化。我们将在这个系统的基础上了解不同mRNA在卵母细胞内定位的机制基础。基于我们最近未发表的结果，我们将测试这个假设，即RNA和运动分子在颗粒中的聚集是mRNA在卵母细胞中的目的地的关键决定因素。这将通过人工构建的rna -蛋白质组装或已知对卵母细胞中特定mrna定位重要的蛋白质来实现。我们还将通过在玻璃表面构建定义的微管模式来测试微管细胞骨架结构对不同RNP物种的运输和锚定的影响。在一种互补的方法中，我们将破坏一个关键的mRNA运输蛋白的“低复杂性”片段，该片段在其他情况下与控制颗粒组装有关，并监测卵母细胞内外的影响。我们小组最近开发的高效果蝇基因组编辑技术将为这些实验提供便利。总的来说，这项工作将提供独特的见解，了解mrna如何在同一细胞中被分类，以及rna和蛋白质组装成颗粒如何影响它们的功能。通过揭示可用于调节动力蛋白运输的策略，我们的研究结果也将有助于了解机动运输其他货物的方式，包括膜结合囊泡和病毒。