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

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 We will test the hypothesis, based on our recent unpublished results, that clustering of RNA and motor molecules in granules is a key determinant of an mRNA's destination in the oocyte.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