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

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We will test the hypothesis, based on our recent unpublished results, that clustering of RNA and motor molecules in granules is a key determinant of an mRNA's destination in the oocyte.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

The goal of this project is to gain molecular insights into the trafficking of mRNAs to specific sites within cells. This process plays important roles in the spatial control of protein function in a variety of contexts, including neuronal morphogenesis, synaptic plasticity, epithelial cell function and embryonic development. Drosophila oogenesis is a striking, and genetically tractable, example of a system that relies on mRNA localisation. mRNAs encoding proteins that pattern the embryonic axes are delivered to discrete sites in the oocyte by processes that depend on the minus end-directed microtubule motor dynein. Genetic screens have identified proteins that are required to direct dynein-associated RNPs to different locations in the oocyte but how they do this is not understood. We will address the long-standing question of how mRNAs are localised in the oocyte by exploiting a new in vitro assay for mRNA transport that uses purified components of the core dynein machinery. Our unpublished data suggest that variation in motor copy number could be key to mRNA accumulation in the dorso-anterior corner of the oocyte, a process essential for dorso-ventral patterning. We will build model RNA-protein assemblies and use single-molecule resolution imaging to reveal the effects and functional implications of motor number and cytoskeletal organisation on mRNA sorting. We will evaluate directly how proteins that affect mRNA localisation patterns in the oocyte influence RNP composition and behavior in vitro. These experiments will be complemented by in vivo dissection of RNP assembly, dynamics and function using live cell imaging and CRISPR genome editing. Collectively, this work will reveal novel strategies used to sort mRNAs, and molecular interactions that regulate RNP assembly and function in vivo.

In addition to the impact of the acquired knowledge on the academic sector, the proposed work will have a number of wider benefits (see 'Pathways to Impact' for details). The project will train a researcher in a range of cutting-edge techniques (molecular biology, multi-protein complex production, in vitro reconstitution, single-molecule microscopy, CRISPR mutagenesis, genetics and live cell imaging) that are of significant value in the academic and commercial private sector. A number of collaborations between academic groups will be cemented as a result of the project, which includes exchange visits of researchers between the UK and USA. This will lead to an enduring sharing of ideas and knowledge on a range of topics. Although the primary scientific goal of the project is to elucidate fundamental mechanisms in cell and developmental biology, we expect the work to also be of interest to those working in pharmaceutical and nanoengineering sectors. Specifically, the ability to modulate RNA localisation in cells is of significant interest to the emerging field of mRNA therapeutics, whereas reconstitution of complex cytoskeletal processes in vitro can inform the development of commercial 'lab-on-chip' diagnostic and sensor devices. To maximise economic and societal benefits of the work the post-doc and PI will meet with the technology transfer teams at LMB and LifeArc (formerly MRC technology), as well as with representatives of the pharmaceutical industry that have contacted the PI to express an interest in mRNA trafficking in the context of mRNA therapeutics. Finally, the vivid nature of mRNA trafficking processes lends itself to engaging students and educators with the beauty and importance of research in cell and developmental biology. The post-doc and PI will therefore develop and deliver a practical workshop on mRNA localisation for college students (16-18 years old). A website describing the workshop and associated background information will be produced so that it can be replicated by others, thus maximising the impact of the project. The PI will also develop a lecture for the general public that incorporates findings of the research project and present it at the Cambridge Science Festival and to non-scientific staff at LMB.