The function and regulation of translationally active RNA granules

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Almost all life forms require the capacity to produce energy and a mechanism to convert the information in genes into chains of different amino acids called proteins. Proteins are the principal effectors of biological function, responsible for catalysing most biochemical reactions including those that produce energy and those required for protein production itself. Genes are translated into protein from an intermediate molecule, messenger RNA (mRNA), in a process that is highly similar across all eukaryotic cells (animals, plants and fungi). Both proteins and mRNAs can be localised in cells to allow the generation of local concentrations of specific proteins, and this plays critical roles in the spatial development of cellular zones such as long projections on nerve cells or membrane protrusions on gut cells. mRNA localisation to such sites involves granules which contain the mRNAs in an inert, repressed state. Inert mRNAs can also become localised during cellular adversity, where two different classes of granule have been identified, 'stress granules' and 'P-bodies'. These granules are thought to play roles in both the storage of useful and destruction of surplus mRNAs. Additionally, their deployment has also been linked to human disease, especially in diseases of the brain and muscles, as well as fundamental roles in the development of multicellular animals, especially development of the embryo. We use the simple single-celled organism, brewer's yeast, as a model to study these fundamental processes. mRNA localisation both to defined polarised regions and to P-bodies or stress granules has been widely studied in yeast to uncover key principles that control protein synthesis. Yeast has served as a paradigm in such studies owing to its relatively simple genome, its level of similarity to human cells and the ease with which genes can be mutated, deleted or tagged in some way. In fact whole yeast strains collections have been constructed where all of the yeast genes have been systematically deleted or tagged, and these have facilitated a range of unbiased screens, where individual strains are tested for activity changes.Our recent studies have uncovered a particularly novel finding in yeast- mRNAs encoding non-localised proteins involved in energy generation and protein synthesis are present in mRNA granules even in actively growing cells. Counter to most mRNA localisation events, these mRNAs are not inert, instead they are translated into protein in these granules. We have extended this work in a number of directions. Remarkably, we have found that almost every mRNA across the two pathways is co-localised to and translated in granules. We have taken advantage of yeast as a system to perform unbiased screens and identify genes that are important for these granules. These genes encode proteins with functions such as RNA binding and protein folding. We have also taken hypothesis driven approaches to identify key determinants involved in the localisation of mRNA to granules. As such, we show that DNA sequences dictating mRNA production called promoters, as well as the chemical modification of mRNA are important in determining the fate of specific mRNAs to granules. In this project, we will decipher the precise determinants of mRNA selection to granules and how this impacts on the physiology of cells. We will examine how the mRNAs are marked for a granular fate and investigate the proteins that decipher these marks. We will also investigate the functional rationale for the localisation focussing on energy and protein production, especially during cell division. These fundamental studies will guide and inform studies in other systems including human, as well as provide alternative mechanisms to tweak industrial biotechnology systems where yeast is commonly used. The studies in this proposal may well allow optimisation at this level, especially for multi-protein biochemical pathways.