Mapping global mRNA fate: integrating translational and spatial dynamics

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The information content of genes in living cells is decoded to produce chains of different amino acids called proteins that dictate the identity and function of a cell. Proteins are the principal effectors of biological function, responsible for catalysing most biochemical reactions, as well as serving numerous structural and regulatory roles. They are translated into protein from an intermediate molecule, messenger RNA (mRNA), by a process that is highly similar across all eukaryotic cells (animals, plants and fungi). It is becoming increasingly clear that this translation process is a key regulatory step in the control of protein level (and hence biological function and cellular state), both by changing the level of translation of specific mRNAs and also by targeting the location. Both proteins and mRNAs can be localised within cells to facilitate the generation of local concentrations of particular proteins, and this plays critical roles in the spatial development of specific cellular zones such as axons on neurons or microvilli on intestinal cells. mRNA localisation to specific sites in cells usually involves granules which contain the mRNAs in an inert, translationally repressed state. mRNAs can also become localised during times of 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. Since mRNA localisation to defined cellular regions has been widely studied in yeast, linking cellular stress to mRNA localisation in both P-bodies and stress granules, we aim to use this simple model organism to uncover the fundamental molecular biology of this process for the control of protein synthesis. Our recent studies have uncovered two novel findings. Surprisingly, mRNAs encoding unlocalised proteins involved in routine pathways such as sugar metabolism and translation itself are present in mRNA granules, even in actively growing cells. Our experiments suggest that mRNA translation into protein occurs in these granules. In a second study, we have found that most of these localised mRNAs do not interact in a classical "closed loop" model of selection for protein synthesis, making it unclear how these mRNAs are translated. In this project we will use cutting-edge molecular technologies to decipher which mRNAs and proteins are present in the granules, find out how they are translated, and explore the biological reasons for their localisation. We will examine how mRNAs become localised and also assess how they are passed on to their daughter cells. Specifically, we will determine whether proteins from the same pathway or complex are co-ordinately produced and regulated at these sites. We will also test the hypothesis that key transcripts are passed on to daughter cells via these granules, as a means to provide a "start-up pack" of key mRNAs for developing progeny.Although yeast is a simple eukaryote, all known mechanisms of translational control utilised in yeast are present in mammalian cells. Hence, our fundamental studies in yeast will guide and inform studies in other systems including human, as well as provide alternative mechanisms to tweak industrial biotechnology expression systems where yeast is commonly used. The studies in this proposal may well allow optimisation at this level, especially for multi-protein complexes.