Dynamics and specificity of RNP granules

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Proteins are the principal effectors of biological function, responsible for catalyzing most biochemical reactions, as well as serving numerous structural and regulatory roles. Proteins are 'translated' from an intermediate molecule, messenger RNA (mRNA), by a complex process that is essentially identical across all eukaryotic life (animals, plants and fungi). It is becoming increasingly clear that the localization of mRNA within the cell is critically important and can play roles in the degradation, storage and the translation process itself. Indeed, we are discovering that these RNA-containing protein bodies or granules are important in a number of neurodegenerative and musculodegenerative diseases such as Fragile X mental retardation, spinal muscular atrophy, Huntington's and Alzheimer's. Recent evidence suggests that the physical properties of the RNA and protein are important in the formation of such granules. These include the likelihood to form aggregates and the capacity to form bodies that have a liquid-like state: these have been called 'liquid droplets' and form by phase separation in a manner analogous to the separation of vinegar and oil from a vinaigrette mix. Critically, these liquid droplets concentrate protein and RNA allowing specific reactions to occur and also seeding the more static aggregation of proteins and RNA.Recently, there has been a huge upsurge in the identification of different RNA containing granules. These granules can have different functions and are observed under different conditions. In the main, such granules have been observed after cells have been switched to unfavourable growth conditions (so-called 'stress' conditions) where the granules appear to play roles in the degradation or storage of mRNA and protein. However, we don't really know which proteins and RNAs are important for the formation of which granule types, nor how and why they form in the first place.Our recent studies have uncovered two novel findings. Firstly, a class of RNA containing granules exist even in 'normal' actively growing cells where mRNA translation into protein can occur. In a second study, we have found that RNA granules formed after stress are remarkably similar to protein aggregates that can be purified by virtue of their capacity to sediment. In this project, we will use cutting-edge 'omic technologies to precisely define the molecular composition of RNA-containing granules using yeast as a model system. We will examine how the composition of these granules changes before and after different stresses and characterize the relationship between the different RNA granule classes to each other and to protein aggregates. Finally, we will examine a key question, which is what are the molecular parameters determining how different proteins and RNAs arrive in different granules to ultimately have distinct fates.Although yeast is a simple eukaryote, all of the RNA granules utilized in yeast are also present in higher 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 optimization at this level, especially where stress conditions prove an important factor in the industrial fermentation.