Functional specialization of RNP granules in RNA metabolism

Although proteins are the principal effectors of all biological function they must first be made from a template molecule known as messenger RNA (mRNA). This process, known as translation, is a complex process that is essentially identical across all eukaryotic life (animals, plants, and fungi). The fate of mRNA molecules is therefore critically important in determining when, where and how much of each specific protein is made. Recent work has shown that many mRNAs are key components of a class of subcellular bodies known as RNA granules which have been postulated to play important roles in mRNA degradation, storage, and even the translation process itself. Indeed, these RNA-containing bodies or granules have recently been shown to play important roles in many neurodegenerative and musculodegenerative diseases such as Fragile X mental retardation, spinal muscular atrophy, Huntington's and Alzheimer's.

Subcellular bodies where mRNA can be stored were first discovered over 30 years ago and are now known to come in different flavours, representing specialised compartments that can segregate specific molecules inside a cell. Unlike other organelles, they have no membranes and often arise as a consequence of a change in the physical properties of specific proteins and RNAs. So rather like oil droplets in water, the proteins and RNAs self-associate into liquid droplets that form microscopically distinct bodies. They can subsequently be distinguished by the conditions under which they form, as well as the nature of the mRNAs and proteins that coalesce into them. Stress granules (SGs) and Processing bodies (PBs) are two such examples which have served as a paradigm for these biomolecular condensates. They are thought to rationalise mRNA content under times of stress- storing useful mRNAs and possibly destroying others. However, we don't really know which mRNAs are stored, which are degraded, and more importantly what determines the individual fates of the mRNAs in these granules. This is a prerequisite to understanding their functional role in helping cells and organisms to adapt to changing conditions.

In this project, we will use cutting-edge large-scale technologies to precisely define the molecular fate of the majority of the ~1400 mRNAs that we have previously shown localize to PBs. We will use a labelling technique to systematically examine the stability of the different mRNAs that are found in PBs following nutrient deprivation. Our hypothesis is that that some mRNAs are rapidly degraded in PBs to remove them from cells whilst others are more stable and provide a source of mRNAs that can rapidly resume protein production once the stress is removed. Mutagenesis approaches will be used to define the cis and trans-acting factors that control the fate of mRNAs in PBs. Whilst it is known that parts of the RNA decay machinery are found in PBs and so may promote RNA degradation, it is unknown how other mRNAs can survive in PBs essentially protected against degradation. Finally, we will examine how PBs and SGs interact to engender functionality during stress conditions, as they have traditionally been considered as distinct entities - despite many molecular components in common.

Yeast is considered a simple eukaryote and so is substantially easier to study than more complex multicellular organisms. Since all of the RNA granules utilised in yeast are also present in multicellular organisms such as animals and plants, our fundamental studies in yeast will guide and inform investigations in these other systems. Therefore, as well as having implications for human disease, this work will provide alternative mechanisms to tweak industrial biotechnology expression systems where yeast and plants are commonly used. The studies in this proposal may well allow optimisation at this level, especially where stress conditions prove an important factor in the industrial scale growth of an organism.

Stress granules (SGs) and processing bodies (PBs) are membrane-less RNP granules that assemble in response to stress. They form through liquid-liquid phase separation that is driven by high local concentrations of key proteins and RNAs to form condensates. PBs were originally hypothesised to be sites of cellular mRNA decay. However, PBs have recently been proposed to also act as storage sites for translationally repressed mRNAs that can exit PBs and re-initiate translation. These two functions are not mutually exclusive and hence this current project aims to characterize the fate of mRNAs that localize to PBs, focussing on what determines whether different mRNAs are destined for degradation versus storage. We will use a non-invasive metabolic labeling method to precisely measure the decay kinetics of mRNAs localized to PBs relative to their cytosolic counterparts. This will enable us to test the hypothesis that PBs provide a specialized microenvironment where certain mRNAs are degraded while others are earmarked for storage as part of an adaptive response to stress. We will define the cis-acting features that determine the individual fate of mRNAs in PBs as well as the associated factors that mediate it by combining genetics with advanced microscopy approaches to follow PB-localized mRNAs in mutants. The molecular mechanisms by which mRNAs are protected from enzymatic degradation within PBs are unclear and we will examine the requirement for recently identified PB proteins in mRNA stability. We will test the hypothesis that PB and SG formation is a continuum and that mRNAs initially stored in PBs can be passed onto SGs for longer term storage during prolonged stress conditions. Together, our studies will elucidate the functional roles of PBs in RNA metabolism and how these specialized microenvironments moderate gene expression to coordinate the cellular outcomes in response stress.