Dynamics and specificity of RNP granules

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.

The focus of this application is the formation and function of RNP granules: key determinants of mRNA fate in eukaryotic cells with wide-ranging roles in post-transcriptional control. We will comprehensively characterize the dynamic membership of RNP granules under non-stress and stress conditions (oxidative stress, heat shock, glucose starvation). This will include mRNA granules (sites of translation during active growth), P-bodies (sites of mRNA decay) and stress granules (sites of mRNA storage). We will also examine protein aggregates which form as a response to protein misfolding and aggregation, since they may relate to stress granules, possibly forming as solid phase particles from stress granules. Innovative quantitative proteomics and RNA-immunoprecipitation sequencing approaches will be used to systematically define the dynamic mRNA and protein constituents of RNP granules. This information will be used to address the driving forces that account for the specificity of protein and mRNA localization to RNP granules. Genetic approaches will be used to test the hypothesis that intrinsically disordered regions (IDRs) drive granule formation and RNA-binding proteins account for mRNA specificity in granules. Finally, a range of established techniques will be used to address the fate (translation, degradation or storage) of candidate mRNAs in different granules under distinct conditions to address the functional significance of granule formation. Thus far, the mechanisms controlling mRNA fate in granules are largely conserved, so a careful dissection in yeast of the composition, specificity and functional importance of RNA granules will provide a framework for future studies in higher eukaryotes. It will also support efficiency improvements for exogenous added genes in synthetic biology and bioprocessing applications. Finally, if mechanisms prove not to be conserved, they can be exploited via targeted approaches against pathogenic fungi.

Who will benefit from this research?
Researchers with wide-ranging interests in cell biology, biochemistry and molecular biology will benefit from the methodology/data generated by this project. This work will be of interest to researchers in medical biosciences, since RNA metabolism is linked with many disease processes. Additionally, the project will benefit industrial researchers interested in maximising protein yields from biological systems for biotechnological applications. Finally, the researchers on the grant will also gain in terms of scientific training and transferable skills, by importing and adapting novel proteomic technologies from collaborators.

How will they benefit from this research?
This research will increase our understanding of how cells alter mRNA/protein localization in response to stress. This may be medically significant, since knowledge of these control mechanisms may lead to a greater understanding of disease/pathology states and potentially lead to novel targets for therapeutics. A more detailed understanding of RNA granules and their role in mRNA fate could have important implications for various diseases including cancer, diabetes, viral infections, as well as aging, memory and rare genetic disorders affecting RNA-binding proteins (eg Fragile X syndrome), ribosomal proteins (eg Diamond Blackfan anaemia) and diseases affecting translation factors [Leukoencephalopathy with vanishing white matter (VWM), X-linked intellectual disability (XLID) syndrome]. A range of drugs also impact on RNA granules e.g. silvestrol acts on eIF4A to induce stress granules and is currently being evaluated for conditions such as transplant rejection, and 5-fluorouracil, a common chemotherapeutic agent, also induces stress granules. The yeast system offers significant advantages in terms of its tractability to genetic and biochemical manipulation and many aspects of mRNA localisation have been conserved form yeast to human cells. However, should RNA granule regulation in yeast rely on fungal-specific factors, then these could become a target for antifungal therapeutic strategies. Systemic infections caused by fungi such as Candida albicans and Aspergillus nidulans are very difficult to treat and often fatal. Therefore it is possible that patients and practitioners could benefit from this project. The project may also have significant industrial implications since yeast fermentations are extensively used in the production of biofuels and biopharmaceuticals, such as insulin (& insulin analogs), human serum albumin, hepatitis vaccines and virus like particles for vaccines. Such industrial yeast strains are subjected to various stress conditions during the course of manufacturing processes and our studies will examine gene expression during changing growth and stress conditions. Therefore, researchers in these industries will benefit from conceptual advances generated during this work.

What will be done to ensure that they benefit from the research?
Results will be disseminated through research seminars / scientific publications. Funding is requested to attend national and international conferences to allow researchers to publicize the research. Resources generated from this project will include yeast strains, plasmids, RIP-seq and proteomics data and will be made available through recognized repositories and upon request. Detailed protocols and primary data will be made available to academic collaborators. Manchester University has a good track record of encouraging public engagement. This includes regular open days to inform school children and the public about University research and tours of the research facilities at Manchester. This will allow the researchers to share their research findings with the public and to raise awareness of the importance of basic research. Manchester University maintains excellent links with the business sector which will allow us to exploit any potential for industry collaboration