The function and regulation of translationally active RNA granules

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.

mRNA granules are critical for localised translation and stress responses: from neuronal synaptic translation to mRNA storage in stress granules. Most granule-localised mRNAs are translationally inert, only becoming active after reaching their destination or following stress relief. We have recently found two different mRNA containing granules, the CoFe granule and TFG, that harbour translationally active mRNAs, glycolytic mRNAs and translation factor mRNAs, respectively. These mRNAs are very highly translated, hence, their concentration to granules may prove critical in achieving the levels of protein required for cellular proliferation.
The overarching aim of this project will be to uncover how the mRNAs are localised and the functional consequences of this localisation. We have combined yeast genetics with high throughput microscopy to identify over 250 deletion strains with altered patterns of CoFe and/or TFGs. This has highlighted a range of functions with RNA binding, chaperone and signalling activities predominating. Hypothesis driven approaches have also identified key determinants in mRNA localisation to granules: promoter elements and mRNA modifying enzymes. We will pursue these observations by studying the fundamental connections linking - RNA binding activities, chaperones, signalling, the nuclear history of a transcript and mRNA modifications - to an mRNA's cytoplasmic fate.
Glycolysis, protein synthesis and the control of mRNA fate are highly conserved and fundamental cellular processes. A careful dissection in yeast of mRNA localisation for such pathways will provide a framework for future studies in higher eukaryotes. It will also support efficiency improvements for exogenously added pathways in synthetic biology. Finally, if the mechanism proves not to be conserved, it can be exploited via a targeted approach as a strategy against pathogenic fungi.

Who will benefit from this research?
The non-academic beneficiaries of this project can be divided into two areas; those in the medical and industrial sectors. In terms of medical impact, mRNA localisation and translation play roles in a host of medically related topics. For instance, various connections exist between mRNA localisation and neurodegenerative and musculodegenerative diseases such as Fragile X mental retardation, spinal muscular atrophy, Huntington's and Alzheimer's. Other conditions are linked to deficiencies in translational control such as the defence against viral infection, iron homeostasis, severe diabetes mellitus and oxidative stress. A range of drugs targeting translation factors (e.g. silvestrol on eIF4A) or signalling molecules linked to translation (e.g. everolimus on mTOR) are currently being evaluated for conditions such as transplant rejection or cancer. Furthermore, the discovery of disease-causing mutations in translation factors (e.g. for VWM and Wolcott-Rallison syndromes) identifies translation as an important clinical target. Since most aspects of mRNA localisation and translation have been conserved form yeast to human cells, it seems likely that the localised translation of glycolytic and translation factor mRNAs will prove important in humans. However, should localised mRNA translation 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.
In terms of industrial beneficiaries, any company using eukaryotic organisms for bioprocessing or synthetic biology will benefit from greater understanding of new mechanisms such as the control of mRNA localisation to allow the coordinated production of proteins across the same pathway.

How will they benefit from this research?
The results of the research proposed here will enrich general understanding of the interplay between mRNA localisation and translation in eukaryotes. This greater understanding could inform and ultimately improve treatments of diseases like cancer and viral infection, as well as advance new models of translational control that factor in cellular location. As a result, this work could lead to increased efficiency in bioprocessing and synthetic biology applications, by supporting the development of advanced models of translation that extend the lifetimes of such systems, reduce cellular stress, and thereby improve product yields.

What will be done to ensure that they benefit from this research?
Results will be disseminated through research seminars, presentations at conferences and publications in scientific journals. Funding is requested to attend national and international research conferences to allow the researchers to publicise this research. Resources generated from this project are likely to include next generation sequencing data (which will be deposited in open repositories), as well as yeast strains and plasmids that will be made available to the scientific community upon request. Detailed protocols and primary data will be made freely 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. This will allow the researchers to share their research findings with the wider public and to raise awareness of the importance of basic research. Manchester University maintains excellent links with the business sector allowing researchers to exploit any potential collaboration with industry. This is managed by the faculty Business Development Team, who provide support and information for staff wishing to develop relationships with business.