Mapping global mRNA fate: integrating translational and spatial dynamics

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.

The location and concentration of the cellular proteome shapes cellular traits and function; hence, the regulation of translation is of fundamental importance to many biological contexts. We have discovered mRNAs that are localised to, and translated in, granules during active yeast growth. These mRNAs encode abundant, heavily translated proteins in key pathways such as glycolysis, yet they interact poorly with the closed loop translation initiation complex making it likely that they are selected for translation in a fundamentally different manner.

This project will uncover the advantage that mRNA translation in granules offers cells. We have collected evidence supporting a view that granular translation allows cells to co-regulate protein production from the same pathway or same multi-protein complex. Hence, we will explore this in much greater depth, investigating mRNAs from specific metabolic pathways and protein complexes. We will study the mechanism of mRNA localisation taking both candidate-gene approaches and identifying co-purifying proteins. We will test how granular translation differs from the standard view of translation, via reduced reliance on the closed loop complex compared to other heavily translated mRNAs. We will also define the extent to which localisation allows cells to donate mRNAs en masse to provide a translationally primed "start-up pack" to their progeny, using affinity purification and next-gen sequencing to identify the attendant mRNA components.

The control of translation and mRNA fate is conserved mechanistically. Therefore, a careful dissection in yeast of the translation and localisation of the mRNAs will provide a framework for future studies in higher eukaryotes. It will also support efficiency improvements for exogenous added pathways in synthetic biology and bioprocessing applications. 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 defense against viral infection, iron homeostasis, severe diabetes mellitus and oxidative stress. A range of drugs targeting translation factors (e.g. silvestrol on eIF4A) or signaling 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 key mRNAs from the same pathway or complex 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 (potential alternative) mechanisms that might allow coordinated production of proteins from specific classes of mRNA.

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 and proteomics 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.