Specialised ribosomes facilitating cellular responses to oxidative stress

We study how cells control the conversion of nutrients (or food) into the new proteins that are required for life and how cells moderate these processes in response to environmental cues. Termed 'protein synthesis' this process occurs within relatively large and complex molecular machines called ribosomes that decode instructions relayed from the genome within intermediary molecules called messenger RNAs (mRNAs). Human cells each contain over a million ribosomes. mRNA decoding by ribosomes is made possible by the concerted action of 'helpers': protein synthesis factors and transfer RNAs (tRNAs). In concert they bring the necessary amino acids together with the instructions to ensure the correct proteins are made at the right time. Making the right proteins at the right time is critical when organisms have to respond to changing environments, especially those containing toxins or other harmful agents. How cells sense the changes and control their responses is critical to many areas of biology.
Until relatively recently it was assumed that ribosomes simply translated all mRNAs equally and that mRNA levels were a good proxy for the expression of genes. However increasingly accurate measurements of mRNA and protein levels in cells show that there can be a wide discrepancy between mRNA and protein levels. Translational control is the term used to describe a major contributor to these differences. Translational controls also allow changes in protein levels to be generated very rapidly in response to different signals. One stress we are studying here is the cellular responses to oxidative damage inducing agents which causes widespread repression of protein synthesis, but specifically allows translation of a subset of mRNAs, including antioxidant enzymes needed to overcome the stress imposed. At present how mRNAs overcome the general stress induced repression of protein synthesis and remain actively translated is not clear, but our previous work has provided strong clues and this is the focus of our proposal.
Ribosomes interact with many accessory proteins, which are present in lower amounts and can moderate ribosome activity. In this way ribosome interacting proteins may act to generate specialised ribosomes, for example ribosomes instructed to translate particular mRNAs. We have identified one family of these proteins for study in this proposal. These proteins are called La Related proteins or LARPs. As their name suggests, they are related to a protein called La, which was identified in the study of autoimmune disease. La and LARP proteins are RNA binding proteins that contain a conserved RNA binding domain called the La motif or LaM. The LARPs we are studying also bind active ribosomes. We will study two LARPs that our preliminary studies suggest function to give different translation outcomes. One is a potential activator of protein synthesis that allows targeted mRNAs to escape translational repression in response to oxidative stress. In this proposal we wish to uncover how it interacts with both ribosomes and mRNAs and how these interactions lead to enhanced protein synthesis of targets and their continued translation during oxidative stress. As such this proposal is primarily a basic science proposal. However the outcomes of the work may also be of interest to companies that produce specific proteins as drug therapeutics or commercial products. Improved understanding of protein synthesis mechanisms will assist in the design of optimized commercial protein expression or fermentation systems that are used to make advanced products and medicines.

Control of protein synthesis facilitates differential relative mRNA and protein abundances in eukaryotic cells and enables rapid reprogramming of cellular activities in response to environmental inputs. Oxidative stress rapidly represses global protein synthesis while allowing preferential translation of stress response mRNAs. Mechanisms enabling mRNAs to escape from stress-induced translational repression are currently poorly understood. Our preliminary work suggests one mechanism is the interaction of specific mRNA-binding proteins with ribosomes to create specialised ribosomes to promote or repress protein synthesis on target mRNAs. Specifically, our work examining translational control of yeast cells in response to oxidative stress identifies the La Related Proteins (LARPs) Slf1 and Sro9 as critical for translational control. LARPs are evolutionarily-conserved RNA-binding proteins. La is a human protein implicated in auto-immune disease. The yeast LARPs each bind to both specific mRNAs and actively translating ribosomes and remain ribosome-bound following oxidative stress. Loss of either factor confers oxidant sensitivity. Slf1 is required for production of stress-specific proteins and provide clues to its mechanism of action as an activator of protein synthesis. Sro9 appears functionally distinct. Our hypothesis is that by interacting with ribosomes these RNA-binding proteins create 'specialised ribosomes' with altered properties to control protein synthesis on specific mRNAs. We will test this hypothesis by studying two LARPs Slf1 and Sro9 that mediate mRNA-ribosome interactions to facilitate cellular responses to oxidative stress. Our work program will generate LARP mutants with altered properties. It will identify where on the ribosome and on target mRNA each LARP binds and will address the mechanisms of translational control using a range of molecular and yeast genetics techniques.

Researchers with wide-ranging interests in cell biology, biochemistry and molecular biology will benefit from the methodology/ data generated by this research project. The research will benefit industrial researchers, interested in maximising protein production from biological systems for biotechnological and biopharmaceutical applications. Additionally, this work will be of interest to researchers in medical biosciences, since alterations in protein synthesis and folding are implicated in numerous disease processes. Finally, the research staff on the grant will also gain in terms of scientific training and transferable skills.

This research will increase our understanding of how cells alter gene expression patterns to respond to oxidative stress conditions. All organisms must respond to changes in their external environment and the translation of mRNA into protein is a fundamental component of the gene expression pathway. Our yeast studies will provide a framework for researchers in medical and disease-related fields to understand these changes. This will potentially benefit researchers in the study of diverse diseases and pathologies which involve reactive oxygen species. The yeast system offers significant advantages in terms of its tractability to genetic and biochemical manipulation. An added benefit is that studies using the yeast model system provide an alternative to animal-based studies. Living with environmental change is a priority area for the BBSRC and our research will uncover mechanisms used by cells to cope with changes in oxidative stress. This research will provide information and tools to increase the knowledge needed to enable organisms to build resilience and adapt to environmental change. The project may have significant industrial implications since yeast fermentations are extensively used in baking and brewing as well as for the production of biofuels and biopharmaceuticals. Industrial yeast strains are subjected to various stress conditions during the course of manufacturing processes. Our findings will increase understanding of the yeast oxidative stress responses, which may be extended to industrial usage of yeast in commercial settings. We have a letter of support from one protein-expression company, Novozymes, that is interested in our findings. Researchers in these industries will benefit from conceptual advances generated during this work.

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 publicize this research. Resources generated from this project are likely to include yeast strains, plasmids and sequencing data and 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 includes tours of the research facilities at Manchester. This allows 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, which will allow us to exploit any potential for collaboration with industry. This is managed by the faculty Business Development Team, which provides support and information to help academics develop relationships with business.