NEW / CURRENT: Systems modelling of a translational negative feedback loop: an in vivo toolkit to dissect ribosomal termination and mRNA surveillance

Recent years have seen a host of genome sequences being completed, including of course the human genome. Each gene in a genome is used to direct the synthesis of a specific protein. It is the proteins that are the functional agents in a cell, for example acting as catalysts to speed individual chemical reactions within the cell. Information in the gene, coded as different sequences of the bases A, T, C and G, is used to make a protein in a two-stage process. First, the gene information is copied into a similar chain of bases in the form of a messenger RNA (mRNA). The mRNA, a long chain-like molecule, is then used as an information store to direct the assembly of a protein, consisting of a chain of amino acids, in a process called translation. The precise sequence of amino acids (directed by the mRNA base sequence) determines the eventual function of the protein. The amino acid sequence is defined by the mRNA sequence, which in turn is defined by the gene sequence, thus linking gene to protein. The process of translation forms the focus of this research proposal. During translation, small particles called ribosomes (themselves made of RNA and protein) travel along the mRNA, sequentially adding amino acids to make the final protein. This production line process is stopped (terminated) in response to a specific sequence of bases in the mRNA, causing the release of the completed protein. Termination is crucial for ensuring the protein made is of the correct length. It is now known that following termination, ribosomes are directed back to the beginning of the mRNA, effectively recycling them on the mRNA chain. This makes the translation process more efficient, but generates very complex ribosome traffic flow on the mRNA production line. For this reason, mathematical modelling of ribosome flow will be used in this research alongside the biochemical experimentation to help unravel the mechanisms by which translation is controlled. This proposal seeks to study translation termination for two important reasons. First, in many human genetic diseases, the affected gene (e.g cystic fibrosis, Duchenne muscular dystrophy) is mutated because it contains an additional stop codon early in the gene sequence. This has the effect of prematurely terminating translation, resulting in a shortened, non-functional protein. There is increasing interest in developing drugs that would make translation termination less accurate. This would allow the ribosome to bypass the early stop codon and reach the natural stop codon to make correct length protein. Research into the molecular mechanisms of termination, as this proposal describes, can provide crucial insight used directly in the development of drugs to treat some forms of human genetic disease. Termination is also important because associated with this process is the recycling of the ribosomes on the mRNA. After termination at the end of the message, ribosomes are actively returned to the beginning of the mRNA to make a new protein from the same template, forming a type of circular ribosomal race track; each circuit of the track results in a new protein being made. The recycling process is very poorly understood, and yet it is key to protein synthetic efficiency. By understanding how recycling works, it may be possible to boost the efficiency of protein synthesis in cells, which is crucial for the manufacture of drugs like hepatitis B vaccine and insulin, to name but two. In summary then, the process of translation termination is crucially important in the expression of genes in every cell, and thus has fundamental 'pure' research interest. It is however also a key to understand how proteins can be made efficiently in biotechnological processes important in drug manufacture, and is also an attractive target for drugs that can treat a range of extremely debilitating human genetic diseases.

Ribosomal protein synthesis involves the translation of the mRNA template in a process that can be considered as comprising three distinct stages; initiation, elongation and termination. In eukaryote translation, during the termination phase, stop codons are recognised by a release factor complex to trigger release of the nascent polypeptide. It is increasingly recognised however that the release factors do not simply terminate translation, but also act as a protein hub to co-ordinate a number of other processes central both to the control of mRNA stability, and to the maintenance of efficient mRNA translation. This is achieved through interactions between the release factors and (i) poly(A) binding protein (ii) Upf1, the nonsense-mediated mRNA decay factor, and (iii) Rli1, the ribosome recycling factor. These interactions involve the dynamic remodelling of the termination complex, and depend upon competitive interactions between these proteins and the release factors. This project will use an integrated systems analysis of the dynamic flux of ribosomes along the mRNA, coupled with a model of the termination process, to identify how mRNA stability is dictated by the interplay between natural mRNA decay, nonsense mediated decay, and translational activity. Since the release factor complex also coordinates ribosome recycling, this proposal will also investigate the role of termination in governing the recycling of ribosomes on the same mRNA. Systems biology approaches will integrate modelling and experimentation to dissect the functional consequences of termination complex remodelling, and to define how stop codon position and translational efficiency govern protein productivity and stability of an mRNA.

The process of translation termination is carried out by the release factor complex, which forms the hub of a dynamic protein complex that regulates nonsense-mediated decay (NMD), normal mRNA decay, and ribosome recycling. Translation termination and NMD represent important potential drug targets for the treatment of human genetic diseases caused by premature stop codons, such as cystic fibrosis and Duchenne muscular dystrophy. Targeting these processes could enhance stop codon readthrough, or stabilise otherwise unstable mRNAs, thus relieving clinical symptoms. This principle has already been established with the treatment of some genetic conditions using drugs that target these processes, using aminoglycosides as well as a new drug, PTC124. One key beneficiary of this research will therefore be pharmaceutical companies, as the research here potentially identifies new drug targets. Treatments for diseases such as cystic fibrosis have large potential global markets, and thus in the longer term, this research has the potential to enhance UK competitiveness in the pharmaceutical sector. The processes of mRNA turnover, and importantly, ribosome recycling on the mRNA, are crucial determinants of translational efficiency, and regulators of the efficiency of gene expression. Thus research into how these central processes are controlled will help understand how recycling of ribosomes on mRNAs may be regulated, and thus enhanced. This research will therefore have as key beneficiaries biotechnology companies seeking to express proteins at high level, including vaccine components, therapeutic proteins including anti-cancer therapeutics and antibodies, as well as other industrially-used proteins. Thus medicine and the biotechnology industry will be key beneficiaries of this research, which has the potential to enhance health, identify new treatments for disease, and enhance industrial competitiveness. In the longer term, members of the public may benefit from this research, as developments of new pharmaceuticals, and the industrial production of heterologous proteins become available as products that enhance health and the quality of life. The UK trained workforce will benefit from this proposal through the training of two PDRA researchers, both of whom will acquire an ability increasingly in demand, that of the ability to work across discipline boundaries as part of pharmaceutical research, and systems and synthetic biology research. As part of teaching in a research led environment, undergraduates and masters students will also benefit from teaching imparted by researchers on this project. Part of this teaching will involve tutoring undergraduates, through their participation in iGEM, the leading synthetic biology competition organised from MIT, Boston; both Aberdeen and Imperial regularly participate in iGEM. This will impact upon the supply of the new generation of graduates with skills in the synthetic biology field, with its huge potential for future industrial application. The general public will benefit from this research as part of the University of Aberdeen's vibrant outreach programme, involving Café Scientifique and TechFest programmes, the latter an annual science festival for the general public. IS regularly gives research talks aimed at a lay audience at these events, as well as visiting on average 4 schools per year to give science talks connected with his research. Additionally, key research findings that may be of general interest to the public will be communicated to both local and national media outlets via press releases.