A systems analysis of the translational release factor as a coordinator of termination mRNA stability and ribosome recycling
Lead Research Organisation:
University of Aberdeen
Department Name: School of Medical Sciences
Abstract
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.
Technical Summary
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.
Planned Impact
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.
Organisations
Publications
Betney R
(2012)
Regulation of release factor expression using a translational negative feedback loop: a systems analysis.
in RNA (New York, N.Y.)
Bonnin P
(2017)
Novel mRNA-specific effects of ribosome drop-off on translation rate and polysome profile.
in PLoS computational biology
Ciandrini L
(2013)
Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.
in PLoS computational biology
Gorgoni B
(2016)
Identification of the mRNA targets of tRNA-specific regulation using genome-wide simulation of translation.
in Nucleic acids research
Gorgoni B
(2014)
Controlling translation elongation efficiency: tRNA regulation of ribosome flux on the mRNA.
in Biochemical Society transactions
Kemp AJ
(2013)
A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation.
in Molecular microbiology
Marshall E
(2014)
Ribosome recycling induces optimal translation rate at low ribosomal availability.
in Journal of the Royal Society, Interface
McFarland MR
(2020)
The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels.
in Nucleic acids research
Description | Project findings: A stochastic mathematical model has been developed to study the interaction between translation, recycling of ribosomes on the mRNA (whereby ribosomes that have terminated translation can 'go back to the beginning' and re-translae the same mRNA,) and mRNA turnover. That model has been experimentally validated, and is the subject of a key publication in this area (Gorgoni et al 2016). The experimental approaches allowed us to validate a model that predicts the translation of proteins across the complete transcriptome of a cell using a mathematical model, in response to changes in tRNA abundance. This was experimentally validated using cell mutants (in yeast) with altered tRNA abundance, which change ribosome queuing dynamics on the mRNA, and thus, protein synthesis efficiencies. The research showed that it is the codon composition of an mRNA, together with the codon confuguration, which is a key determinant of transaltional efficiency, through the formation of ribosome queues on the mRNA. Importantly, this key research output from this project has showed that the transaltion system is extremely robust to changes in tRNA concentration, and that only rare tRNAs have the propensity to significantly regulate ribosome flux along the mRNA. We have extended the Total Asymmetric Exclusion Process mathematical models of translation we have previously developed, to include a description of recycling of ribosomes. The integrated model is now being used to predict the effect of alternative stop codon positions within the mRNA to replicate the effect of premature termination. The model analysis was the subject of another publication emerging from this research, Marshall et al 2014. We have established the decay rates for a series of yeast PGK1 mRNAs that have either normally positioned stop codons, or premature stop codons engineered at various codon positions. The reearch shows that very early stop codons have an mRNA destabilising influence on mRNA stability that is independent of the nonsense-mediated mRNA decay pathway. Crucially, we also demonstrated that this destabilisation pathway is independent of the ribodsome recycling pathway, since mutants of the Rli1 recycling factor do not stabilise these early stop codon mutants. This work is currently being written for publication. We have developed a system of regulating tRNA expression using regulated tetR expression to control tRNA gene transcription. Doxycycline can thus be used to titrate tRNA expression. Using this system, we have varied expression of a suppressor tRNA to give between 0 and 3% readthrough on mRNAs with early stop codons to examine the effect on mRNA stability. We are now integrating model predictions and experiment. The TASEP model with recycling has been used analytically to show that there are counterintuitive relations between initiation rate, and translation rate. This prediction can be tested experimentally, by varying the translation initiation rate, to either switch recycling on or off, and this work is ongoing. Overall, the research has shown that in eukaryotes, the binding of poly(A) binding protein (PAB) to the poly(A) tail is central to maintaining mRNA stability. PABP interacts with the translation termination apparatus, and with eIF4G to maintain 3'-5' mRNA interactions as part of an mRNA closed loop. It is however unclear how ribosome recycling on a closed loop mRNA is influenced by the proximity of the stop codon to the poly(A) tail, and how post-termination ribosome recycling affects mRNA stability. We show that in a yeast disabled for nonsense mediated mRNA decay, mRNAs with early stop codons in the first 5% of the reading frame are still highly unstable, and that this instability cannot be significantly countered even when 50% stop codon readthrough is triggered. Early stop codons on mRNAs therefore drive instability of transcript in part through failure to deliver a bulk flow of ribosomes to the 3' natural stop codon position. In mutant yeast defective for Rli1-mediated ribosome recycling, reporter alleles with 3' proximal stop codons could not be rendered unstable, inferring defective Rli1 recycling is insufficient to trigger mRNA instability. Mathematical modelling of a translation system including the effect of ribosome recycling and poly(A) tail shortening supports the hypothesis that impaired ribosome recycling from 5' proximal stop codons may compromise initiation processes and thus destabilise the mRNA. A model is proposed wherein ribosomes undergo a maturation process during early elongation steps, and acquire competency to re-initiate on the same mRNA as translation elongation progresses beyond the very 5' proximal regions of the mRNA. |
Exploitation Route | Optimisation of translation is of considerable interest to biotechnology. We are applying the knowledge gained during this proposal to understand how best to optimise gene expression and translation through interactions with a biotechnology company, now the subject of a successful TSB proposal. |
Sectors | Manufacturing including Industrial Biotechology |
Description | The research on this project, dealing with the optimisation of protein synthesis. has led directly to the development of a research interaction with an SME biotechnology company, Ingenza Ltd., which then led directly to the successful application for a Technology Strategy Board grant with that company. |
First Year Of Impact | 2013 |
Sector | Manufacturing, including Industrial Biotechology |
Impact Types | Economic |
Description | Cafe Scientifique outreach talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | 40 members of the public attended a Cafe Scientifique presentation at a local bookshop on the subject of the application of synthetic biology to the diagnosis of African Sleeping sickness |
Year(s) Of Engagement Activity | 2015 |
URL | https://www.abdn.ac.uk/events/6584/ |
Description | Talk at World Fungus Day |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | A talk describing the industrial, medical and research impacts of fungi, andtheir role in everyday life. The talk was designed to inform, and change the views of the general public about the importance of fungi. |
Year(s) Of Engagement Activity | 2016 |