The Mitochondrial End Game : How key proteins control more than just translation termination

Summary Context of the research All cells in the body need fuel to live. The fuel production is controlled by a sub-compartment in every cell, an organelle called the mitochondrion. This organelle is extremely specialised and is the only part of the cell other than the nucleus that contains the genetic material, DNA. The 'code' in this DNA can be stored temporarily as 'message' molecules. These messages are used to dictate production of 13 proteins that are essential for fuel production. Each message contains signals that recruit the correct machinery required to make the protein and more specifically where to start or stop reading the code in the message. The principal part of this protein synthesis machinery is called a ribosome and is itself composed of ~80 independent proteins that have to be correctly assembled. After assembly is complete the ribosome has to interact with other components/accessory proteins in a particular sequence to ensure that the messages are correctly translated into proteins and accurately assembled to create functional protein complexes that generate fuel for the cell. Research has clearly shown that specific mutations in this mitochondrial DNA or malfunctions in any of the processes that help make and assemble the encoded proteins can lead to mitochondrial disease. These diseases primarily affect the muscles, nerves and brain, are progressive and are presently untreatable. Aims, objectives, potential applications, benefits The machinery needed to convert the messages in the mitochondrial DNA into functional proteins is considerably more complex than described above. From the little that is currently known about the human mitochondrial machinery, we know it is different to the machinery in the rest of the cell and different again to the bacterial and yeast systems. Even after decades of work on the latter 3 systems, where the key players in this translation process are known, there remains a great deal to be learned about new interactors and modes of regulation. Contrastingly, in human mitochondria, many of the key players have yet to be identified and this needs to be done in order to understand the basic mechanisms and the subtleties of regulation. We have now identified 3 proteins that are critical in the final stage of protein synthesis. Two are release factors that recognise the STOP signals in these 'messages'. The third is a ribosome recycling factor believed to assist in dis-assembling the ribosome ready for a new round of protein synthesis. As part of this work we will further characterise these, identify their structures and so clarify the mechanism by which they operate. A further aim of this proposal is to use technology already established in our group to trap, isolate and identify the cohort of proteins responsible for this critical function of mitochondrial protein synthesis. Using as 'bait' proteins that we have already shown are involved in synthesis and assembly, we can isolate currently unidentified proteins that interact very strongly. These are likely to be involved in or modulate, translation or assembly. We will then identify how these components contribute to organellar function, and how malfunction might cause disease. By generating a more complete picture of the key players in these processes governing protein synthesis and assembly we will gain a better understanding of how these proteins function. These are all proteins that are encoded by the nuclear DNA but are part of the mitochondrion. It is important to know how each of these newly identified proteins works normally to allow us to recognise how changes in them may cause disease. There is currently no therapy available for diseases caused by defects in the mitochondrially encoded proteins so this research is important as it will identify nuclear encoded proteins that crucial for healthy mitochondria that could be targets for future gene therapies and so help develop strategies to avoid mitochondrial disease.

Technical summary: Mitochondria are essential eukaryotic organelles. They perform many critical functions for the cell, one of which is to house the five multi-subunit complexes that couple ATP synthesis to cellular respiration, OXPHOS. Vital to this process is the synthesis of thirteen polypeptides in the mitochondrial matrix that are encoded by the organelles genome, mtDNA. Defects in the expression of mtDNA cause a wide variety of human disease and have been implicated in the ageing process. Given the importance of mitochondrial gene expression for cell viability, our relative lack of in depth knowledge of this process is concerning and it is important to address this. In our proposal, we intend to identify and characterise key players in one major aspect of mitochondrial gene expression - post-transcriptional mechanisms such as mRNA metabolism, translation termination, membrane insertion facilitators and OXPHOS complex integrators. In particular, we intend to test our hypothesis that the spatio-temporal coordination of mitochondrial translation and membrane insertion is exquisitely important for correct OXPHOS complex assembly. We intend to build on our recent work on translation termination and our substantial preliminary data to identify critical factors and characterise the molecular mechanisms underlying post-transcriptional gene expression. We will determine the exact mechanisms of translation termination using in vivo and in vitro assays. We will utilise our FLAG tag system to identify proteins associated with mitoribosomes and transcript poly(A) tails. Finally, we will assess the roles of several additional proteins whose yeast orthologues have been shown to facilitate membrane insertion and assembly of nascent mitochondrial polypeptides. These studies will greatly advance our current knowledge of the players and mechanism that underlie mitochondrial gene expression in man.