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ESR 4 Project: Impact of inaccurate mitochondrial translation on stress signaling and ageing

Partner: Martin Ott

Institution: Stockholm University, Sweden

Duration: 36 months

Objectives: To date, nothing is known about fidelity of translation in mitochondria, one of the most important features of the process by which genetic information is translated into function. Mitochondrial ribosomes, which evolved from the translation apparatus of the eubacterial ancestor of the organelle, were traditionally considered to function essentially identical to the very wellstudied bacterial ribosomes. Recent structural as well as proteomic data strengthen the view that mitochondrial ribosomes instead are surprisingly different. To date, no in vitro assay is available to directly dissect and analyze the different steps and fidelity of translation by mitochondrial ribosomes. In this project, we will combine the power of yeast mitochondrial genetics with wellestablished biochemical and cell biological assays to investigate mitochondrial translation accuracy. Our strategy will be to generate novel mitochondrially encoded reporter genes that will allow direct readout of how many mis-incorparations of amino acids into mitochondrial translation products occur in the cell. These reporter constructs will allow us for the first time to directly determine translational accuracy and efficiency in the cell. In addition, we attempt to establish a basic in vitro system to stuy mitochondrial translation relying on our novel methods to purify mitoribosomes. Importantly, we will test how mitochondrial translation is affected by changes in cellular physiology or when cells are exposed to toxins. We will combine these efforts with the characterization of mutations in mitoribosomal proteins that are conserved between bacteria and the organelle and that have previously been shown to alter fidelity of translation by bacterial ribosomes. Because an imbalance of mitochondrial and nuclear gene expression modulates the ageing process, we will extend these studies to the analyses of yeast replicative life span and how it is affected in mutants affected in mitochondrial translation accuracy. In turn, again employing our mitochondrially encoded reporter constructs, we will assess how accurate mitochondrial translation is in young and aged cells.

Expected results: These projects will shed new light on mitochondrial translation. The generation of novel, innovative reporter constructs integrated into the mitochondrial DNA and a basic in vitro assay for mitochondrial translation will allow detailed studies of mitochondrial translation. The creation of mitoribosomal protein mutants affecting translational accuracy will enable us to analyze in detail how an imbalance of mitochondrial and nuclear gene expression affects cellular physiology and the ageing process. This project will therefore increase our knowledge on mitochondrial translation and shed light on the physiological consequences of a dysfunctional mitochondrial translation system.