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ESR 13 Project: Characterization of the quality control mechanisms that operate in human mitochondrial protein synthesis.

Partner: Zofia Chrzanowska-Lightowlers 

Institution: Newcastle University, UK

Duration: 36 months

Objectives: Despite the importance of mitochondria in cell physiology, there is still a paucity of knowledge concerning the players and mechanisms involved in gene expression in human mitochondria. This is particularly true for how mitochondrial mRNA is translated and how mitochondria are able to assure quality control of mitochondrial translation. In eubacteria and the eukaryotic cytosol there are clearly defined mechanisms that control the quality of protein synthesis. Ribosomes can stall for a number of reasons. This may reflect that the RNA has strong secondary structures or has been damaged, a change in binding proteins as a consequence of altered physiological conditions, stalls that occur naturally to ensure that the emerging polypeptide can correctly associate with chaperones before elongation continues, or a rare codon may be encountered and the cognate tRNA is limiting. Whatever the cause of the stall / pause there are mechanisms that function to dismantle stalled ribosome / mRNA complexes to prevent aminoacylated tRNAs becoming limiting and further interrupting elongation. A number of these mechanisms have been identified and characterized, but crucially none have been recorded in human mitochondria, despite the obvious need. Human mitochondria harbour 4 translational termination factor family members, all of which contain a GGQ motif that is regarded as characteristic of a ribosome-dependent peptidyl tRNA hydrolase. Although all 4 are essential for cell viability, only 1 of these members, mtRF1a, is involved in the canonical mechanism to terminate protein synthesis. It is our belief that the other members are involved in quality control of mitochondrial protein synthesis. The impressive technique of ribosome profiling generates data on the ribosomal occupancy of all translated transcripts and can do so with a nucleotide resolution (http://www.sciencemag.org/content/324/5924/218.abstract). This means that each transcript can be assessed for a number of parameters including the relative translation rate, the frame context, and naturally occurring stall positions. This technique uses next generation RNA sequencing to generate large data sets corresponding to the short RNA sequences that have been protected by ribosomes. We have established a modified protocol to analyse mitoribosomal profiling and have preliminary data that demonstrates we have the capacity to do this in our lab. In this project we aim to use this method to determine the roles of these translation termination factor family proteins in rescuing stalled mitoribosomes during protein synthesis, through which we will gain new insights into mechanisms regulating gene expression in the human organelle. Main objectives of this project are: (i) To investigate the mechanisms that control the quality of protein synthesis in mammalian mitochondria; (ii) To determine the roles of human mitochondrial translational termination factor family members in rescuing stalled mitoribosomes during protein synthesis.

Expected results: Even if a number of mechanisms that control the quality of protein synthesis have been identified and characterized in eubacteria and the eukaryotic cytosol, none have been recorded in human mitochondria. With this project, we expect to determine the role of some translational termination factor family proteins in quality control of mitochondrial protein synthesis.