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ESR 14 Project - Characterizing the coordinated regulation and localization of mitochondrial protein synthesis

Work Package 7: Identification of novel factor governing mitochondrial ribosomal assembly and protein synthesis.

Partner: Robert Lightowlers

Institution: Newcastle University, UK

Duration: 36 months

Objectives: It is estimated that > 150 nuclear encoded proteins need to be imported to facilitate mitochondrial protein synthesis. Defects in a wide array of these proteins have already been associated with mitochondrial disease, but in many cases the exact function of the pathogenic protein is unknown. Approximately 80 of these proteins are mitoribosomal proteins. Studying this is very important as mammalian mitochondrial ribosomes differ substantially from both eubacterial 70S and eukaryotic 80S particles. The most striking difference is the change in RNA to protein ratio. Both 70S and 80S ribosomes share a characteristic ratio of 70% RNA: 30% protein, whilst mammalian mitoribosomes have completely reversed this to approximately 70% protein : 30% RNA, to generate a 55S particle. This change in ratio is not universal to mitochondria from all species, even yeast that are often used a model organism for studies of human mitochondria are more similar to eukaryotic 80S or bacterial 70S ribosomes. This change in mitochondrial content has resulted in approximately 50% of the mitochondrial ribosomal proteins (MRPs) having no orthologues to help us define their function. One of the aims of this project is to characterize the roles of a subset of these human MRPs for which there is no function assigned or no bacterial homologue. Assembly of a fully functional ribosome is a demanding but critical part of cell metabolism, reflecting the fundamental requirement for protein synthesis. In most organisms the ribosome is composed of 50 or more components that have to be correctly assembled to generate a small and large subunit, each with the appropriate panel of modifications made to either the RNA or polypeptides. For yeast cytosolic ribosomes over 200 assembly factors have been identified that are involved in this process, whilst bacterial ribosome biogenesis appears to require only a few including the GTPases Era. Similarly, the factors reported to date that are required for mitoribosome assembly are few eg. EraL1, MTERF4/NSUN4, and Noa1. One approach to identify further candidates for mitoribosomes assembly is by analogy to bacterial assembly factors. We have identified a number of proteins that we believe are involved in either assembly or disassembly of mitochondrial ribosomes. Therefore, the second aim of this project is to characterize assembly factors that are critical in the generation of a fully functional human mitoribosomes. We also have the advantage that whole exome sequencing of patients with defects in mitochondrial protein synthesis is being undertaken in Newcastle. Several candidate pathogenic mutations have now been identified. This may allow the student to begin to dissect the function of one of these proteins using an array of biochemical, cell biological and molecular biological techniques that have been established in our laboratory. Therefore the main objectives of this project are: (i) To study the roles of human mitochondrial ribosomal proteins having no bacterial orthologues; (ii) To characterize assembly factors, which are required for the generation of human mitoribosomes.

Expected results: This project aims to characterize novel factors involved in the correct assembly of human mitochondrial ribosomal proteins to facilitate normal protein synthesis, as well as to determine the molecular mechanisms that govern the assembly process.