Developing mitoribosomal profiling to investigate quality control of human mitrochondrial protein synthesis

Abstract

Mitochondria are essential organelles of nucleated cells that have the ability to conduct intra-organellar protein synthesis. Many aspects of this process are poorly understood especially the quality control and the rescue of stalled mitoribosomes. My project was focused on investigating potential candidates involved in quality control and ribosome rescue, including the release factor protein C12orf65. The importance of this protein in mt-translation was confirmed in a patient harbouring a mutation in C12orf65 gene, which displayed a general decrease in de novo mt-translation with subsequent disruption in assembly of OXPHOS complexes I, IV and V. I investigated the consequences on mitochondrial homeostasis in such a cell line. However, for a molecular understanding of the mechanism of this protein’s function a different, more focused approach was needed. To this end I applied ribosome profiling to mitochondria. Ribosome profiling provides a genome-wide analysis of protein synthesis by deep-sequencing of the mRNA fragments protected by ribosomes. It allows monitoring of progression of translation in vivo and can be used to identify contributions by regulating factors. I optimized the protocol for use in human mitochondria, initially on a cell line with a mutation in mt-tRNAVal. The decrease in stability of the uncharged tRNAVal resulted in an increase in mitoribosomal density over the valine codons, consistent with ribosomal stalling. I then used the final protocol, proven effective to study defects in mitoribosome progression, on cells with siRNA depleted C12orf65. Reducing transcript levels to 30% of control gave modest differences in mitoribosomal profiles. The control profiles, however, allowed normal features of mitochondrial translation to be identified. Although the exact function of C12orf65 remains unknown its involvement in mitochondrial protein translation is clear. Further applications of mitoribosome profiling to depleted or mutant cell lines should elucidate mechanisms that rescue stalled mitoribosomes and the potential role of C12orf65.