Investigation of human C12orf65 and mtRF1, members of the mitochondria translation release factor family

Abstract

Mitochondria contain their own DNA (mtDNA) and protein synthesis machinery. Human mtDNA encodes for two rRNAs, 22 tRNAs and 13 polypeptides. Those polypeptides are subunits of complexes involved in oxidative phosphorylation and their translation is driven by the mitoribosome. Translation consists of four phases - initiation, elongation, termination and mitoribosome recycling- with translation factors required at each step. My work focused on termination, which requires the action of release factors. In human mitochondria, there are four proteins in the release factor family: mRF1a, ICT1, mtRF1 and C12orf65. Termination occurs once a release factor recognises a stop codon (UAA or UAG) in the A site of the mitoribosome. This changes the mitoribosome conformation, inducing the polypeptide’s release. Only mtRF1a recognizes stop codons and terminates translation for all the 13 mtDNA-encoded polypeptides. However, all four members contain the GGQ motif involved in peptide release. ICT1 is a component of the mitoribosome large subunit. My research focused on the functions of mtRF1 and C12orf65 which are yet unknown, although it was observed that patients with C12orf65 pathogenic variants show impaired mitochondria translation. My research hypothesis was that C12orf65 and mtRF1 initiate termination in mitoribosomes that stall during translation. Stalling has multiple causes including mRNA pseudoknot structures, defective tRNA or insufficient amino acids. Release factors would recognise mRNA targets to the same effect as stop codons. To test this, I aimed to identify mRNA targets specific to mtRF1. I also aimed to detect proteins C12orf65 interacted with to confirm whether it was involved in termination. I used cross-linking and immunoprecipitation of the mitoribosome, BioID2 and CRISPR-Cas9 techniques. Thus, I obtained and characterized two BioID2 cell lines that helped locate C12orf65 interactors involved in translation. Moreover, I established a C12orf65 knockout cell line that presents a growth defect, further demonstrating C12orf65’s involvement in cell viability.