Ribosome assembly, proteolysis and pathogenesis in human mitochondria

Abstract

The human mitochondrial genome is approximately 16.6kbp and encodes 22 tRNAs, 2 rRNAs and 13 polypeptides. These polypeptides are synthesised by the mitoribosome, composed of a large (39S) and small (28S) subunit. Although recently published cryo-EM structures of the human, porcine and yeast mitoribosomes have identified a number of significant differences including new components with uncharacterised functions. One such component was mS38 (AURKAIP1) as a part of the mt-SSU of the mitoribosome. This was consistent with previous work in my host laboratory using a siRNA screen of candidate mitochondrial proteins, which identified AURKAIP1 as having a role in mitochondrial gene expression. I continued this work and confirmed levels of AURKAIP1 are tightly controlled within the cell. Overexpression of both AURKAIP1 and AURKAIP1-FLAG caused disruption to the mitoribosome and mitochondrial translation, and depletion of AURKAIP1 impaired cell growth and resulted in cell death. Although the exact function of AURKAIP1 is still elusive, AURKAIP1 may have a role as an RNA chaperone. When overexpressed, AURKAIP1 was cleaved by the mitochondrial matrix protease LONP1, from a 25kDa to a 15kDa species by two cleavage events, although this could not be determined in the endogenous species. This work led to the characterisation of LONP1 variants that were believed to be pathogenic. Whole exome sequencing identified two novel mutations (p.Tyr565His; p.Glu733Lys) in LONP1 in a paediatric patient with a severe mitochondrial disease. By cloning and sequencing LONP1 from patient samples, I showed compound heterozygosity in the patient for 2 independent mutations, confirming the disorder to be autosomal recessive. Skeletal muscle showed severe defects in OXPHOS complexes I, III and IV, reduced mtDNA copy number and a decrease in the steady state levels of TFAM. Steady state levels of LONP1 remained unaffected. The p.Tyr565His mutation is localised to a highly conserved aromatic-hydrophobic (Ar-φ) motif present in hexameric ATP-dependent proteases and in vitro assays indicated a retained ability to hydrolyse ATP but a loss of proteolytic activity. This suggests that the mutation is likely to prevent the substrate from reaching the proteolytic site.