AFG3L2 supports mitochondrial protein synthesis and Purkinje cell survival.

Mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia type 28 and spastic ataxia-neuropathy syndrome in humans; however, the pathogenic mechanism is still unclear. AFG3L2 encodes a subunit of the mitochondrial m-AAA protease, previously implicated in quality control of misfolded inner mitochondrial membrane proteins and in regulatory functions via processing of specific substrates. Here, we used a conditional Afg3l2 mouse model that allows restricted deletion of the gene in Purkinje cells (PCs) to shed light on the pathogenic cascade in the neurons mainly affected in the human diseases. We demonstrate a cell-autonomous requirement of AFG3L2 for survival of PCs. Examination of PCs prior to neurodegeneration revealed fragmentation and altered distribution of mitochondria in the dendritic tree, indicating that abnormal mitochondrial dynamics is an early event in the pathogenic process. Moreover, PCs displayed features pointing to defects in mitochondrially encoded respiratory chain subunits at early stages. To unravel the underlying mechanism, we examined a constitutive knockout of Afg3l2, which revealed a decreased rate of mitochondrial protein synthesis associated with impaired mitochondrial ribosome assembly. We therefore propose that defective mitochondrial protein synthesis, leading to early-onset fragmentation of the mitochondrial network, is a central causative factor in AFG3L2-related neurodegeneration.

NOA1 is an essential GTPase required for mitochondrial protein synthesis.

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1⁻/⁻ cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1⁻/⁻ cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis.

Translation termination in human mitochondrial ribosomes.

Mitochondria are ubiquitous and essential organelles for all nucleated cells of higher eukaryotes. They contain their own genome [mtDNA (mitochondrial DNA)], and this autosomally replicating extranuclear DNA encodes a complement of genes whose products are required to couple oxidative phosphorylation. Sequencing of this human mtDNA more than 20 years ago revealed unusual features that included a modified codon usage. Specific deviations from the standard genetic code include recoding of the conventional UGA stop to tryptophan, and, strikingly, the apparent recoding of two arginine triplets (AGA and AGG) to termination signals. This latter reassignment was made because of the absence of cognate mtDNA-encoded tRNAs, and a lack of tRNAs imported from the cytosol. Each of these codons only occurs once and, in both cases, at the very end of an open reading frame. The presence of both AGA and AGG is rarely found in other mammals, and the molecular mechanism that has driven the change from encoding arginine to dictating a translational stop has posed a challenging conundrum. Mitochondria from the majority of other organisms studied use only UAA and UAG, leaving the intriguing question of why human organelles appear to have added the complication of a further two stop codons, AGA and AGG, or have they? In the present review, we report recent data to show that mammalian mitochondria can utilize a -1 frameshift such that only the standard UAA and UAG stop codons are required to terminate the synthesis of all 13 polypeptides.

A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome.

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.