Solution structure and siRNA‐mediated knockdown analysis of the mitochondrial disease‐related protein C12orf65

Abstract

Loss of function of the c12orf65 gene causes a mitochondrial translation defect, leading to encephalomyopathy. The C12orf65 protein is thought to play a role similar to that of ICT1 in rescuing stalled mitoribosomes during translation. Both proteins belong to a family of Class I peptide release factors (RFs), all characterized by the presence of a GGQ motif. Here, we determined the solution structure of the GGQ-containing domain (GGQ domain) of C12orf65 from mouse by NMR spectroscopy, and examined the effect of siRNA-mediated knockdown of C12orf65 on mitochondria in HeLa cells using flow cytometry. The GGQ domain, comprising residues 60-124 of the 184-residue full-length protein, forms a structure with a 3(10) -β1-β2-β3-α1 topology that resembles the GGQ domain structure of RF more closely than that of ICT1. Thus, the GGQ domain structures of this protein family can be divided into two types, depending on the region linking β2 and β3; the C12orf65/RF type having a 6-residue π-HB turn and the ICT1 type having an α-helix. Knockdown of C12orf65 resulted in increased ROS production and apoptosis, leading to inhibition of cell proliferation. Substantial changes in mitochondrial membrane potential and mass in the C12orf65-knockdown cells were observed compared with the control cells. These results indicate that the function of C12orf65 is essential for cell vitality and mitochondrial function. Although similar effects were observed in ICT1-downregulated cells, there were significant differences in the range and pattern of the effects between C12orf65- and ICT1-knockdown cells, suggesting different roles of C12orf65 and ICT1 in rescuing stalled mitoribosomes.

Optic Atrophy and a Leigh-Like Syndrome Due to Mutations in the C12orf65 Gene

“…Kogure et al (2) found that knockdown of the C12orf65 protein in cell culture assays resulted in an increase in reactive oxygen species and apoptosis. Mitochondrial dysfunction was demonstrated by impaired cytochrome c oxidase activity.…”

“…The C12orf65 gene encodes a mitochondrial matrix protein that is critical for the release of newly synthesized proteins from mitochondrial ribosomes. Loss-of-function mutations impair the translation of mitochondrial proteins, with a resultant decrease in the protein complexes necessary for effective oxidative phosphorylation (1,2). The COXPD7 phenotype has been described in 2 distinct pedigrees by Antonicka et al (1).…”

Delineation of C12orf65-related phenotypes: a genotype–phenotype relationship

“…6 The entire group of the organellar release-factor family is characterized by the presence of a conserved peptidyl-hydrolase domain containing the universally conserved Gly-Gly-Gln (GGQ) motif, 7 which interacts with the large ribosomal subunit to release the polypeptide chain from the P-site bound peptidyl-tRNA. This GGQ-containing domain stretching from amino acid 57 to 121 in the human 166-residue full-length protein 8 is not affected by the mutations that lead to a relatively mild phenotype. However, both in family B and in the previously described patients with the severe phenotype 2 the mutation interrupts this highly conserved domain C12orf65 and ICT1, in contrast to other class I release factors, share C-terminal region comprising 20 basic residues.…”

“…However, both in family B and in the previously described patients with the severe phenotype 2 the mutation interrupts this highly conserved domain C12orf65 and ICT1, in contrast to other class I release factors, share C-terminal region comprising 20 basic residues. 8 Gagnon et al 9 showed that in the bacterial ICT1 homolog, YaeJ, the C-terminal tail functions as a sensor to discriminate between stalled and actively translating ribosomes. The mutations leading to a mild phenotype are expected to cause, in addition to a shortened C terminus, a less basiccharged tail, which may reduce the effectiveness of the interaction with the phosphate backbone of the ribosomal RNA that forms the walls of the ribosomal channel.…”

Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons

“…These insertions consist of three amino acids (Gly-Leu-Ser) in the stop codon recognition loop and two residues (Arg-Thr) in the short loop preceding the a5 helix. The other two homologues of the bacterial release factors (RFs), ICT1 and C12orf65, are both mitochondrial proteins with critical functions for cell viability and have been proposed to be involved in rescuing stalled ribosomes 9,15,16 . ICT1 has further been shown to be an integral part of the mitoribosome 9 .…”