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.

SummaryVarious specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the mitochondrial matrix. GRSF1 (G-rich sequence factor 1) is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the mitochondrial matrix. These foci are composed of nascent mitochondrial RNA and also contain RNase P, an enzyme that participates in mitochondrial RNA processing. GRSF1 was found to interact with RNase P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of mitochondrially encoded proteins and mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNase P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures “mitochondrial RNA granules.”

The molecular mechanisms by which mutations in the gene for Cu/Zn superoxide dismutase (SOD1) lead to the selective death of motor neurones in familial amyotrophic lateral sclerosis (FALS) remain incompletely understood. Previous evidence has indicated that mitochondrial abnormalities may develop during motor neurone injury, but several important questions remain unanswered. We have developed a cell culture model of FALS in which a motor neurone cell line (NSC34) has been stably transfected to express normal or mutant human SOD1 at levels approximating to those seen in the human disease. The aims of the study were to: (i) investigate whether morphological mitochondrial abnormalities occur at expression levels of mutant SOD1 close to physiological levels; and (ii) determine whether the presence of mutant SOD1 causes abnormalities of mitochondrial respiratory chain function and changes in cellular bioenergetic parameters in motor neuronal cells. Using this cellular model, we demonstrate that the presence of mutant SOD1 results in the development of abnormally swollen and pale staining mitochondria. These morphological changes are accompanied by biochemical abnormalities with specific decreases in the activities of complexes II and IV of the mitochondrial electron transfer chain. These same complexes are inhibited when control NSC34 cells are subjected to oxidative stress induced by serum withdrawal. The decrease in respiratory chain complex activity in the presence of mutant SOD1 was not accompanied by decreased expression of representative proteins present in these complexes. Motor neuronal cells expressing mutant SOD1 showed increased cell death when exposed to oxidative stress by serum withdrawal, whereas the presence of normal human SOD1 exerted a protective effect. Under basal, unstressed culture conditions, no change in the ATP : ADP ratio was observed in the presence of mutant SOD1. However, the mitochondrial changes associated with the presence of mutant SOD1 clearly had adverse cellular bioenergetic consequences as shown by increased cell death in the presence of pharmacological inhibition of the glycolytic pathway. We conclude that one important mechanism by which mutant SOD1 causes motor neurone injury involves inhibition of specific components of the mitochondrial electron transfer chain. Therapeutic measures aimed at protecting mitochondrial respiratory chain function may be useful in SOD1 related familial and possibly other forms of amyotrophic lateral sclerosis.