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Abstract: Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2–q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent. [Copyright &y& Elsevier]

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mttRNA Met, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression. [ABSTRACT FROM AUTHOR]