Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA 'plus' phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.

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We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor B2 (d-mt-TFB2), a protein that plays a role in mitochondrial transcription and mitochondrial DNA (mtDNA) replication in Drosophila. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB2 to 5% of its normal level in Schneider cells. RNAi knock-down of d-mtTFB2 reduces the abundance of specific mitochondrial RNA transcripts 2-to 8-fold and decreases the copy number of mtDNA ϳ3-fold. In a corollary manner, we find that overexpression of d-mtTFB2 increases both the abundance of mitochondrial RNA transcripts and the copy number of mtDNA. In a comparative experiment, we find that overexpression of Drosophila mitochondrial transcription factor A (d-TFAM) increases mtDNA copy number with no significant effect on mitochondrial transcripts. This argues for a direct role for mtTFB2 in mitochondrial transcription and suggests that, if TFAM serves a role in transcription, its endogenous level limits mtDNA copy number but not transcription. Furthermore, we suggest that mtTFB2 increases mtDNA copy number by increasing the frequency of initiation of DNA replication, whereas TFAM serves to stabilize and package mtDNA in mitochondrial nucleoids. Our work represents the first study to document the function of mtTFB2 in vivo, establishing a dual role in regulation of both transcription and replication, and provides a benchmark for comparative biochemical studies in various animal systems.The mitochondria of eukaryotic cells utilize a number of organelle-specific factors in DNA and RNA metabolism. In Saccharomyces cerevisiae, mitochondrial transcription is mediated by the yeast mtRNA 1 polymerase, Rpo41p (1-3), and a specificity factor Mtf1p (4 -6), also known as mitochondrial transcription factor B (sc-mtTFB) (7). Deficiency in sc-mtTFB lowers the abundance of mitochondrial transcripts and reduces mtDNA copy number (5, 7). sc-mtTFB facilitates specific binding of mitochondrial RNA polymerase at numerous promoter sites in the yeast mitochondrial genome (4,8,9). Although sc-mtTFB is functionally similar to bacterial sigma factor (9 -11), the two proteins do not share amino acid sequence (7,12) or structural homology (13). Rather, the structure of sc-mtTFB is homologous to bacterial rRNA methyltransferases (13).Mammalian mitochondrial transcription utilizes mitochondrial RNA polymerase, and three distinct transcription factors: transcription factor A (TFAM; formerly referred to as mtTFA) and two proteins homologous to sc-mtTFB, transcription factors mtTFB1 and mtTFB2 (3, 14 -17). TFAM contains two high mobility group boxes and was shown in organello to bind nonspecifically at regularly phased intervals to the control region of human mtDNA (18); it has recently been shown to package mtDNA in nucleoids (19,20). h-TFAM was also shown to be required for specific initiation at mitochondrial promoters in vitro (14 -17). The yeast homologue of TFAM is Abf2p, an abundant protein whose primary role is to stabilize and c...

Objective To evaluate the influence of the mitochondrial DNA (mtDNA) haplogroups in the risk of incident knee osteoarthritis (OA) and to explain the functional consequences of this association to identify potential diagnostic biomarkers and therapeutic targets. Methods Two prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 2579 subjects of the incidence subcohort, and the cohort hip and cohort knee (CHECK) included 635, both with 8-year follow-up. The analysis included the association of mtDNA haplogroups with the rate of incident knee OA in subjects from both cohorts followed by a subsequent meta-analysis. Transmitochondrial cybrids harbouring haplogroup J or H were constructed to detect differences between them in relation to physiological features including specific mitochondrial metabolic parameters, reactive oxygen species production, oxidative stress and apoptosis. Results Compared with H, the haplogroup J associates with decreased risk of incident knee OA in subjects from OAI (HR=0.680; 95% CI 0.470 to 0.968; p<0.05) and CHECK (HR=0.728; 95% CI 0.469 to 0.998; p<0.05). The subsequent meta-analysis including 3214 cases showed that the haplogroup J associates with a lower risk of incident knee OA (HR=0.702; 95% CI 0.541 to 0.912; p=0.008). J cybrids show a lower free radical production, higher cell survival under oxidative stress conditions, lower grade of apoptosis as well as lower expression of the mitochondrially related pro-apoptotic gene BCL2 binding component 3 (BBC3). In addition, J cybrids also show a lower mitochondrial respiration and glycolysis leading to decreased ATP production. Conclusions The physiological effects of the haplogroup J are beneficial to have a lower rate of incident knee OA over time. Potential drugs to treat OA could focus on emulating the mitochondrial behaviour of this haplogroup.

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3' downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.

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SummaryDictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA -mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology.

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knockdown of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.Mitochondrial number and DNA content vary widely depending on cellular energy requirements, which are met in large part by ATP production by the oxidative phosphorylation pathway. Expression of the 13 polypeptides involved in oxidative phosphorylation that are encoded in the mtDNA 1 genome is essential for this process. Transcription in animal mitochondria is thought to involve mitochondrial RNA polymerase and three distinct transcription factors (1, 2). Mitochondrial transcription factor A (formerly referred to as mtTFA) contains two HMG boxes and was shown in organello to bind nonspecifically at regularly phased intervals to the control region of human mtDNA (3) and to package mtDNA in nucleoids (4, 5). Human mitochondrial transcription factor A was also shown to be required for specific initiation at mitochondrial promoters in vitro (6 -9). Two additional human transcription factors, mtTFB1/TFB1M and mtTFB2/TFB2M, have also been shown to activate transcription from mitochondrial promoters in the presence of mitochondrial transcription factor A and mitochondrial RNA polymerase in vitro, and h-mtTFB2 is more active in promoting transcription than h-mtTFB1 (6, 10). Recent studies show that h-mtTFB1 has rRNA adenine dimethyltransferase activity when expressed in bacteria (11) and that its in vitro transcriptional activation and methylase activities can be inactivated differentially by mutation (12).Although in vitro studies show that both mtTFB1 and mtTFB2 support transcription from human mitochondrial promoters (6), their relative importance and specific physiological roles are not well understood. In a recent study (13), we showed that RNAi knockdown of d-mtTFB2 reduces the abundance of specific mitochondrial RNA transcripts and decreases the copy number of mtDNA in Drosophila cultured cells. This finding suggests that endogenous d-mtTFB1 cannot complement a deficiency in d-mtTFB2 and thus is not functionally redundant with d-mtTFB2, ...