The mitochondrial R-loop

The DNA in mitochondria contributes essential components of the organelle’s energy producing machinery that is essential for life. In 1971, many mitochondrial DNA molecules were found to have a third strand of DNA that maps to a region containing critical regulatory elements for transcription and replication. Forty-five years later, a third strand of RNA in the same region has been reported. This mitochondrial R-loop is present on thousands of copies of mitochondrial DNA per cell making it potentially the most abundant R-loop in nature. Here, I assess the discovery of the mitochondrial R-loop, discuss why it remained unrecognized for almost half a century and propose for it central roles in the replication, organization and expression of mitochondrial DNA, which if compromised can lead to disease states

Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model

All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17−/− liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17−/− mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology

strategies for fighting mitochondrial diseases

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Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large‐scale deletions or point mutations

We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site‐specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the “common deletion” (CD), a 4977‐bp repeat‐flanked deletion associated with adult‐onset chronic progressive external ophthalmoplegia and, less frequently, Kearns‐Sayre and Pearson's marrow pancreas syndromes. Expression of mtZFNs led to a reduction in mutant mtDNA haplotype load, and subsequent repopulation of wild‐type mtDNA restored mitochondrial respiratory function in a CD cybrid cell model. This study constitutes proof‐of‐principle that, through heteroplasmy manipulation, delivery of site‐specific nuclease activity to mitochondria can alleviate a severe biochemical phenotype in primary mitochondrial disease arising from deleted mtDNA species

Kearns-Sayre's syndrome developing in a boy who survived Pearson's syndrome caused by mitochondrial DNA deletion

Documenta Ophthalmologica 1992, Volume 82, Issue 1-2, pp 73-79 Kearns-Sayre's syndrome developing in a boy who survived Pearson's syndrome caused by mitochondrial DNA deletion Dr H. J. Simonsz, K. Bärlocher, A. Rötig … show all 3 hide » Download PDF (2,322 KB) Abstract A 7-year-old boy presented with bilateral ptosis and atypical retinitis pigmentosa. Before age two, he had had an Fe-refractory anemia, with neutropenia and thrombopenia. Just prior to the ophthalmic examination, the patient developed lactate acidosis, muscular hypotonia, ataxia and increased protein in the spinal fluid. Pancytopenia, pancreas dysfunction and growth retardation are the main features of Pearson's syndrome, most children not surviving beyond age three. The cause of Pearson's syndrome in our patient turned out to be a 5 kb deletion in the mitchondrial DNA. Similar deletions have been described in the Kearns-Sayre syndrome. It seems that children who survive the initial phase of Pearson's syndrome, may develop Kearns-Sayre syndrome

Serotonergic modulation of the activity of GLP-1 producing neurons in the nucleus of the solitary tract in mouse.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) and 5-HT are potent regulators of food intake within the brain. GLP-1 is expressed by preproglucagon (PPG) neurons in the nucleus tractus solitarius (NTS). We have previously shown that PPG neurons innervate 5-HT neurons in the ventral brainstem. Here, we investigate whether PPG neurons receive serotonergic input and respond to 5-HT. METHODS: We employed immunohistochemistry to reveal serotonergic innervation of PPG neurons. We investigated the responsiveness of PPG neurons to 5-HT using in vitro Ca²⁺ imaging in brainstem slices from transgenic mice expressing the Ca²⁺ indicator, GCaMP3, in PPG neurons, and cell-attached patch-clamp recordings. RESULTS: Close appositions from 5-HT-immunoreactive axons occurred on many PPG neurons. Application of 20 μM 5-HT produced robust Ca²⁺ responses in NTS PPG dendrites but little change in somata. Dendritic Ca²⁺ spikes were concentration-dependent (2, 20, and 200 μM) and unaffected by blockade of glutamatergic transmission, suggesting 5-HT receptors on PPG neurons. Neither activation nor blockade of 5-HT₃ receptors affected [Ca²⁺]i. In contrast, inhibition of 5-HT₃ receptors attenuated increases in intracellular Ca²⁺ and 5-HT₂c receptor activation produced Ca²⁺ spikes. Patch-clamp recordings revealed that 44% of cells decreased their firing rate under 5-HT, an effect blocked by 5-HT₁ᴀ receptor antagonism. CONCLUSIONS: PPG neurons respond directly to 5-HT with a 5-HT₂c receptor-dependent increase in dendritic [Ca²⁺]i. Electrical responses to 5-HT revealed additional inhibitory effects due to somatic 5-HT₁ᴀ receptors. Reciprocal innervation between 5-HT and PPG neurons suggests that the coordinated activity of these brainstem neurons may play a role in the regulation of food intake.This study was supported by grants MR/J013293/2 from the MRC, UK (ST) and Project Grant #1025031 from NHMRC Australia (ILS). MKH holds a UCL Graduate Research Scholarship. FR and FMG are supported by the Wellcome Trust (106262/Z/14/Z, 106263/Z/14/Z) and the MRC (MRC_MC_UU_12012/3)

Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.

The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.Medical Research Council (MRC); Biotechnology and Biological Sciences Research Council (BBSRC); European Union; Academy of Finland (to H.M.C.). Funding for open access charge: MRC

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC); Intramural program of the National Institutes of Health, National Heart, Lung and Blood Institute (to K.N.); Academy of Finland (to H.M.C.). Funding for open access charge: MRC

Sequence Homology at the Breakpoint and Clinical Phenotype of Mitochondrial DNA Deletion Syndromes

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (<6 years old) showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (<6 years old) carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data