The mechanisms of mitochondrial DNA replication have been hotly debated for a decade. The strand‐displacement model states that lagging‐strand DNA synthesis is initiated from the origin of ...light‐strand DNA replication (OriL), whereas the strand‐coupled model implies that OriL is dispensable. Mammalian mitochondria cannot be transfected and the requirements of OriL in vivo have therefore not been addressed. We here use in vivo saturation mutagenesis to demonstrate that OriL is essential for mtDNA maintenance in the mouse. Biochemical and bioinformatic analyses show that OriL is functionally conserved in vertebrates. Our findings strongly support the strand‐displacement model for mtDNA replication.
Saturation mutagenesis in mice shows that an origin of light‐strand DNA replication (OriL) is essential for mitochondrial DNA (mtDNA) maintenance in vivo. OriL is further functionally conserved in vertebrates, which lends strong support to the strand displacement model for mtDNA replication.
Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression ...of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.
Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine‐rich pentatricopeptide repeat containing ...(LRPPRC) protein regulates mitochondrial mRNA stability and an amino‐acid substitution of this protein causes the French‐Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue‐specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady‐state levels of most mitochondrial mRNAs. LRPPRC forms an RNA‐dependent protein complex that is necessary for maintaining a pool of non‐translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post‐transcriptional level.
LRPPRC regulates mitochondrial mRNA stability and it is implicated in a neurodegenerative disorder. A heart‐specific mouse knockout now reveals that LRPPRC is necessary for mRNA polyadenylation and stability, and for coordinated mitochondrial translation, resulting in respiratory chain defects and cardiomyopathy.
Mammals develop age-associated clonal expansion of somatic mtDNA mutations resulting in severe respiratory chain deficiency in a subset of cells in a variety of tissues. Both mathematical modeling ...based on descriptive data from humans and experimental data from mtDNA mutator mice suggest that the somatic mutations are formed early in life and then undergo mitotic segregation during adult life to reach very high levels in certain cells. To address whether mtDNA mutations have a universal effect on aging metazoans, we investigated their role in physiology and aging of fruit flies. To this end, we utilized genetically engineered flies expressing mutant versions of the catalytic subunit of mitochondrial DNA polymerase (DmPOLγA) as a means to introduce mtDNA mutations. We report here that lifespan and health in fruit flies are remarkably tolerant to mtDNA mutations. Our results show that the short lifespan and wide genetic bottleneck of fruit flies are limiting the extent of clonal expansion of mtDNA mutations both in individuals and between generations. However, an increase of mtDNA mutations to very high levels caused sensitivity to mechanical and starvation stress, intestinal stem cell dysfunction, and reduced lifespan under standard conditions. In addition, the effects of dietary restriction, widely considered beneficial for organismal health, were attenuated in flies with very high levels of mtDNA mutations.
Mitochondrial transcription factor A (TFAM) is compacting mitochondrial DNA (dmtDNA) into nucleoids and directly controls mtDNA copy number. Here, we show that the TFAM-to-mtDNA ratio is critical for ...maintaining normal mtDNA expression in different mouse tissues. Moderately increased TFAM protein levels increase mtDNA copy number but a normal TFAM-to-mtDNA ratio is maintained resulting in unaltered mtDNA expression and normal whole animal metabolism. Mice ubiquitously expressing very high TFAM levels develop pathology leading to deficient oxidative phosphorylation (OXPHOS) and early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle leading to strong repression of mtDNA expression and OXPHOS deficiency. In the heart, increased mtDNA copy number results in a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In liver, induction of LONP1 protease and mitochondrial RNA polymerase expression counteracts the silencing effect of high TFAM levels. TFAM thus acts as a general repressor of mtDNA expression and this effect can be counterbalanced by tissue-specific expression of regulatory factors.
Skeletal muscle often shows a delayed force recovery after fatiguing stimulation, especially at low stimulation frequencies.
In this study we focus on the role of reactive oxygen species (ROS) in ...this fatigue-induced prolonged low-frequency force
depression. Intact, single muscle fibres were dissected from flexor digitorum brevis (FDB) muscles of rats and wild-type and
superoxide dismutase 2 (SOD2) overexpressing mice. Force and myoplasmic free Ca 2+ (Ca 2+ i ) were measured. Fibres were stimulated at different frequencies before and 30 min after fatigue induced by repeated tetani.
The results show a marked force decrease at low stimulation frequencies 30 min after fatiguing stimulation in all fibres.
This decrease was associated with reduced tetanic Ca 2+ i in wild-type mouse fibres, whereas rat fibres and mouse SOD2 overexpressing fibres instead displayed a decreased myofibrillar
Ca 2+ sensitivity. The SOD activity was â¼50% lower in wild-type mouse than in rat FDB muscles. Myoplasmic ROS increased during
repeated tetanic stimulation in rat fibres but not in wild-type mouse fibres. The decreased Ca 2+ sensitivity in rat fibres could be partially reversed by application of the reducing agent dithiothreitol, whereas the decrease
in tetanic Ca 2+ i in wild-type mouse fibres was not affected by dithiothreitol or the antioxidant N -acetylcysteine. In conclusion, we describe two different causes of fatigue-induced prolonged low-frequency force depression,
which correlate to differences in SOD activity and ROS metabolism. These findings may have clinical implications since ROS-mediated
impairments in myofibrillar function can be counteracted by reductants and antioxidants, whereas changes in SR Ca 2+ handling appear more resistant to interventions.
Next,we performed western blot analyses with an antibody directed against a peptide in the carboxy terminus of mouse POLRMT, but could not find the previously reported5 nuclear protein isoform of ...,110 kDa inmouse heart, muscle, kidney, spleen, liver or brain,whereas a,140-kDa protein corresponding to mitochondrially localizedPOLRMT was present in all tissues (Fig. 2a).Wealso studied the subcellular localization of POLRMT in mouse heart, and in HeLa, 143B and 143B rho0 human cell lines and found no nuclear spRNAP-IV isoformon western blot analysis (Fig. 2b and Appendix Fig. 3) and no nuclear signal on confocal microscopy (n545 cells; Fig. 2c and Appendix Fig. 4) with a POLRMTantibody recognizing both protein isoforms (Appendix Fig. 5).
Mitochondria are the structures that produce the bulk part of the cellular energy currency ATP, which drives numerous energy requiring processes in the cell. This process involves a series of large ...enzyme complexes--the respiratory chain--that couples the transfer of electrons to the creation of a concentration gradient of protons across the inner mitochondrial membrane, which drives ATP synthesis. Complex I (or NADH-quinone oxidoreductase) is the largest and by far the most complicated of the respiratory chain enzyme complexes. The molecular mechanism whereby it couples electron transfer to proton extrusion has remained mysterious until very recently. Low-resolution X-ray structures of complex I have, surprisingly, suggested that electron transfer in the hydrophilic arm, protruding into the mitochondrial matrix, causes movement of a coupling rod that influences three putative proton pumps within the hydrophobic arm embedded in the inner mitochondrial membrane. In this Primer, we will briefly introduce the recent progress made in this area and highlight the road ahead that likely will unravel the detailed molecular mechanisms of complex I function.
The mtDNA mutator mice have high levels of point mutations and linear deletions of mtDNA causing a progressive respiratory chain dysfunction and a premature aging phenotype. We have now performed ...molecular analyses to determine the mechanism whereby these mtDNA mutations impair respiratory chain function. We report that mitochondrial protein synthesis is unimpaired in mtDNA mutator mice consistent with the observed minor alterations of steady-state levels of mitochondrial transcripts. These findings refute recent claims that circular mtDNA molecules with large deletions are driving the premature aging phenotype. We further show that the stability of several respiratory chain complexes is severely impaired despite normal synthesis of the corresponding mtDNA-encoded subunits. Our findings reveal a mechanism for induction of aging phenotypes by demonstrating a causative role for amino acid substitutions in mtDNA-encoded respiratory chain subunits, which, in turn, leads to decreased stability of the respiratory chain complexes and respiratory chain deficiency.
Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription ...factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light‐strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position −1 to −2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT‐dependent promoter recognition.
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