Mitochondria are subject to unique genetic control by both nuclear DNA and their own genome, mitochondrial DNA (mtDNA), of which each mitochondrion contains multiple copies. In humans, mutations in ...mtDNA can lead to devastating, heritable, multi-system diseases that display different tissue-specific presentation at any stage of life. Despite rapid advances in nuclear genome engineering, for years, mammalian mtDNA has remained resistant to genetic manipulation, hampering our ability to understand the mechanisms that underpin mitochondrial disease. Recent developments in the genetic modification of mammalian mtDNA raise the possibility of using genome editing technologies, such as programmable nucleases and base editors, for the treatment of hereditary mitochondrial disease.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Mitochondria are the major source of ATP in the cell. Five multi-subunit complexes in the inner membrane of the organelle are involved in the oxidative phosphorylation required for ATP production. ...Thirteen subunits of these complexes are encoded by the mitochondrial genome often referred to as mtDNA. For this reason, the expression of mtDNA is vital for the assembly and functioning of the oxidative phosphorylation complexes. Defects of the mechanisms regulating mtDNA gene expression have been associated with deficiencies in assembly of these complexes, resulting in mitochondrial diseases. Recently, numerous factors involved in these processes have been identified and characterized leading to a deeper understanding of the mechanisms that underlie mitochondrial diseases.
Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own ...multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.
Mammalian mitochondria contain their own genome that encodes mRNAs for thirteen essential subunits of the complexes performing oxidative phosphorylation as well as the RNA components (two rRNAs and ...22 tRNAs) needed for their translation in mitochondria. All RNA species are produced from single polycistronic precursor RNAs, yet the relative concentrations of various RNAs differ significantly. This underscores the essential role of post-transcriptional mechanisms that control the maturation, stability and translation of mitochondrial RNAs. The present review provides a detailed summary on the role of RNA maturation in the regulation of mitochondrial gene expression, focusing mainly on messenger RNA polyadenylation and stability control. Furthermore, the role of mitochondrial ribosomal RNA stability, processing and modifications in the biogenesis of the mitochondrial ribosome is discussed.
The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a ...mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo-electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.
Mitochondrial DNA (mtDNA) encodes a subset of genes which are essential for oxidative phosphorylation. Deletions in the mtDNA can ablate a number of these genes and result in mitochondrial ...dysfunction, which is associated with bona fide mitochondrial disorders. Although mtDNA deletions are thought to occur as a result of replication errors or following double-strand breaks, the exact mechanism(s) behind deletion formation have yet to be determined. In this review we discuss the current knowledge about the fate of mtDNA following double-strand breaks, including the molecular players which mediate the degradation of linear mtDNA fragments and possible mechanisms of recircularization. We propose that mtDNA deletions formed from replication errors versus following double-strand breaks can be mediated by separate pathways.
Mitochondrial DNA deletions lead to bona fide mitochondrial disorders but the mechanisms behind their formation is unknown.
It has been observed that deletions can form from errors in replication or following double-strand breaks.
Analysis of deletion breakpoints reveal breakpoints flanked by either direct repeats or imperfect/micro-homologies, implicating two different mechanisms for deletion formation.
Mitochondrial DNA is rapidly degraded following double-strand breaks, and recent studies have identified some of the molecular players which mediate this degradation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the ...discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease.
Organelle-targeted nucleases allow specific destruction of mutant mtDNA.In combination with adeno-associated viruses, these nucleases allow organ-specific changes in mtDNA heteroplasmy.Recent data have provided early proof of principle for gene therapy of incurable mtDNA disorders using these engineered enzymes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Perturbation of mitochondrial DNA (mtDNA) gene expression can lead to human pathologies. Therefore, a greater appreciation of the basic mechanisms of mitochondrial gene expression is desirable to ...understand the pathophysiology of associated disorders. Although the purpose of the mitochondrial gene expression machinery is to provide only 13 proteins of the oxidative phosphorylation (OxPhos) system, recent studies have revealed its remarkable and unexpected complexity. We review here the latest breakthroughs in our understanding of the post-transcriptional processes of mitochondrial gene expression, focusing on advances in analyzing the mitochondrial epitranscriptome, the role of mitochondrial RNA granules (MRGs), the benefits of recently obtained structures of the mitochondrial ribosome, and the coordination of mitochondrial and cytosolic translation to orchestrate the biogenesis of OxPhos complexes.
The genetic system required for mitochondrial gene expression is housed within the mitochondrial matrix, with all the necessary RNAs being provided by transcription of the mtDNA itself.
Our understanding of the extent and nature of post-transcriptional modifications of mtRNA, the epitranscriptome, is rapidly expanding. Several required nucleus-encoded enzymes have recently been identified.
mtRNA maturation factors localize in distinct foci, termed mtRNA granules, with newly transcribed RNA. These foci may allow spatiotemporal control of mtRNA processing.
Recent high-resolution structures obtained via cryo-electron microscopy have rapidly advanced our understanding of the specialized adaptations of the mitochondrial ribosome.
Production of respiratory complexes requires tight coordination between the cytoplasmic and mitochondrial translation systems.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this ...task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5'-3'exonuclease MGME1, elimination of the 3'-5'exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.
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.
Synopsis
Mutations and rearrangements of mitochondrial DNA (mtDNA) are a common cause of human disease, where they often co‐exist with wild‐type mtDNA within a single cell. Mitochondrially targeted engineered zinc finger nucleases (mtZFNs) can phenotypically rescue a severe mtDNA‐mediated dysfunction and show future therapeutic potential.
Previously reported mtZFN constructs were redesigned, greatly improving target specificity and allowing their safe use in human mitochondria.
The capacity of the novel mtZFN design was validated by selectively degrading point mutant mtDNA associated with neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) and maternally inherited Leigh syndrome (MILS).
The use of the novel mtZFNs was expanded by selectively degrading mtDNA harbouring a pathogenic large‐scale deletion associated with adult‐onset chronic progressive external ophthalmoplegia (CPEO) and, less frequently, Kearns‐Sayre syndrome (KSS) and Pearson's marrow pancreas syndrome.
Data are provided demonstrating that elimination of deleted, pathogenic mtDNA molecules by mtZFNs is sufficient for full recovery of oxidative phosphorylation in a disease model cell line.
Mutations and rearrangements of mitochondrial DNA (mtDNA) are a common cause of human disease, where they often co‐exist with wild‐type mtDNA within a single cell. Mitochondrially targeted engineered zinc finger nucleases (mtZFNs) can phenotypically rescue a severe mtDNA‐mediated dysfunction and show future therapeutic potential.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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