Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease

Kornblum, Cornelia; Nicholls, Thomas J.; Haack, Tobias B.; Schöler, Susanne; Peeva, Viktoriya; Danhauser, Katharina; Hallmann, Kerstin; Zsurka, Gábor; Rorbach, Joanna; Iuso, Arcangela; Wieland, Thomas; Sciacco, Monica; Ronchi, Dario; Comi, Giacomo P.; Moggio, Maurizio; Quinzii, Catarina M.; DiMauro, Salvatore; Calvo, Sarah E.; Mootha, Vamsi K.; Klopstock, Thomas; Strom, Tim M.; Meitinger, Thomas; Minczuk, Michal; Kunz, Wolfram S.; Prokisch, Holger

doi:10.1038/ng.2501

Cited by 206 publications

(265 citation statements)

References 33 publications

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“…Alternatively, EXOG might be more important in maintaining mtDNA integrity during DNA replication in proliferating cells, rather than damage repair per se. Such a function has recently been shown for another mitochondrial exonuclease MGME1 (9) and may explain why proliferating cells are more prone to EXOG depletion.…”

Section: Discussionmentioning

confidence: 99%

Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy

Tigchelaar

Jong

et al. 2015

American Journal of Physiology-Cell Physiology

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Tigchelaar W, Yu H, de Jong AM, van Gilst WH, van der Harst P, Westenbrink BD, de Boer RA, Silljé HH. Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy. Am J Physiol Cell Physiol 308: C155-C163, 2015. First published November 5, 2014 doi:10.1152/ajpcell.00227.2014.-Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy.cardiomyocytes; hypertrophy; mitochondria; mitochondrial respiration; ROS THE HEART IS ONE OF THE MOST energy-consuming organs in the human body. This energy, in the form of ATP, is used to maintain proper contractile function and is mainly produced by cellular respiration, in particular by oxidative phosphorylation (OXPHOS) in the mitochondria. There is a strict correlation between energy production (supply) and energy utilization (demand). As the heart has limited energy storage capacity, ATP-generating systems must respond proportionally to fluctuations in demand. This energetic status of the heart is a delicate balance and is often disturbed in cardiovascular diseases, including heart failure.As a byproduct of oxidative phosphorylation, the mitochondria produce reactive oxygen species (ROS). Low levels of ROS may be protective (7, 23) and affect signaling pathways that may stimulate growth (5). However, an imbalance between ROS production and the normal cellular antioxidant defense system will lead to oxidative stress and potentially in DNA damage (10). Therefore, cells have develo...

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Section: Discussionmentioning

confidence: 99%

Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy

Tigchelaar

Jong

et al. 2015

American Journal of Physiology-Cell Physiology

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“…However, RNase H1 cannot remove every last ribonucleotide of an RNA/ DNA hybrid, and therefore other factors should also be required, such as those implicated in Okazaki fragment processing in the nucleus, that have also been found in mitochondria (25)(26)(27), and, possibly, MGME1 (28).…”

Section: Resultsmentioning

confidence: 99%

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication

Holmes

Akman²,

Wood

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.H uman mitochondrial DNA (mtDNA) is most frequently organized as 16.5-kb circles of duplex DNA, whose two strands are called heavy (H) and light (L), based on their nucleotide composition. The human mitochondrial genome is gene dense, with only one substantial noncoding region (NCR) of approximately 1 kb (1). Based on electron microscopy (2), 5′ end mapping of DNA ends (3-5) and later 2D agarose gel electrophoresis (2D-AGE) studies (6, 7) it was inferred that replication of mammalian mtDNA is frequently unidirectional, commencing within the NCR.In mammalian mitochondria, initiation of DNA synthesis occurs preferentially at or near two specific sites. On the leading strand, initiation has been assigned to a prominent free 5′ end of DNA, designated as Ori-H, located at nucleotide position 16,034 on the map of the mouse mitochondrial genome. Ori-H lies downstream of the light-strand promoter (LSP), and RNA species spanning from LSP to approximately Ori-H have been detected in mammalian mitochondria, albeit not covalently linked with DNA (8). LSP has been robustly mapped both in vivo (9) and in vitro (10) and is assumed to be used by mitochondrial RNA polymerase (POLRMT) to synthesize polycistronic transcripts of the light strand and to generate much shorter products terminating in the NCR, which serve as primers for leading (H-)strand DNA synthesis. POLRMT is also implicated in the synthesis of a primer at a short spacer DNA amid a cluster of five tRNA genes (termed Ori-L) that is a major site of initiation of lagging-strand DNA synthesis (11,12). A role for encoding ribonuclease H1 (RNase H1) in generating, processing, or removing primers at Ori-H and Ori-L has been inferred but has not been experimentally tested.Other data indicate that the initiation of mtDNA replication is more complex, because the NCR contains a second putative origin of replication, termed cluster II, or Ori-b (7, 13). Moreover, other sites of second-strand DNA synthesis than Ori-L have been inferred both by atomic force microscopy (14) and neutral 2D agarose gel electrophoresis (7, 13). Mitochondrial DNA rep...

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“…Because R-loop numbers are low in the cells of a patient with mutant RNase H1 and mtDNA abnormalities, investigation of the abundance and behavior of the mitochondrial R-loop in the context of pathological mutants of Twinkle DNA helicase (37), mitochondrial genome maintenance exonuclease 1 (MGME1) (38), FARS2 (39), FBXL4 (40), and POLG (41) could prove informative. Human cultured cells are an appropriate model system for the study of this and many other aspects of mtDNA replication and maintenance.…”

Section: Discussionmentioning

confidence: 99%

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria

Akman

Desai

Bailey

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The genetic information in mammalian mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its replication and expression. Many molecules of mitochondrial DNA bear a third strand of DNA, known as "7S DNA," which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for mitochondrial DNA replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with mitochondrial disease, R-loops are of low abundance, and there is mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.RNase H1 | R-loop | mitochondrial DNA | DNA segregation | mitochondrial disease

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Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease

Cited by 206 publications

References 33 publications

Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy

Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria

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