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

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation

Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration

The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries. Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria. To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl4) administration. TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation. The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl4 exposure. Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS). Steady-state levels of ATP, O2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl4-treated KO mice. To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide. Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion. Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation

Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late-onset disorder of mitochondrial DNA maintenance

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. While dominantly-inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remain challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G > C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G > C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the nineteenth (19th) locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes

The Cepheid Distance to the Narrow-Line Seyfert 1 Galaxy NGC 4051

We derive a distance of $D = 16.6 \pm 0.3$~Mpc ($\mu=31.10\pm0.04$~mag) to the archetypal narrow-line Seyfert 1 galaxy NGC 4051 based on Cepheid Period--Luminosity relations and new Hubble Space Telescope multiband imaging. We identify 419 Cepheid candidates and estimate the distance at both optical and near-infrared wavelengths using subsamples of precisely-photometered variables (123 and 47 in the optical and near-infrared subsamples, respectively). We compare our independent photometric procedures and distance-estimation methods to those used by the SH0ES team and find agreement to 0.01~mag. The distance we obtain suggests an Eddington ratio $\dot{m} \approx 0.2$ for NGC 4051, typical of narrow-line Seyfert 1 galaxies, unlike the seemingly-odd value implied by previous distance estimates. We derive a peculiar velocity of $-490\pm34$~km~s$^{-1}$ for NGC 4051, consistent with the overall motion of the Ursa Major Cluster in which it resides. We also revisit the energetics of the NGC 4051 nucleus, including its outflow and mass accretion rates.Comment: 15 pages, 12 figures, 6 tables, accepted for publication in Ap

Space Telescope and Optical Reverberation Mapping project. XI. Disk-wind characteristics and contributions to the very broad emission lines of NGC 5548

Funding: Support for HST program number GO-13330 was provided by NASA through a grant from the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS5-26555. We thank NSF (1816537, 1910687), NASA (17-ATP17-0141, 19-ATP19-0188), and STScI (HST-AR-15018, HST-AR-14556). MC acknowledges support from NASA through STScI grant HST-AR-14556.001-Aand NASA grant 19-ATP19-0188, and also support from National Science Foundation through grant AST-1910687.M.D. and G.F. and F. G. acknowledge support from the NSF (AST-1816537), NASA (ATP 17-0141), and STScI (HST-AR-13914, HST-AR-15018), and the Huffaker Scholarship. M.M. is supported by the Netherlands Organization for Scientific Research (NWO) through the Innovational Research Incentives Scheme Vidi grant 639.042.525. J.M.G. gratefully acknowledges support from NASA under the ADAP award 80NSSC17K0126. MV gratefully acknowledges support from the Independent Research Fund Denmark via grant number DFF 8021-00130.In 2014 the NGC 5548 Space Telescope and Optical Reverberation Mapping campaign discovered a two-month anomaly when variations in the absorption and emission lines decorrelated from continuum variations. During this time the soft X-ray part of the intrinsic spectrum had been strongly absorbed by a line-of-sight (LOS) obscurer, which was interpreted as the upper part of a disk wind. Our first paper showed that changes in the LOS obscurer produces the decorrelation between the absorption lines and the continuum. A second study showed that the base of the wind shields the broad emission-line region (BLR), leading to the emission-line decorrelation. In that study, we proposed the wind is normally transparent with no effect on the spectrum. Changes in the wind properties alter its shielding and affect the spectral energy distribution (SED) striking the BLR, producing the observed decorrelations. In this work we investigate the impact of a translucent wind on the emission lines. We simulate the obscuration using XMM-Newton, NuSTAR, and Hubble Space Telescope observations to determine the physical characteristics of the wind. We find that a translucent wind can contribute a part of the He ii and Fe Kα emission. It has a modest optical depth to electron scattering, which explains the fainter far-side emission in the observed velocity-delay maps. The wind produces the very broad base seen in the UV emission lines and may also be present in the Fe Kα line. Our results highlight the importance of accounting for the effects of such winds in the analysis of the physics of the central engine.Publisher PDFPeer reviewe

Prototype ATLAS IBL Modules using the FE-I4A Front-End Readout Chip

The ATLAS Collaboration will upgrade its semiconductor pixel tracking detector with a new Insertable B-layer (IBL) between the existing pixel detector and the vacuum pipe of the Large Hadron Collider. The extreme operating conditions at this location have necessitated the development of new radiation hard pixel sensor technologies and a new front-end readout chip, called the FE-I4. Planar pixel sensors and 3D pixel sensors have been investigated to equip this new pixel layer, and prototype modules using the FE-I4A have been fabricated and characterized using 120 GeV pions at the CERN SPS and 4 GeV positrons at DESY, before and after module irradiation. Beam test results are presented, including charge collection efficiency, tracking efficiency and charge sharing.Comment: 45 pages, 30 figures, submitted to JINS

CLUH couples mitochondrial distribution to the energetic and metabolic status

Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status