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1.
Annu Rev Biochem ; 2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38594940

ABSTRACT

Mammalian mitochondrial DNA (mtDNA) is replicated and transcribed by phage-like DNA and RNA polymerases, and our understanding of these processes has progressed substantially over the last several decades. Molecular mechanisms have been elucidated by biochemistry and structural biology and essential in vivo roles established by cell biology and mouse genetics. Single molecules of mtDNA are packaged by mitochondrial transcription factor A into mitochondrial nucleoids, and their level of compaction influences the initiation of both replication and transcription. Mutations affecting the molecular machineries replicating and transcribing mtDNA are important causes of human mitochondrial disease, reflecting the critical role of the genome in oxidative phosphorylation system biogenesis. Mechanisms controlling mtDNA replication and transcription still need to be clarified, and future research in this area is likely to open novel therapeutic possibilities for treating mitochondrial dysfunction.

2.
Nat Rev Mol Cell Biol ; 25(2): 119-132, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37783784

ABSTRACT

The expression of mitochondrial genes is regulated in response to the metabolic needs of different cell types, but the basic mechanisms underlying this process are still poorly understood. In this Review, we describe how different layers of regulation cooperate to fine tune initiation of both mitochondrial DNA (mtDNA) transcription and replication in human cells. We discuss our current understanding of the molecular mechanisms that drive and regulate transcription initiation from mtDNA promoters, and how the packaging of mtDNA into nucleoids can control the number of mtDNA molecules available for both transcription and replication. Indeed, a unique aspect of the mitochondrial transcription machinery is that it is coupled to mtDNA replication, such that mitochondrial RNA polymerase is additionally required for primer synthesis at mtDNA origins of replication. We discuss how the choice between replication-primer formation and genome-length RNA synthesis is controlled at the main origin of replication (OriH) and how the recent discovery of an additional mitochondrial promoter (LSP2) in humans may change this long-standing model.


Subject(s)
DNA Replication , Transcription, Genetic , Humans , DNA Replication/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Mitochondria/genetics , Mitochondria/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism
3.
EMBO Mol Med ; 15(5): e16775, 2023 05 08.
Article in English | MEDLINE | ID: mdl-37013609

ABSTRACT

Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.


Subject(s)
Mitochondrial Diseases , Muscular Diseases , Humans , Mitochondria/genetics , DNA, Mitochondrial/genetics , Mitochondrial Diseases/genetics , Syndrome , Genomic Instability
4.
Nat Commun ; 14(1): 1009, 2023 02 23.
Article in English | MEDLINE | ID: mdl-36823193

ABSTRACT

Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.


Subject(s)
Transcription Factors , Zebrafish , Child , Animals , Humans , Transcription Factors/genetics , RNA, Mitochondrial , Zebrafish/genetics , Zebrafish/metabolism , DNA, Mitochondrial/genetics , Transcription, Genetic , Mutation , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism
5.
Mol Cell ; 82(19): 3646-3660.e9, 2022 10 06.
Article in English | MEDLINE | ID: mdl-36044900

ABSTRACT

The human mitochondrial genome must be replicated and expressed in a timely manner to maintain energy metabolism and supply cells with adequate levels of adenosine triphosphate. Central to this process is the idea that replication primers and gene products both arise via transcription from a single light strand promoter (LSP) such that primer formation can influence gene expression, with no consensus as to how this is regulated. Here, we report the discovery of a second light strand promoter (LSP2) in humans, with features characteristic of a bona fide mitochondrial promoter. We propose that the position of LSP2 on the mitochondrial genome allows replication and gene expression to be orchestrated from two distinct sites, which expands our long-held understanding of mitochondrial gene expression in humans.


Subject(s)
Genome, Mitochondrial , Adenosine Triphosphate/metabolism , DNA, Mitochondrial/metabolism , Humans , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Transcription, Genetic
6.
Cell ; 185(13): 2309-2323.e24, 2022 06 23.
Article in English | MEDLINE | ID: mdl-35662414

ABSTRACT

The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.


Subject(s)
DNA, Mitochondrial , Mitochondrial Proteins , Animals , DNA, Mitochondrial/genetics , DNA-Directed RNA Polymerases/metabolism , Dimerization , Humans , Mammals/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , RNA/metabolism , RNA, Mitochondrial , RNA, Small Cytoplasmic , Signal Recognition Particle , Transcription, Genetic
7.
Nat Commun ; 13(1): 3701, 2022 06 28.
Article in English | MEDLINE | ID: mdl-35764627

ABSTRACT

Stress granules (SGs) are non-membranous organelles facilitating stress responses and linking the pathology of age-related diseases. In a genome-wide imaging-based phenomic screen, we identify Pab1 co-localizing proteins under 2-deoxy-D-glucose (2-DG) induced stress in Saccharomyces cerevisiae. We find that deletion of one of the Pab1 co-localizing proteins, Lsm7, leads to a significant decrease in SG formation. Under 2-DG stress, Lsm7 rapidly forms foci that assist in SG formation. The Lsm7 foci form via liquid-liquid phase separation, and the intrinsically disordered region and the hydrophobic clusters within the Lsm7 sequence are the internal driving forces in promoting Lsm7 phase separation. The dynamic Lsm7 phase-separated condensates appear to work as seeding scaffolds, promoting Pab1 demixing and subsequent SG initiation, seemingly mediated by RNA interactions. The SG initiation mechanism, via Lsm7 phase separation, identified in this work provides valuable clues for understanding the mechanisms underlying SG formation and SG-associated human diseases.


Subject(s)
Biochemical Phenomena , Saccharomyces cerevisiae Proteins , Cytoplasmic Granules/metabolism , Humans , Poly(A)-Binding Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Stress Granules
8.
Nucleic Acids Res ; 50(2): 989-999, 2022 01 25.
Article in English | MEDLINE | ID: mdl-35018464

ABSTRACT

Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.


Subject(s)
DNA Polymerase gamma/metabolism , DNA/metabolism , Mitochondria/genetics , RNA, Mitochondrial/metabolism , Ribonucleotides/metabolism , DNA Replication , Escherichia coli/genetics , Humans
9.
Nature ; 602(7898): 664-670, 2022 02.
Article in English | MEDLINE | ID: mdl-35016195

ABSTRACT

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody1. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab2, S2X2593 and S2H974. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Antigenic Drift and Shift/immunology , Broadly Neutralizing Antibodies/immunology , Neutralization Tests , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antigenic Drift and Shift/genetics , COVID-19 Vaccines/immunology , Cell Line , Convalescence , Epitopes, B-Lymphocyte/immunology , Humans , Immune Evasion , Mice , SARS-CoV-2/chemistry , SARS-CoV-2/classification , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vesiculovirus/genetics
10.
bioRxiv ; 2021 Dec 20.
Article in English | MEDLINE | ID: mdl-34931194

ABSTRACT

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb 1 , retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab 2 , S2X259 3 and S2H97 4 , neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

11.
Sci Adv ; 7(27)2021 07.
Article in English | MEDLINE | ID: mdl-34215584

ABSTRACT

We report a role for the mitochondrial single-stranded DNA binding protein (mtSSB) in regulating mitochondrial DNA (mtDNA) replication initiation in mammalian mitochondria. Transcription from the light-strand promoter (LSP) is required both for gene expression and for generating the RNA primers needed for initiation of mtDNA synthesis. In the absence of mtSSB, transcription from LSP is strongly up-regulated, but no replication primers are formed. Using deep sequencing in a mouse knockout model and biochemical reconstitution experiments with pure proteins, we find that mtSSB is necessary to restrict transcription initiation to optimize RNA primer formation at both origins of mtDNA replication. Last, we show that human pathological versions of mtSSB causing severe mitochondrial disease cannot efficiently support primer formation and initiation of mtDNA replication.


Subject(s)
DNA Replication , DNA-Binding Proteins , Animals , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Mammals/genetics , Mice , Mitochondria/genetics , Mitochondria/metabolism
13.
Nat Commun ; 12(1): 1135, 2021 02 18.
Article in English | MEDLINE | ID: mdl-33602924

ABSTRACT

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.


Subject(s)
DNA-Directed RNA Polymerases/genetics , Mitochondria/genetics , Mutation/genetics , Nervous System Diseases/genetics , Transcription, Genetic , Adolescent , Adult , Child , DNA, Mitochondrial/genetics , DNA-Directed RNA Polymerases/chemistry , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Infant , Male , Nervous System Diseases/pathology , Oxidative Phosphorylation , Pedigree , Protein Domains , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Young Adult
14.
Nature ; 588(7839): 712-716, 2020 12.
Article in English | MEDLINE | ID: mdl-33328633

ABSTRACT

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.


Subject(s)
Mitochondria/drug effects , Mitochondria/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transcription, Genetic/drug effects , Animals , Cell Proliferation/drug effects , Cryoelectron Microscopy , DNA, Mitochondrial/drug effects , DNA, Mitochondrial/genetics , DNA-Directed RNA Polymerases/metabolism , Down-Regulation/drug effects , Enzyme Stability/drug effects , Female , Gene Expression Regulation/drug effects , Genes, Mitochondrial/drug effects , Humans , Male , Mice , Neoplasms/drug therapy , Neoplasms/pathology , Substrate Specificity/drug effects , Xenograft Model Antitumor Assays
15.
PLoS Genet ; 16(12): e1009242, 2020 12.
Article in English | MEDLINE | ID: mdl-33315859

ABSTRACT

Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.


Subject(s)
DNA, Mitochondrial/genetics , Gene Deletion , Gene Duplication , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNA/methods , Animals , DNA, Mitochondrial/chemistry , High-Throughput Nucleotide Sequencing/standards , Mice , Reproducibility of Results , Sequence Analysis, DNA/standards
16.
Crit Rev Biochem Mol Biol ; 55(6): 509-524, 2020 12.
Article in English | MEDLINE | ID: mdl-32972254

ABSTRACT

Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome (mtDNA) that codes for subunits of the oxidative phosphorylation machinery. Mutations in mtDNA cause a number of rare, human disorders and are also associated with more common conditions, such as neurodegeneration and biological aging. In this review, we discuss our current understanding of mtDNA replication in mammalian cells and how this process is regulated. We also discuss how deletions can be formed during mtDNA replication.


Subject(s)
DNA, Mitochondrial/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Animals , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Replication/genetics , DNA Replication/physiology , DNA, Mitochondrial/genetics , Humans , Mitochondrial Proteins/genetics
17.
PLoS Genet ; 16(5): e1008798, 2020 05.
Article in English | MEDLINE | ID: mdl-32469861

ABSTRACT

Alterations in epigenetic silencing have been associated with ageing and tumour formation. Although substantial efforts have been made towards understanding the mechanisms of gene silencing, novel regulators in this process remain to be identified. To systematically search for components governing epigenetic silencing, we developed a genome-wide silencing screen for yeast (Saccharomyces cerevisiae) silent mating type locus HMR. Unexpectedly, the screen identified the mismatch repair (MMR) components Pms1, Mlh1, and Msh2 as being required for silencing at this locus. We further found that the identified genes were also required for proper silencing in telomeres. More intriguingly, the MMR mutants caused a redistribution of Sir2 deacetylase, from silent mating type loci and telomeres to rDNA regions. As a consequence, acetylation levels at histone positions H3K14, H3K56, and H4K16 were increased at silent mating type loci and telomeres but were decreased in rDNA regions. Moreover, knockdown of MMR components in human HEK293T cells increased subtelomeric DUX4 gene expression. Our work reveals that MMR components are required for stable inheritance of gene silencing patterns and establishes a link between the MMR machinery and the control of epigenetic silencing.


Subject(s)
MutL Protein Homolog 1/genetics , MutL Proteins/genetics , MutS Homolog 2 Protein/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/metabolism , Acetylation , DNA Mismatch Repair , Epigenesis, Genetic , Gene Silencing , Genes, Mating Type, Fungal , Heredity , Histones/metabolism , Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/metabolism , Telomere/genetics
18.
Proc Natl Acad Sci U S A ; 117(13): 7524-7535, 2020 03 31.
Article in English | MEDLINE | ID: mdl-32184324

ABSTRACT

Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.


Subject(s)
Cell Respiration/physiology , Fermentation/physiology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Ethanol/metabolism , Gene Expression Regulation, Fungal/genetics , Glucose/metabolism , Mass Spectrometry/methods , Proteome/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
20.
Mol Cell ; 76(5): 784-796.e6, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31588022

ABSTRACT

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.


Subject(s)
14-3-3 Proteins/metabolism , Biomarkers, Tumor/metabolism , Exoribonucleases/metabolism , Mitochondria/enzymology , Oligonucleotides/metabolism , Promoter Regions, Genetic , RNA Stability , RNA, Mitochondrial/metabolism , 14-3-3 Proteins/deficiency , 14-3-3 Proteins/genetics , Animals , Biomarkers, Tumor/genetics , Exoribonucleases/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Humans , Mice, Inbred C57BL , Mice, Knockout , RNA, Mitochondrial/genetics , Sf9 Cells , Spodoptera
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