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1.
EMBO Rep ; 20(6)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31036713

RESUMO

Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.

2.
Nat Commun ; 10(1): 759, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770810

RESUMO

Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.


Assuntos
DNA Mitocondrial/genética , Deleção de Sequência/genética , Southern Blotting , Replicação do DNA/genética , Humanos , Proteínas Mitocondriais/genética , Mutação/genética
3.
PLoS Genet ; 15(1): e1007781, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605451

RESUMO

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3'-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.


Assuntos
Replicação do DNA/genética , DNA Mitocondrial/biossíntese , Mitocôndrias/genética , Ribonuclease H/genética , Animais , Polimerase do DNA Mitocondrial/genética , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Origem de Replicação/genética
4.
Trends Biochem Sci ; 43(11): 869-881, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30224181

RESUMO

Cells contain thousands of copies of the mitochondrial genome. These genomes are distributed within the tubular mitochondrial network, which is itself spread across the cytosol of the cell. Mitochondrial DNA (mtDNA) replication occurs throughout the cell cycle and ensures that cells maintain a sufficient number of mtDNA copies. At replication termination the genomes must be resolved and segregated within the mitochondrial network. Defects in mtDNA replication and segregation are a cause of human mitochondrial disease associated with failure of cellular energy production. This review focuses upon recent developments on how mitochondrial genomes are physically separated at the end of DNA replication, and how these genomes are subsequently segregated and distributed around the mitochondrial network.

5.
Mol Cell ; 69(1): 9-23.e6, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29290614

RESUMO

How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3α (Top3α) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3α-RMI1-RMI2 (BTR) complex. Our data indicate that mtDNA replication termination occurs via a hemicatenane formed at the origin of H-strand replication and that Top3α is essential for resolving this structure. Decatenation is a prerequisite for separation of the segregating unit of mtDNA, the nucleoid, within the mitochondrial network. The importance of this process is highlighted in a patient with mitochondrial disease caused by biallelic pathogenic variants in TOP3A, characterized by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3α as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.

7.
Mol Biol Cell ; 28(13): 1738-1744, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28515143

RESUMO

The multiprotein Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Mediator contains the Cdk8 regulatory subcomplex, which directs periodic transcription and influences cell cycle progression in fission yeast. Here we investigate the role of CycC, the cognate cyclin partner of Cdk8, in cell cycle control. Previous reports suggested that CycC interacts with other cellular Cdks, but a fusion of CycC to Cdk8 reported here did not cause any obvious cell cycle phenotypes. We find that Cdk8 and CycC interactions are stabilized within the Mediator complex and the activity of Cdk8-CycC is regulated by other Mediator components. Analysis of a mutant yeast strain reveals that CycC, together with Cdk8, primarily affects M-phase progression but mutations that release Cdk8 from CycC control also affect timing of entry into S phase.


Assuntos
Ciclina C/metabolismo , Complexo Mediador/metabolismo , Pontos de Checagem do Ciclo Celular , Divisão Celular , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Regulação Fúngica da Expressão Gênica , Mitose/genética , Mitose/fisiologia , Fosforilação , RNA Polimerase II/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo
8.
PLoS Genet ; 13(3): e1006620, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28267784

RESUMO

Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A>T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNAPhe, tRNALeu1 and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNAPhe (m.616T>C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNAPhe.


Assuntos
DNA Mitocondrial/genética , Nefropatias/genética , Túbulos Renais/patologia , Mutação , Acetilglucosaminidase/urina , Biópsia , Feminino , Fibroblastos/metabolismo , Ligação Genética , Humanos , Leucina/química , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Linhagem , Fenótipo , Fenilalanina/química , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Músculo Quadríceps/patologia , RNA de Transferência/genética
9.
SLAS Discov ; 22(4): 378-386, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28328323

RESUMO

Mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which under aerobic conditions produces the bulk of cellular adenosine triphosphate (ATP). The mitochondrial genome encodes key components of the OXPHOS system, and it is transcribed by the mitochondrial RNA polymerase, POLRMT. The levels of mitochondrial transcription correlate with the respiratory activity of the cell. Therefore, compounds that can increase or decrease mitochondrial gene transcription may be useful for fine-tuning metabolism and could be used to treat metabolic diseases or certain forms of cancer. We here report the establishment of a novel high-throughput assay technology that has allowed us to screen a library of 430,000 diverse compounds for effects on mitochondrial transcription in vitro. Following secondary screens facilitated by the same assay principle, we identified 55 compounds that efficiently and selectively inhibit mitochondrial transcription and that are active also in cell culture. Our method is easily adaptable to other RNA or DNA polymerases and varying spectroscopic detection technologies.

10.
PLoS Genet ; 13(2): e1006628, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28207748

RESUMO

Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.


Assuntos
Reparo do DNA/genética , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , Ribonucleotídeos/genética , DNA/biossíntese , Polimerase do DNA Mitocondrial , Replicação do DNA/genética , Fibroblastos , Genoma Mitocondrial , Células HeLa , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , RNA/biossíntese , Ribonucleases/genética
11.
J Biol Chem ; 292(7): 2637-2645, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28028173

RESUMO

The mitochondrial transcription initiation machinery in humans consists of three proteins: the RNA polymerase (POLRMT) and two accessory factors, transcription factors A and B2 (TFAM and TFB2M, respectively). This machinery is required for the expression of mitochondrial DNA and the biogenesis of the oxidative phosphorylation system. Previous experiments suggested that TFB2M is required for promoter melting, but conclusive experimental proof for this effect has not been presented. Moreover, the role of TFB2M in promoter unwinding has not been discriminated from that of TFAM. Here we used potassium permanganate footprinting, DNase I footprinting, and in vitro transcription from the mitochondrial light-strand promoter to study the role of TFB2M in transcription initiation. We demonstrate that a complex composed of TFAM and POLRMT was readily formed at the promoter but alone was insufficient for promoter melting, which only occurred when TFB2M joined the complex. We also show that mismatch bubble templates could circumvent the requirement of TFB2M, but TFAM was still required for efficient initiation. Our findings support a model in which TFAM first recruits POLRMT to the promoter, followed by TFB2M binding and induction of promoter melting.


Assuntos
Metiltransferases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Genética , Pegada de DNA , Humanos , Fosforilação
12.
Sci Adv ; 2(8): e1600963, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27532055

RESUMO

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA(+) Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.


Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Transcrição Genética , Animais , Replicação do DNA/genética , Regulação da Expressão Gênica , Genoma Mitocondrial , Camundongos
13.
Nucleic Acids Res ; 44(12): 5861-71, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27220468

RESUMO

Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3'-end strand. This is dependent on the 3'-5' exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3'-5' degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5'-end processing of nascent DNA at OriH, and DNA repair.


Assuntos
Replicação do DNA , DNA Mitocondrial/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Sistema Livre de Células/metabolismo , Clonagem Molecular , Clivagem do DNA , Polimerase do DNA Mitocondrial , DNA Mitocondrial/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/metabolismo , Expressão Gênica , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
Nucleic Acids Res ; 44(12): 5732-42, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27112570

RESUMO

During replication of nuclear ribosomal DNA (rDNA), clashes with the transcription apparatus can cause replication fork collapse and genomic instability. To avoid this problem, a replication fork barrier protein is situated downstream of rDNA, there preventing replication in the direction opposite rDNA transcription. A potential candidate for a similar function in mitochondria is the mitochondrial transcription termination factor 1 (MTERF1, also denoted mTERF), which binds to a sequence just downstream of the ribosomal transcription unit. Previous studies have shown that MTERF1 prevents antisense transcription over the ribosomal RNA genes, a process which we here show to be independent of the transcription elongation factor TEFM. Importantly, we now demonstrate that MTERF1 arrests mitochondrial DNA (mtDNA) replication with distinct polarity. The effect is explained by the ability of MTERF1 to act as a directional contrahelicase, blocking mtDNA unwinding by the mitochondrial helicase TWINKLE. This conclusion is also supported by in vivo evidence that MTERF1 stimulates TWINKLE pausing. We conclude that MTERF1 can direct polar replication fork arrest in mammalian mitochondria.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , DNA Helicases/genética , Replicação do DNA , DNA Mitocondrial/genética , DNA Ribossômico/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA Helicases/metabolismo , DNA Mitocondrial/metabolismo , DNA Ribossômico/metabolismo , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
15.
Annu Rev Biochem ; 85: 133-60, 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-27023847

RESUMO

Mammalian mitochondrial DNA (mtDNA) encodes 13 proteins that are essential for the function of the oxidative phosphorylation system, which is composed of four respiratory-chain complexes and adenosine triphosphate (ATP) synthase. Remarkably, the maintenance and expression of mtDNA depend on the mitochondrial import of hundreds of nuclear-encoded proteins that control genome maintenance, replication, transcription, RNA maturation, and mitochondrial translation. The importance of this complex regulatory system is underscored by the identification of numerous mutations of nuclear genes that impair mtDNA maintenance and expression at different levels, causing human mitochondrial diseases with pleiotropic clinical manifestations. The basic scientific understanding of the mechanisms controlling mtDNA function has progressed considerably during the past few years, thanks to advances in biochemistry, genetics, and structural biology. The challenges for the future will be to understand how mtDNA maintenance and expression are regulated and to what extent direct intramitochondrial cross talk between different processes, such as transcription and translation, is important.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , Transcrição Genética , Animais , Evolução Biológica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Replicação do DNA , DNA Mitocondrial/metabolismo , Transporte de Elétrons/genética , Regulação da Expressão Gênica , Mamíferos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Fosforilação Oxidativa , Transporte Proteico , Transdução de Sinais
16.
Biochim Biophys Acta ; 1859(2): 339-47, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26608234

RESUMO

Mediator is a co-regulator of RNA polymerase II (Pol II), transducing signals from regulatory elements and transcription factors to the general transcription machinery at the promoter. We here demonstrate that Med20 influences ribosomal protein expression in fission yeast. In addition, loss of Med20 leads to an accumulation of aberrant, readthrough tRNA transcripts. These transcripts are polyadenylated and targeted for degradation by the exosome. Similarly, other non-coding RNA molecules, such as snRNA, snoRNA and rRNA, are also enriched in the polyadenylate preparations in the absence of Med20. We suggest that fission yeast Mediator takes part in a regulatory pathway that affects Pol III-dependent transcripts.


Assuntos
Complexo Mediador/genética , RNA de Transferência/biossíntese , RNA não Traduzido/biossíntese , Transcrição Genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Sequências Reguladoras de Ácido Nucleico/genética , Schizosaccharomyces/genética , Fatores de Transcrição/genética
17.
Nucleic Acids Res ; 43(19): 9262-75, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26253742

RESUMO

The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5'-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3'-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Mitocondrial/biossíntese , DNA Mitocondrial/química , Proteínas Mitocondriais/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Células HeLa , Humanos , Sequências Repetidas Invertidas , Camundongos , Motivos de Nucleotídeos , RNA Citoplasmático Pequeno/química , RNA Citoplasmático Pequeno/genética , Sequências Reguladoras de Ácido Nucleico , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genética , Terminação da Transcrição Genética , Vertebrados/genética
18.
PLoS Genet ; 11(6): e1005333, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26125550

RESUMO

Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication-coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3' to 5' direction. Our study gives insights into mtDNA function in cancer and answers questions regarding mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems.


Assuntos
Mitocôndrias/genética , Mutação , Neoplasias/genética , Alelos , DNA Mitocondrial , DNA de Neoplasias/genética , Genoma Mitocondrial , Humanos , RNA Neoplásico , RNA de Transferência/genética , Análise de Sequência de RNA
19.
Nucleic Acids Res ; 43(15): 7306-14, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26138482

RESUMO

The Med2, Med3 and Med15 proteins form a heterotrimeric subdomain in the budding yeast Mediator complex. This Med15 module is an important target for many gene specific transcription activators. A previous proteome wide screen in yeast identified Med3 as a protein with priogenic potential. In the present work, we have extended this observation and demonstrate that both Med3 and Med15 form amyloid-like protein aggregates under H2O2 stress conditions. Amyloid formation can also be stimulated by overexpression of Med3 or of a glutamine-rich domain present in Med15, which in turn leads to loss of the entire Med15 module from Mediator and a change in stress response. In combination with genome wide transcription analysis, our data demonstrate that amyloid formation can change the subunit composition of Mediator and thereby influence transcriptional output in budding yeast.


Assuntos
Amiloide/metabolismo , Complexo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/fisiologia , Peróxido de Hidrogênio/farmacologia , Complexo Mediador/química , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química
20.
Nat Commun ; 6: 7743, 2015 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-26205790

RESUMO

Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin, but how they target chromatin remains poorly understood. We have used chromatin RNA immunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAs enriched in repressive chromatin from breast cancer cells. Using one of the chromatin-interacting lncRNAs, MEG3, we explore the mechanisms by which lncRNAs target chromatin. Here we show that MEG3 and EZH2 share common target genes, including the TGF-ß pathway genes. Genome-wide mapping of MEG3 binding sites reveals that MEG3 modulates the activity of TGF-ß genes by binding to distal regulatory elements. MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA-DNA triplex formation. We have found that RNA-DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-ß pathway genes. Our findings suggest that RNA-DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs.


Assuntos
Regulação da Expressão Gênica , RNA Longo não Codificante/metabolismo , Linhagem Celular Tumoral , DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Complexo Repressor Polycomb 2/metabolismo , Fator de Crescimento Transformador beta/metabolismo
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