RESUMO
FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms-that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3' region of FAD1 mRNA was analyzed by 3'RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3'UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3'UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3'UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.
RESUMO
The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.
RESUMO
ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders, and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement, we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients. We documented impaired activity of purified DNMT1 mutant proteins, which in fibroblasts results in increased DNMT1 amount. We demonstrated that DNMT1 is not localized within mitochondria, but it is associated with the mitochondrial outer membrane. Concordantly, mitochondrial DNA failed to show meaningful CpG methylation. Strikingly, we found activated mitobiogenesis and OXPHOS with significant increase of H2O2, sharply contrasting with a reduced ATP content. Metabolomics profiling of mutant cells highlighted purine, arginine/urea cycle and glutamate metabolisms as the most consistently altered pathways, similar to primary mitochondrial diseases. The most severe mutations showed activation of energy shortage AMPK-dependent sensing, leading to mTORC1 inhibition. We propose that DNMT1 RFTS mutations deregulate metabolism lowering ATP levels, as a result of increased purine catabolism and urea cycle pathways. This is associated with a paradoxical mitochondrial hyper-function and increased oxidative stress, possibly resulting in neurodegeneration in non-dividing cells.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Predisposição Genética para Doença , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Degeneração Neural/genética , Ataxias Espinocerebelares/genética , Metilação de DNA/genética , Surdez/genética , Surdez/fisiopatologia , Feminino , Fibroblastos/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/fisiopatologia , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação/genética , Narcolepsia/genética , Narcolepsia/fisiopatologia , Degeneração Neural/fisiopatologia , Fosforilação Oxidativa , Fenótipo , Processamento de Proteína Pós-Traducional/genética , Ataxias Espinocerebelares/fisiopatologiaRESUMO
The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII2, yielding a small amount of active CI+CIII2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH2-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII2, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII2 and CIV2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.
Assuntos
Trifosfato de Adenosina/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Consumo de Oxigênio , Animais , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Deleção de Genes , Mitocôndrias/genética , Oxirredução , Rotenona/farmacologiaRESUMO
Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Ouriços-do-Mar/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Transferência Ressonante de Energia de Fluorescência , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
GST fusion proteins expressed in bacteria often tend to form aggregates and are inefficiently purified by standard procedures, which employ a mixture of detergents that compromise the binding efficiency to the affinity resin and the biological activity of the recombinant proteins. Moreover, the binding to the resin is negatively affected by the molecular weight of the fusion protein. Here we report a simple and efficient method to purify active large GST-tagged proteins, which uses high ionic strength buffer to solubilize the protein aggregates in a bacterial lysate. Affinity-chromatography purification is achieved by adopting two columns connected in series, which facilitate the binding of large GST fused molecules. This approach was applied to purify the 180-kDa GST-tagged mitochondrial RNA polymerase. We also report conditions for simple and efficient GST tag removal from the eluted protein. Finally we demonstrate that the recombinant enzyme is capable to catalyze RNA synthesis.
Assuntos
Glutationa Transferase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Cromatografia de Afinidade , Glutationa Transferase/isolamento & purificação , Concentração Osmolar , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.
Assuntos
DNA Mitocondrial/genética , Renovação Mitocondrial/genética , Atrofia Óptica Hereditária de Leber/diagnóstico , Atrofia Óptica Hereditária de Leber/genética , Penetrância , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Adulto JovemRESUMO
Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial transcription we have characterized the DNA binding properties of the mitochondrial transcription factor A (TFAM). The protein contains two HMG box domains but, differently from vertebrates, displays a very short C-terminal tail. Phylogenetic analysis showed that the distribution of tail length is mixed in the different lineages, indicating that it is a trait that undergoes rapid changes during evolution. Homology modeling suggests that the protein adopts the same configuration of the human counterpart and possibly a similar mode of binding to DNA. DNase I footprinting showed that TFAM specifically contacts mtDNA at a fixed distance from three AT-rich consensus sequences that were supposed to act as transcriptional initiation sites. Bound sequences are homologous and contain an inverted repeat motif, which resembles that involved in the intercalation of human TFAM in LSP DNA. The here reported data indicate that sea urchin TFAM specifically binds mtDNA. The protein could intercalate residues at the DNA inverted motif and, despite its short tail, might have a role in mitochondrial transcription.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ouriços-do-Mar/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Pegada de DNA , Proteínas de Ligação a DNA/química , Variação Genética , Proteínas Mitocondriais/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/químicaRESUMO
DREF [DRE (DNA replication-related element)-binding factor] controls the transcription of numerous genes in Drosophila, many involved in nuclear DNA (nDNA) replication and cell proliferation, three in mitochondrial DNA (mtDNA) replication and two in mtDNA transcription termination. In this work, we have analysed the involvement of DREF in the expression of the known remaining genes engaged in the minimal mtDNA replication (d-mtDNA helicase) and transcription (the activator d-mtTFB2) machineries and of a gene involved in mitochondrial mRNA translation (d-mtTFB1). We have identified their transcriptional initiation sites and DRE sequences in their promoter regions. Gel-shift and chromatin immunoprecipitation assays demonstrate that DREF interacts in vitro and in vivo with the d-mtDNA helicase and d-mtTFB2, but not with the d-mtTFB1 promoters. Transient transfection assays in Drosophila S2 cells with mutated DRE motifs and truncated promoter regions show that DREF controls the transcription of d-mtDNA helicase and d-mtTFB2, but not that of d-mtTFB1. RNA interference of DREF in S2 cells reinforces these results showing a decrease in the mRNA levels of d-mtDNA helicase and d-mtTFB2 and no changes in those of the d-mtTFB1. These results link the genetic regulation of nuclear DNA replication with the genetic control of mtDNA replication and transcriptional activation in Drosophila.
Assuntos
DNA Helicases/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Animais , Western Blotting , Núcleo Celular , Imunoprecipitação da Cromatina , DNA Helicases/metabolismo , Proteínas de Drosophila/genética , Ensaio de Desvio de Mobilidade Eletroforética , Luciferases , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
The MTERF protein family comprises members from Metazoans and plants. All the Metazoan MTERF proteins characterized to date, including the mitochondrial transcription termination factors, play a key role in mitochondrial gene expression. In this study we report the characterization of Drosophila MTERF5 (D-MTERF5), a mitochondrial protein existing only in insects, probably originated from a duplication event of the transcription termination factor DmTTF. D-MTERF5 knock-down in D.Mel-2 cells alters transcript levels with an opposite pattern to that produced by DmTTF knock-down. D-MTERF5 is able to interact with mtDNA at the same sites contacted by DmTTF, but only in the presence of the termination factor. We propose that the two proteins participate in the transcription termination process, with D-MTERF5 engaged in relieving the block exerted by DmTTF. This hypothesis is supported also by D-MTERF5 homology modeling, which suggests that this protein contains protein-protein interaction domains. Co-regulation by DREF (DNA Replication-related Element binding Factor) of D-MTERF5 and DmTTF implies that expression of the two factors needs to be co-ordinated to ensure fine modulation of Drosophila mitochondrial transcription.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Drosophila , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Proteínas Mitocondriais/genética , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de AminoácidosRESUMO
The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.
Assuntos
RNA Polimerases Dirigidas por DNA/isolamento & purificação , Glutationa Transferase/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent K(m) and V(max) values for FMN equal to 1.3+/-0.7 microM and 4.4+/-1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 microM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria.
Assuntos
Mitocôndrias/química , Nucleotidiltransferases/análise , Animais , Western Blotting , Linhagem Celular , Cricetinae , Humanos , Isoenzimas/análise , Cinética , Fígado/química , Microscopia Confocal , Microscopia de Fluorescência , Peso Molecular , Transporte Proteico , RatosRESUMO
The MTERF family is a large protein family, identified in metazoans and plants, which consists of four subfamilies, MTERF1, 2, 3 and 4. Mitochondrial localisation was predicted for the vast majority of MTERF family members and demonstrated for the characterised MTERF proteins. The main structural feature of MTERF proteins is the presence of a modular architecture, based on repetitions of a 30-residue module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes transcription termination factors: human mTERF, sea urchin mtDBP and Drosophila DmTTF. In addition to terminating transcription, they are involved in transcription initiation and in the control of mtDNA replication. This multiplicity of functions seems to flank differences in the gene organisation of mitochondrial genomes. MTERF2 and MTERF3 play antithetical roles in controlling mitochondrial transcription: that is, mammalian and Drosophila MTERF3 act as negative regulators, whereas mammalian MTERF2 functions as a positive regulator. Both proteins contact mtDNA in the promoter region, perhaps establishing interactions, either mutual or with other factors. Regulation of MTERF gene expression in human and Drosophila depends on nuclear transcription factors NRF-2 and DREF, respectively, and proceeds through pathways which appear to discriminate between factors positively or negatively acting in mitochondrial transcription. In this emerging scenario, it appears that MTERF proteins act to coordinate mitochondrial transcription.
RESUMO
In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.
Assuntos
DNA Mitocondrial/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação da Expressão Gênica , Proteínas/genética , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação/genética , Western Blotting , Imunoprecipitação da Cromatina , DNA Helicases/genética , DNA Polimerase beta/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Fator de Transcrição de Proteínas de Ligação GA/genética , Células HeLa , Humanos , Proteínas Mitocondriais , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.
Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Mitocôndrias/enzimologia , Regiões Terminadoras Genéticas/genética , Transcrição Gênica , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ouriços-do-MarRESUMO
The MTERF family is a wide protein family, identified in Metazoa and plants, which consists of 4 subfamilies named MTERF1-4. Proteins belonging to this family are localized in mitochondria and show a modular architecture based on repetitions of a 30 amino acid module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes the characterized transcription termination factors human mTERF, sea urchin mtDBP and Drosophila DmTTF. In vitro and in vivo studies show that these factors play different roles which are not restricted to transcription termination, but concern also transcription initiation and the control of mtDNA replication. The multiplicity of functions could be related to the differences in the gene organization of the mitochondrial genomes. Studies on the function of human and Drosophila MTERF3 factor showed that the protein acts as negative regulator of mitochondrial transcription, possibly in cooperation with other still unknown factors. The complete elucidation of the role of the MTERF family members will contribute to the unraveling of the molecular mechanisms of mtDNA transcription and replication.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Humanos , Insetos/genética , Mitocôndrias/genética , Proteínas Mitocondriais , Fatores de Terminação de Peptídeos/genética , Ouriços-do-Mar/genética , Transcrição Gênica , Vertebrados/genéticaRESUMO
The DREF [DRE (DNA replication-related element)-binding factor], which regulates the transcription of a group of cell proliferation-related genes in Drosophila, also controls the expression of three genes involved in mtDNA (mitochondrial DNA) replication and maintenance. In the present study, by in silico analysis, we have identified DREs in the promoter region of a gene participating in mtDNA transcription, the DmTTF (Drosophila mitochondrial transcription termination factor). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DREs control DmTTF transcription; moreover, gel-shift and ChIP (chromatin immunoprecipitation) assays demonstrated that the analysed DRE sites interact with DREF in vitro and in vivo. Accordingly, DREF knock-down in S2 cells by RNAi (RNA interference) induced a considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the other hand, mtRNApol (mitochondrial RNA polymerase) lacks DREs in its promoter and is not regulated in vivo by DREF. In situ RNA hybridization studies showed that DmTTF was transcribed almost ubiquitously throughout all stages of Drosophila embryogenesis, whereas mtRNApol was efficiently transcribed from stages 11-12. Territories where transcription occurred mostly were the gut and Malpighi tubes for DmTTF, and the gut, mesoderm, pharyngeal muscle and Malpighi tubes for mtRNApol. The partial overlapping in the temporal and spatial mRNA expression patterns confirms that transcription of the two genes is differentially regulated during embryogenesis and suggests that DmTTF might play multiple roles in the mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional , Animais , Sequência de Bases , Células Cultivadas , Sequência Conservada , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , RNA Polimerase I/fisiologia , Elementos de Resposta , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Paracentrotus/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , Regulação da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/química , Dados de Sequência Molecular , Paracentrotus/enzimologia , Alinhamento de Sequência , Spodoptera/citologiaRESUMO
The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved "mTERF-motifs", three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein-protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.
Assuntos
Sequência Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas/genética , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Proteínas Mitocondriais/química , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , RNA/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial , Alinhamento de SequênciaRESUMO
DmTTF is a Drosophila mitochondrial DNA-binding protein, which recognizes two sequences placed at the boundary of clusters of genes transcribed in opposite directions. To obtain in vivo evidences on the role of DmTTF, we characterized a DmTTF knock-down phenotype obtained by means of RNA interference in D.Mel-2 cells. By a combination of RNase protection and real-time RT-PCR experiments we found that knock-down determines remarkable changes in mitochondrial transcription. In particular, protein depletion increases not only the level of (+) and (-)strand RNAs mapping immediately after of the two protein-binding site, but also that of transcripts located further downstream. Unexpectedly, depletion of the protein also causes the decrease in the content of those transcripts mapping upstream of the protein target sites, including the two rRNAs. The changes in transcript level do not depend on a variation in mitochondrial DNA (mtDNA) content, since mtDNA copy number is unaffected by DmTTF depletion. This work shows conclusively that DmTTF arrests in vivo the progression of the mitochondrial RNA polymerase; this is the first ever-obtained evidence for an in vivo role of an animal mitochondrial transcription termination factor. In addition, the reported data provide interesting insights into the involvement of DmTTF in transcription initiation in Drosophila mitochondria.