RESUMO
The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI's capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.
Assuntos
5-Metilcitosina , Adenosina , Análise de Sequência de RNA , Transcriptoma , Adenosina/análogos & derivados , Adenosina/metabolismo , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Humanos , Metilação , Análise de Sequência de RNA/métodos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA/metabolismo , RNA/genéticaRESUMO
miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.
Assuntos
Hipertrofia/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Biossíntese de Proteínas/genética , Animais , Regulação da Expressão Gênica , Humanos , Camundongos , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Poliadenilação/genética , Processamento Pós-Transcricional do RNARESUMO
The cap-binding complex elF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: elF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865-4874). Here we exploit this approach to assess which of the other subunits of elF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the lambda phage antiterminator protein N to generate the chimeric proteins lambda4A, lambda4E-102 (abolished cap binding), and lambda4E-73-102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific lambda-N binding site (boxB) in the intercistronic space. We show that lambda4E-102, but neither the double mutant lambda4E-73-102 nor lambda4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all lambda-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, lambda4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.
Assuntos
Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Capuzes de RNA/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Fator de Iniciação 4E em Eucariotos , Genes Reporter , Células HeLa , Humanos , Cinética , Luciferases/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Subunidades Proteicas , Capuzes de RNA/química , Proteínas Recombinantes/metabolismo , Transfecção , beta-Galactosidase/genéticaRESUMO
We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.
Assuntos
Regiões 5' não Traduzidas , Fases de Leitura Aberta , Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro , Sequência de Aminoácidos , Sequência de Bases , Sistema Livre de Células , Centrifugação com Gradiente de Concentração , Cloranfenicol O-Acetiltransferase/metabolismo , Códon , Genes de Plantas , Genes Reporter , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Protoplastos , Ribossomos/metabolismo , Rosales/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex. Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm. It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function. Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction. Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.
Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Núcleo Celular/metabolismo , Fator de Iniciação Eucariótico 4G , Proteínas Fúngicas/genética , Genes Fúngicos , Mutação , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , Proteínas de Ligação ao Cap de RNA , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Sistema Livre de Células , Regulação Viral da Expressão Gênica , Iniciação Traducional da Cadeia Peptídica , Picornaviridae/genética , Poli A/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , Vírus da Encefalomiocardite/genética , Células HeLa , Hepatovirus/genética , Humanos , Poliovirus/genética , Poli A/química , Capuzes de RNA , RNA Mensageiro/química , RNA Viral/química , Ribossomos/fisiologiaRESUMO
Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.
Assuntos
Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila , Drosophila/embriologia , Proteínas Nucleares/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Animais , Sítios de Ligação/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Poli A/genética , Capuzes de RNA/genéticaRESUMO
Biochemical and genetic studies are revealing a network of interactions between eukaryotic translation initiation factors, further refining or redefining perceptions of their function. The notion of translated mRNA as a 'closed-loop' has gained support from the identification of physical and functional interactions between the two mRNA ends and their associated factors. Translational control mechanisms are beginning to unravel in sufficient detail to pinpoint the affected step in the initiation pathway.
Assuntos
Células Eucarióticas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/metabolismoRESUMO
Most eukaryotic mRNAs possess a 5' cap structure (m(7)GpppN) and a 3' poly(A) tail which promote translation initiation by binding the eukaryotic translation initiation factor (eIF)4E and the poly(A) binding protein (PABP), respectively. eIF4G can bridge between eIF4E and PABP, and-through eIF3-is thought to establish a link to the small ribosomal subunit. We fused the C-terminal region of human eIF4GI lacking both the eIF4E- and PABP-binding sites, to the IRE binding protein IRP-1. This chimeric protein suffices to direct the translation of the downstream cistron of bicistronic mRNAs bearing IREs in their intercistronic space in vivo. This function is preserved even when translation via the 5' end is inhibited. Deletion analysis defined the conserved central domain (amino acids 642-1091) of eIF4G as an autonomous 'ribosome recruitment core' and implicated eIF4A as a critical binding partner. Our data reveal the sufficiency of the conserved eIF4G ribosome recruitment core to drive productive mRNA translation in living cells. The C-terminal third of eIF4G is dispensable, and may serve as a regulatory domain.
Assuntos
Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , Ribossomos/genética , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Relação Dose-Resposta a Droga , Fator de Iniciação Eucariótico 4G , Regulação da Expressão Gênica , Genes/genética , Genes Reporter/genética , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs. Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.
Assuntos
Proteínas de Ligação a DNA , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Regiões Terminadoras Genéticas/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Ligação Competitiva , Cloranfenicol O-Acetiltransferase/imunologia , Proteínas Fúngicas/genética , Proteína 1 Reguladora do Ferro , Proteínas Reguladoras de Ferro , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta/genética , Testes de Precipitina , Proteínas Quinases/genética , Capuzes de RNA/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica , UridinaRESUMO
Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N-terminal part, and binds eIF3 and eIF4A through the central and C-terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs. Although this stimulation is cap-independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of eIF4G-mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.
Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Escherichia coli/genética , Fator de Iniciação Eucariótico 4G , Humanos , Proteínas Reguladoras de Ferro , Proteínas Ferro-Enxofre , Oligonucleotídeos Antissenso , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA , Proteínas Recombinantes/metabolismoRESUMO
One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3' end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2 strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate of dim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGK promoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.
Assuntos
Metiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Alelos , Aminoglicosídeos , Antibacterianos/farmacologia , Nucléolo Celular/enzimologia , Núcleo Celular/enzimologia , Regulação Enzimológica da Expressão Gênica , Genes Letais , Metilação , Metiltransferases/genética , Mutação , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
The messenger RNA 3' poly(A) tail critically affects the initiation and control of translation in eukaryotes. By analogy to elements involved in transcription initiation, the poly(A) tail has been described as a 'translational enhancer' that enhances the 'translational promoter' activity of the mRNA 5'-cap structure. Elongation or shortening of the poly(A) tail regulates translation during development. Here we show, using cell-free and in vivo translation analyses in Saccharomyces cerevisiae, that the poly(A) tail can act as an independent 'translational promoter', delivering ribosomes to uncapped mRNAs even if their 5' end is blocked. When mRNAs compete for ribosome binding, neither the cap structure nor the poly(A) tail alone is enough to drive efficient translation, but together they synergize and direct ribosome entry to the 5' end. The cap structure both promotes ribosome recruitment, together with the poly(A) tail, and tethers recruited ribosomes to the 5' end. Correct choice of translation initiation codons and the function of translational regulators acting on the 5' untranslated region are thus ensured by the functional interaction of the poly(A) tail with the cap structure.
Assuntos
Poli A/metabolismo , Biossíntese de Proteínas , Capuzes de RNA/fisiologia , RNA Fúngico/fisiologia , Saccharomyces cerevisiae/genética , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Códon de Iniciação , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ribossomos/metabolismo , Saccharomyces cerevisiae/fisiologia , Relação Estrutura-AtividadeRESUMO
Expression of the liver-type isopeptides of cytochrome c oxidase is regulated post-transcriptionally. An RNA-binding activity has been found in only those cells where the liver-type subunits are detected. This binding protein, termed COLBP, recognises sequences or structures within the 3'-untranslated regions of transcripts encoding these liver-type isopeptides and has been implicated in the modulation of mRNA expression. We now show by subcellular fractionation, immunocompetition, UV-crosslinking and shift-Western studies that the metabolic enzyme glutamate dehydrogenase, previously reported as being able to bind RNA, is the cytochrome c oxidase transcript-binding protein, COLBP.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Glutamato Desidrogenase/metabolismo , Fígado/enzimologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Animais , Bovinos , Regulação da Expressão Gênica , Técnicas In Vitro , Mitocôndrias Hepáticas/química , RNA Mensageiro/genética , RatosRESUMO
The switching of the subunit VIa isoforms of cytochrome c oxidase has been followed in heart tissue during bovine development both by transcript levels and in terms of the incorporation of L- (liver) and H- (heart) polypeptides into mitochondria. In early fetuses, e.g., 60-days development, there are high levels of VIaL transcript and high levels of the VIaL polypeptide incorporated into mitochondria. In late fetuses (after 200 days), the levels of VIaL transcript are still high, with less but still significant amounts of VIaL polypeptide present in comparison to adult heart in which the amount of this isoform is negligible. As the proportion of VIaL transcript is reduced, the proportion of VIaH transcript increases along with the amount of the VIaH isoform in mitochondria. These data indicate isoform switching during late fetal development. The presence of COLBP (cytochrome oxidase liver isoform binding protein) (Preiss, T. and Lightowlers, R.N. (1993) J. Biol. Chem. 268, 10659-10667) was examined at different developmental stages. COLBP binding activity was observed in hearts of late fetuses but not found in adult heart tissue, providing a correlation between the presence of this factor and the presence of the VIaL polypeptide in mitochondria.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Isoenzimas/biossíntese , Envelhecimento , Animais , Sequência de Bases , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Desenvolvimento Embrionário e Fetal , Intestinos/enzimologia , Isoenzimas/química , Mitocôndrias Cardíacas/enzimologia , Dados de Sequência Molecular , Ligação ProteicaRESUMO
To investigate the molecular basis of nuclear-mitochondrial communication, we have been studying the effect of mitochondrial stress (stimulated by inhibition of mitochondrial protein synthesis) on the homeostasis of transcripts encoding nuclear and mitochondrial gene products. We report that in cells treated with the inhibitor thiamphenicol, nuclear-encoded respiratory gene transcripts were dramatically stabilized. A concomitant up-regulation in the activity of the only known respiratory transcript binding protein, cytochrome c oxidase L-form transcript binding protein (COLBP), was also noted in thiamphenicol-treated cells, demonstrating a potential mechanism for the increased transcript protection. In contradistinction, stability of all mitochondrial RNAs was unaffected by the inhibitor, as were the nuclear-encoded beta-actin, alpha-tubulin mRNAs and total cytosolic RNA. Steady state levels of all nuclear-encoded transcripts tested remained constant after inhibition of mitochondrial protein synthesis, whereas a generalized increase in the levels of processed mitochondrial mRNA was noted. We conclude that thiamphenicol induces (i) an increase in steady state levels of mitochondrial mRNA, (ii) a selective protection of nuclear respiratory gene transcripts against degradation, and (iii) an up-regulation in activity of the respiratory transcript binding protein COLBP, consistent with this protein mediating increased transcript stability. Our results demonstrate a coordinated series of intracellular responses to thiamphenicol-induced mitochondrial stress, regulated at both the pre- and post-transcriptional levels.
Assuntos
Glutamato Desidrogenase/metabolismo , Mitocôndrias Hepáticas/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Estresse Fisiológico/metabolismo , Tianfenicol/farmacologia , Núcleo Celular/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Mitocôndrias Hepáticas/efeitos dos fármacos , RNA Mitocondrial , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The influence of progesterone receptor (PR) and glucocorticoid receptor (GR) on transcription from the mouse mammary tumour virus (MMTV) promoter was analyzed using cell-free transcription of DNA templates with a G-free cassette. Preincubation of the templates with either PR or GR stimulates the rate of transcription initiation 10-50 fold, whereas the recombinant DNA binding domain of GR is inactive. Mutations that inactivate the nuclear factor I (NFI) binding site, or NFI depletion of the nuclear extract, decrease basal transcription without influencing receptor-dependent induction. Recombinant NFI, but not its DNA-binding domain, restores efficient basal transcription of the depleted extract. Recombinant OTF1 or OTF2, but not the POU domain of OTF1, enhance MMTV transcription independently of NF1. In agreement with this finding, NFI and OTF1 do not cooperate, but rather compete for binding to the wild type MMTV promoter, though they have the potential to bind simultaneously to properly oriented sites. Our results imply the existence of two independent pathways for MMTV transcription: one initiated by NFI and the other dependent on octamer transcription factors. Only the second pathway is stimulated by steroid hormone receptors in vitro.