RESUMO
The deadenylation of maternal mRNAs in the Xenopus embryo is a sequence-specific process. One cis element that targets maternal mRNAs for deadenylation after fertilization is the embryo deadenylation element (EDEN). This element, composed of U/R repeats, is specifically bound by a protein, EDEN-BP. In the present study we show that the rate at which an RNA containing an EDEN is deadenylated can be increased by the presence of an additional cis element composed of three AUU repeats. This effect was observed for a natural EDEN (c-mos) and two synthetic EDENs. Hence, the enhancement of EDEN-dependent deadenylation conferred by the (AUU)3 motif is not due to an interaction with a particular EDEN sequence. Mutation of the (AUU)3 motif abrogated the enhancement of EDEN-dependent deadenylation. These data indicate that the rate at which a specific maternal mRNA is deadenylated in Xenopus embryos is probably defined by a cross talk between multiple cis elements.
Assuntos
Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico/genética , Proteínas de Xenopus , Xenopus/embriologia , Animais , Reagentes de Ligações Cruzadas/metabolismo , DNA Recombinante/genética , Elementos Facilitadores Genéticos/genética , Genes mos/genética , Globinas/genética , Oligorribonucleotídeos/genética , Proteínas de Ligação a RNA/genética , Raios UltravioletaRESUMO
Although the maternal Xenopus laevis Eg mRNAs are deadenylated after fertilization, they are not immediately degraded and they persist in the embryos as poly(A)- transcripts. The degradation of these RNAs is not detected until the blastula stage of development (6 to 7 h postfertilization). To understand the basis for this delay between deadenylation and degradation, it is necessary to identify the cis-acting element(s) required to trigger degradation in blastula stage embryos. To this end, several chimeric RNAs containing different portions of the 3' untranslated region of Eg2 mRNA were injected into two-cell X. laevis embryos. We observed that only the RNAs that contained the cis-acting elements that confer rapid deadenylation were subsequently degraded at the blastula stage. This suggested that deadenylation may be sufficient to trigger degradation. By injecting chimeric RNAs devoid of Eg sequence information, we further showed that only deadenylated RNAs were degraded in X. laevis embryos. Last, introduction of a functional cytoplasmic polyadenylation element into a poly(A)- RNA, thereby causing its polyadenylation after injection into embryos, protected the RNA from degradation. Hence, in X. laevis embryos, the postfertilization deadenylation of maternal Eg mRNAs is sufficient to cause the degradation of an mRNA, which, however, only becomes apparent at the blastula stage. Possible causes for this delay between deadenylation and degradation are discussed in the light of these results.
Assuntos
Blastocisto/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Monofosfato de Adenosina , Animais , Globinas/genética , Poli A/metabolismo , RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Xenopus laevis/embriologiaRESUMO
By injecting the appropriately constructed plasmids into one-cell Xenopus embryos, we determined that the 3' region of the maternal Xenopus Eg2 mRNA confers instability on the chimeric mRNA transcribed from these plasmids. This instability, like that of the maternal Eg2 transcript, was abolished by treatment of the embryos with cycloheximide. Analysis of the polysome distribution of the maternal Eg2 mRNA in cycloheximide-treated and untreated embryos showed that Eg2 mRNA was released from polysomes after fertilization and that the stabilization caused by cycloheximide treatment was not due to a reloading of ribosomes onto the mRNA. Insertion of a stable hairpin loop (delta G = -50 kcal/mol) 5' to the reporter gene in the injected plasmid caused a 10- to 20-fold decrease in translation from the transcribed mRNAs. This decrease in translation did not abolish the instability conferred by the 3' Eg2 region. Therefore, the degradation of these chimeric mRNAs in Xenopus embryos requires the translation of another maternal mRNA coding for a trans-acting factor involved in mRNA degradation. Further restriction of the 3' Eg2 region, placed 3' to the reporter gene, showed that a cis-acting instability-conferring sequence is contained in a 497-nucleotide fragment.
Assuntos
Embrião não Mamífero/fisiologia , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/metabolismo , Animais , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cicloeximida/farmacologia , Feminino , Fertilização , Regulação da Expressão Gênica , Cinética , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Plasmídeos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Transcrição Gênica , Xenopus laevisRESUMO
The maternal Xenopus Eg mRNAs are adenylated and translated in the mature oocyte and then, after fertilization, are deadenylated and released from polysomes. Therefore, after fertilization, a change occurs in the cellular mechanisms that control mRNA adenylation. In the study reported here, we show that the 3' untranslated region of Eg2 mRNA contains a cis-acting element that is required for the deadenylation of chimeric RNAs after fertilization. This cis-acting element is contained within a single 17-nucleotide portion of the Eg2 mRNA. Disruption of this deadenylation element allows adenylation of the chimeric transcripts in the embryo. Therefore, this cis-acting element is part of the sequence information required for the developmental switch from adenylation to deadenylation of the maternal Eg2 mRNA in Xenopus embryos.
Assuntos
Regulação da Expressão Gênica , Oócitos/fisiologia , Poli A/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Xenopus laevis/genética , Animais , Sequência de Bases , Fertilização , Dados de Sequência Molecular , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Deleção de Sequência , Relação Estrutura-Atividade , Xenopus laevis/embriologiaRESUMO
The first 12 cell divisions of Xenopus laevis embryos do not require gene transcription. This means that the regulation of gene expression during this period is controlled at post transcriptional levels and makes Xenopus early development a potentially interesting biological system with which to study the mechanisms involved. We describe here the stability characteristics of several maternal Xenopus mRNAs which are deadenylated soon after fertilisation (J. Paris and M. Philippe, Dev. Biol., in press). We show that these mRNAs were only degraded in the embryo after the midblastula transition (MBT), when gene transcription was initiated. The kinetics with which the deadenylated maternal mRNAs decreased in the post-MBT embryos showed sequence specificity. The degradation of these mRNAs after the MBT was inhibited by cycloheximide but was not affected by dactinomycin. Therefore, the destabilization of these mRNAs does not appear to be initiated by new embryonic gene transcripts. Sequence comparisons of the 3' untranslated region of these mRNAs identified several motifs which may be involved in the posttranscriptional control of these gene products.
Assuntos
Embrião não Mamífero/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/genética , Transcrição Gênica , Animais , Sequência de Bases , Northern Blotting , Divisão Celular , Embrião não Mamífero/citologia , Feminino , Cinética , Dados de Sequência Molecular , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Xenopus laevisRESUMO
Friend erythroleukemic cells can be induced to differentiate by chemicals such as dimethyl sulfoxide and hexamethylene bisacetamide (HMBA) in a dose-dependent manner. Others have shown that dexamethasone and other steroid hormones can inhibit the differentiation induced by dimethyl sulfoxide. We show here that dexamethasone has a dual mode of action on the HMBA-induced differentiation of a Friend cell line, DS19. At concentrations above 10(-10) M, dexamethasone inhibits the HMBA-induced differentiation in DS19 cells. We further found that as the concentration of HMBA is reduced, the amount of dexamethasone required to inhibit differentiation increases. Such inhibition seems to act primarily by decreasing the probability that a cell will become committed to differentiate. Besides, dexamethasone does not inhibit hemoglobin synthesis of cells which are committed in its absence. In addition, we show that sensitivity to the inhibition by dexamethasone is inversely related to the inducibility of cell populations. The second action of dexamethasone, observed at concentrations between 10(-10) and 10(-13) M, is to increase the proportion of hemoglobin-containing cells relative to that for cells cultured in its absence. The degree of this synergistic effect for induced differentiation is inversely related to the level of induction in the absence of dexamethasone and thus is best observed in the cells cultured at low levels of HMBA. We present evidence that this synergistic effect may be due to an increased viability of the induced cells and possibly an increase in the proliferative capacity of these cells.
Assuntos
Acetamidas/farmacologia , Dexametasona/farmacologia , Diaminas/farmacologia , Eritrócitos/efeitos dos fármacos , Leucemia Eritroblástica Aguda/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Interações Medicamentosas , Sinergismo Farmacológico , Camundongos , Coloração e RotulagemRESUMO
Glucocorticoids are known to inhibit the induced differentiation of murine erythroleukemia cells. I show here that the binding of glucocorticoid receptors to the intact nuclei of these cells is reduced by exogenous extracellular pyridoxine (1-4 mM). This reduction of glucocorticoid-receptor binding did not abrogate the inhibition by dexamethasone of the induced differentiation.
Assuntos
Dexametasona/toxicidade , Leucemia Experimental/fisiopatologia , Piridoxina/farmacologia , Receptores de Glucocorticoides/fisiologia , Receptores de Esteroides/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Dexametasona/metabolismo , Hemoglobinas/análise , Cinética , Camundongos , Receptores de Glucocorticoides/efeitos dos fármacosRESUMO
During Xenopus early development, the length of the poly(A) tail of maternal mRNAs is a key element of translational control. Several sequence elements (cytoplasmic polyadenylation elements) localized in 3' untranslated regions have been shown to be responsible for the cytoplasmic polyadenylation of certain maternal mRNAs. Here, we demonstrate that the mRNA encoding the catalytic subunit of phosphatase 2A is polyadenylated after fertilization of Xenopus eggs. This polyadenylation is mediated by the additive effects of two cis elements, one being similar to already described cytoplasmic polyadenylation elements and the other consisting of a polycytosine motif. Finally, a candidate specificity factor for polycytosine-mediated cytoplasmic polyadenylation has been purified and identified as the Xenopus homologue of human alpha-CP2.
Assuntos
Fosfoproteínas Fosfatases/genética , RNA Mensageiro , Xenopus/embriologia , Regiões 3' não Traduzidas , Adenina/metabolismo , Sequência de Aminoácidos , Animais , Citoplasma , Fertilização , Humanos , Dados de Sequência Molecular , Oócitos , Proteína Fosfatase 2RESUMO
The biological importance of the various changes in polyamine metabolism that occur during early Xenopus development have been investigated. Incubation of embryos in high salt medium was observed to cause a precocious fall in ornithine decarboxylase activity without affecting development. Similarly, inhibiting ornithine decarboxylase activity with specific inhibitors did not affect development. Injecting spermidine, within physiologically relevant limits, caused a dose-dependent inhibition of mitotic divisions in the injected blastomere. Increasing the intracellular putrescine did not affect cell division or development. Co-injection of both spermidine and putrescine, so that the original molar ratio of these two polyamines was conserved, abrogated the inhibition of cell division observed when spermidine was injected alone. Therefore, in Xenopus embryos the intracellular spermidine concentration must be retained within certain limits relative to that of putrescine to allow normal development.
Assuntos
Poliaminas/metabolismo , Xenopus laevis/embriologia , Animais , Blastômeros/citologia , Blastômeros/efeitos dos fármacos , Blastômeros/metabolismo , Feminino , Microinjeções , Mitose/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Putrescina/farmacologia , Solução Salina Hipertônica , Espermidina/farmacologiaRESUMO
The distribution of four specific RNA species between the poly(A)+ and poly(A)- fractions has been studied during the first hours of Xenopus laevis development, before the mid-blastula transition (MBT). Two of these specific RNA species correspond to clones selected by differential hybridization from a Xenopus egg cDNA library. Another corresponds to Xenopus c-raf mRNA and the last one to RNA revealed by a mouse ornithine decarboxylase probe. We show that two of these RNAs are adenylated after fertilization and remain in the poly(A)+ population. During the same period, the other two RNAs are deadenylated and these new poly(A)- RNAs remain stable at least until the MBT. These results show (i) that polyadenylation of specific RNA species occurs after fertilization in Xenopus and (ii) that, in the absence of transcription, adenylation and deadenylation can occur simultaneously in the fertilized egg.
Assuntos
Blastocisto/fisiologia , Poli A/análise , RNA/análise , Xenopus laevis/embriologia , Animais , Northern Blotting , Clonagem Molecular , DNA/genética , Feminino , Plasmídeos , Proto-Oncogenes , RNA Mensageiro , Transcrição Gênica , Xenopus laevis/genéticaRESUMO
A polyclonal antibody was raised against bacterially produced catalytic alpha subunit of protein phosphatase 2A (PP2AC) cloned from Xenopus ovarian library. The amount of PP2AC in Xenopus oocytes determined by Western blot analysis was 1 ng/microgram of cytosolic protein. The antibody depleted PP2AC from oocyte extracts in association with 6 components (40, 62, 65, 80, 85 and 90 kDa). Prophase- and metaphase-arrested oocytes contained identical amounts of PP2AC. Metaphase oocytes showed one specific change in the 62 kDa protein associated with PP2AC.
Assuntos
Meiose , Oócitos/enzimologia , Fosfoproteínas Fosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Divisão Celular , DNA/genética , Feminino , Biblioteca Gênica , Dados de Sequência Molecular , Oócitos/citologia , Ovário/enzimologia , Fosfoproteínas Fosfatases/análise , Fosfoproteínas Fosfatases/isolamento & purificação , Proteína Fosfatase 2 , Mapeamento por Restrição , XenopusRESUMO
The metabolism of the poly(A) tail is a process important for the translational regulation of maternal mRNAs in Xenopus laevis oocytes and early embryos. Two poly(A) nuclease (PAN) activities have been described in Xenopus embryo or activated egg extracts (Legagneux et al (1995) RNA 1, 1001-1008). These activities (default PAN and EgPAN) are distinguishable by their deadenylation kinetics and their substrate specificities. In this report, we show that these activities display different sensitivities to biochemical treatments. Urea and, to a lesser extent, spermidine, inhibit EgPAN at concentrations which have no effect on default PAN. Heparin activates default PAN but inhibits EgPAN. When extracts are fractionated by ultracentrifugation, the default activity is recovered in one unique fraction, whereas two fractions must be combined to reconstitute the EgPAN activity. Moreover, these two deadenylation activities are separable by size exclusion chromatography under native conditions. We conclude that these two deadenylation activities are mediated by two protein complexes.
Assuntos
Oócitos/enzimologia , Ribonucleases/metabolismo , Animais , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Heparina/farmacologia , Putrescina/farmacologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/isolamento & purificação , Espermidina/farmacologia , Especificidade por Substrato , Proteína Supressora de Tumor p53/metabolismo , Ultracentrifugação , Ureia/farmacologia , Xenopus laevis/embriologiaAssuntos
Pigmentos da Retina , Rodopsina , Cristalografia , Detergentes , Deutério , Micelas , Peso Molecular , NêutronsAssuntos
Pigmentos da Retina , Rodopsina , Animais , Deutério , Conformação Proteica , Espectrofotometria InfravermelhoAssuntos
Pigmentos da Retina , Rodopsina , Hidrogênio , Cinética , Células Fotorreceptoras , Conformação Proteica , Solventes , TrítioAssuntos
Pigmentos da Retina , Animais , Bovinos , Centrifugação com Gradiente de Concentração , Cromatografia , Detergentes , Técnica de Congelamento e Réplica , Hidroxiapatitas , Lipídeos , Substâncias Macromoleculares , Membranas , Microscopia Eletrônica , Fosfatidilcolinas , Fosfolipídeos , Pigmentos da Retina/isolamento & purificação , Espectrofotometria , Tolueno , Água , Difração de Raios XAssuntos
Pigmentos da Retina/análise , Rodopsina/análise , Animais , Detergentes , Deutério , Métodos , Micelas , Nêutrons , Espalhamento de RadiaçãoRESUMO
EDEN (embryo deadenylation element)-dependent deadenylation is a regulatory process that was initially identified in Xenopus laevis early embryos and was subsequently shown to exist in Drosophila oocytes. Recent data showed that this regulatory process is required for somitic segmentation in Xenopus. Inactivation of EDEN-BP (EDEN-binding protein) causes severe segmentation defects, and the expression of segmentation markers in the Notch signalling pathway is disrupted. We showed that the mRNA encoding XSu(H) (Xenopus suppressor of hairless), a protein central to the Notch pathway, is regulated by EDEN-BP. Our data also indicate that other segmentation RNAs are targets for EDEN-BP. To identify new EDEN-BP targets, a microarray analysis has been undertaken.