RESUMO
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.
Assuntos
Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Xenopus laevis/anatomia & histologia , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
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
In most species, transcription is essentially silent during the first mitotic cell cycles that follow fertilization. This means that the regulation of gene expression in early embryos heavily relies on the translational activation or inactivation of maternal mRNAs. In mammals, the mechanisms that control the translation of maternal mRNAs have been mainly studied in the mouse when maternal to zygotic transition occurs after the first mitotic division. In other mammalian species, however, this transition occurs later after several cell cycles, and little is known concerning the regulation of maternal information during this period. To address this question, we have used rabbit pre-implantation embryos to analyze the translational activation and stability of two maternal mRNAs, mm 41 and mm61. During the cleavage period, these mRNAs exhibit distinct kinetics for both their translational activation and degradation. In addition, these mRNAs both undergo cytoplasmic polyadenylation but with different efficiencies. This polyadenylation was functionally correlated with the translational activation of these mRNAs; inhibiting polyadenylation prevented translational activation. The differential efficiency of cytoplasmic polyadenylation, driven by cis-elements in the 3' untranslated region of these mRNAs, was also observed in Xenopus laevis embryos, which emphasizes the high conservation of this mechanism between species.
Assuntos
Regiões 3' não Traduzidas , Desenvolvimento Embrionário , Biossíntese de Proteínas , Animais , Sequência de Bases , Feminino , Hipoxantina Fosforribosiltransferase/genética , Dados de Sequência Molecular , Gravidez , Coelhos , Xenopus laevisRESUMO
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
During Xenopus early development, gene expression is regulated mainly at the translational level by the length of the poly(A) tail of mRNAs. The Eg family and c-mos maternal mRNAs are deadenylated rapidly and translationally repressed after fertilization. Here, we characterize a short sequence element (EDEN) responsible for the rapid deadenylation of Eg5 mRNA. Determining the core EDEN sequence permitted us to localize the c-mos EDEN sequence. The c-mos EDEN confered a rapid deadenylation to a reporter gene. The EDEN-specific RNA-binding protein (EDEN-BP) was purified and a cDNA obtained. EDEN-BP is highly homologous to a human protein possibly involved in myotonic dystrophy. Immunodepleting EDEN-BP from an egg extract totally abolished the EDEN-mediated deadenylation activity, but did not affect the default deadenylation activity. Therefore, EDEN-BP constitutes the first trans-acting factor for which an essential role in the specificity of mRNA deadenylation has been directly demonstrated.
Assuntos
Adenina/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Xenopus , Xenopus/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Dados de Sequência Molecular , Mutagênese , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Proteína Supressora de Tumor p53/metabolismo , Xenopus/embriologiaRESUMO
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
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/embriologiaRESUMO
The poly(A) tail of mRNAs plays an important role in translational control. In Xenopus laevis matured oocytes, maternal mRNAs that contain a cytoplasmic polyadenylation element (CPE) are polyadenylated, whereas CPE deficient mRNAs are deadenylated by a default process. Eg mRNAs are maternal transcripts that are poly(A)+ in matured oocytes and rapidly deadenylated after fertilization. This post-fertilization deadenylation of Eg mRNAs requires specific sequence information. Such a deadenylation element has been identified previously in the 3'UTR of Eg2 mRNA. In this study, we show that cell-free extracts made from embryos or activated eggs contain two kinetically distinct deadenylation activities, with different substrate specificities. One, responsible for the slow deadenylation of RNAs that are devoid of a functional CPE, has the characteristics of a default PAN activity. The other effectuates the rapid deadenylation of RNAs containing a deadenylation element. The in vitro system described here will allow the characterization of factors controlling the deadenylation of Eg mRNAs in embryos.
Assuntos
RNA/metabolismo , Animais , Sequência de Bases , Sistema Livre de Células , Quimera/genética , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Exorribonucleases/metabolismo , Feminino , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Oócitos/metabolismo , Plasmídeos/genética , Poli A/genética , Poli A/metabolismo , Biossíntese de Proteínas , RNA/genética , Especificidade por Substrato , 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 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/farmacologiaAssuntos
Fatores de Alongamento de Peptídeos/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Fatores de Troca do Nucleotídeo Guanina , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , XenopusRESUMO
In the amphibian Xenopus laevis, the elongation factor 1 alpha proteins (EF-1 alpha) synthesised in oocytes and somatic cells correspond to distinct gene products. Furthermore, the somatic EF-1 alpha gene (EF-1 alpha S) produces one of the most highly expressed early zygotic transcripts in the embryo. The functional recycling of EF-1 alpha (conversion of EF-1 alpha-GDP to EF-1 alpha-GTP) is assured by the EF-1 beta gamma complex. We show here that in Xenopus laevis embryos, contrary to the situation for EF-1 alpha, EF-1 beta, and EF-1 gamma mRNAs are transcribed from the same genes in oocytes and somatic cells. In addition, the onset of transcription of the EF-1 beta and EF-1 gamma genes from the zygotic genome occurs several hours after that of the somatic EF-1 alpha S gene. Therefore, during early Xenopus development the expression of these three elongation factors is not co-ordinated at the transcriptional level. The consequences of this uncoupling on the efficiency of translational elongation in the early Xenopus embryo are discussed.
Assuntos
Fatores de Alongamento de Peptídeos/genética , Xenopus laevis/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Desenvolvimento Embrionário e Fetal/genética , Feminino , Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Oócitos/metabolismo , Fator 1 de Elongação de Peptídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Xenopus laevis/embriologia , Xenopus laevis/metabolismoRESUMO
Maternal Xenopus Eg mRNAs have been previously identified as transcripts that are specifically deadenylated after fertilization and degraded after the mid blastula transition. Destabilizing cis sequences were previously localised in the 3' untranslated region of Eg2 mRNA. In order to characterize possible trans-acting factors which are involved in the post-transcriptional regulation of Eg mRNAs, gel-shift and u.v. cross-linking experiments were performed, which allowed the identification of a p53-p55 RNA-binding protein doublet specific for the 3' untranslated regions of Eg mRNAs. These p53-p55 proteins do not bind to the 3' untranslated regions of either ornithine decarboxylase or phosphatase 2Ac mRNAs, which remain polyadenylated in embryos. These novel RNA-binding proteins are distinct from the cytoplasmic polyadenylation element-binding protein that controls the polyadenylation of maternal mRNAs in maturing Xenopus oocytes, and from previously identified thermoresistant RNA-binding proteins present in oocyte mRNP storage particles. The p53-p55 bind a portion of the Eg2 mRNA 3' untranslated region, distinct from the previously identified destabilizing region, that is able to confer the postfertilization deadenylation of CAT-coding chimeric mRNAs. This suggests that the p53-p55 RNA-binding proteins are good candidates for trans-acting factors involved in the deadenylation of Eg mRNAs in Xenopus embryos.
Assuntos
Oócitos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Xenopus laevis/metabolismo , Animais , Autorradiografia , Eletroforese em Gel de Poliacrilamida , Feminino , Processamento Pós-Transcricional do RNA/fisiologiaRESUMO
In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Expressão Gênica , Ornitina Descarboxilase/genética , Processamento Pós-Transcricional do RNA , Animais , Blastocisto/enzimologia , Northern Blotting , Células CHO , Clonagem Molecular , Cricetinae , Cicloeximida/farmacologia , Embrião não Mamífero/enzimologia , Feminino , Microinjeções , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Putrescina/farmacologia , RNA Mensageiro/genética , Xenopus laevis/embriologiaRESUMO
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.