RESUMO
Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs: the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. Here we identify Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. We further identify r2d2 mRNA, encoding a Dicer-2 partner in RNAi, as a Dicer-2 polyadenylation target. Our results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Poliadenilação/fisiologia , Polinucleotídeo Adenililtransferase/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/química , Ribonuclease III/metabolismo , Receptores Toll-Like/química , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Polinucleotídeo Adenililtransferase/genética , Biossíntese de Proteínas/genética , Sinais de Poliadenilação na Ponta 3' do RNA/genética , RNA Helicases/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Piwi-interacting RNAs (piRNAs) and PIWI proteins play a crucial role in germ cells by repressing transposable elements and regulating gene expression. In Drosophila, maternal piRNAs are loaded into the embryo mostly bound to the PIWI protein Aubergine (Aub). Aub targets maternal mRNAs through incomplete base-pairing with piRNAs and can induce their destabilization in the somatic part of the embryo. Paradoxically, these Aub-dependent unstable mRNAs encode germ cell determinants that are selectively stabilized in the germ plasm. Here we show that piRNAs and Aub actively protect germ cell mRNAs in the germ plasm. Aub directly interacts with the germline-specific poly(A) polymerase Wispy, thus leading to mRNA polyadenylation and stabilization in the germ plasm. These results reveal a role for piRNAs in mRNA stabilization and identify Aub as an interactor of Wispy for mRNA polyadenylation. They further highlight the role of Aub and piRNAs in embryonic patterning through two opposite functions.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Padronização Corporal/genética , Padronização Corporal/fisiologia , Drosophila melanogaster/embriologia , Células Germinativas Embrionárias/metabolismo , Feminino , Hibridização in Situ Fluorescente , Masculino , Metilação , Estabilidade de RNARESUMO
Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCFFbw7 ubiquitin ligase. In human cancers bearing FBXW7-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in Xenopus eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Proteínas F-Box/metabolismo , Proteína Quinase C/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína 7 com Repetições F-Box-WD , Humanos , Fosforilação , Xenopus laevisRESUMO
The Piwi-interacting RNA (piRNA) pathway plays an essential role in the repression of transposons in the germline. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. Here, we perform iCLIP with the PIWI protein Aubergine (Aub) and identify hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants.
Assuntos
Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Fatores de Iniciação de Peptídeos/genética , Estabilidade de RNA , RNA Interferente Pequeno/genética , Animais , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Células Germinativas/citologia , Fatores de Iniciação de Peptídeos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoAssuntos
Elementos de DNA Transponíveis/genética , Desenvolvimento Embrionário/genética , Pequeno RNA não Traduzido/fisiologia , Animais , Elementos de DNA Transponíveis/fisiologia , Drosophila/embriologia , Drosophila/genética , Drosophila/fisiologia , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Modelos Biológicos , Poliadenilação/fisiologia , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , Pequeno RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologiaRESUMO
Piwi-associated RNAs (piRNAs), a specific class of 24- to 30-nucleotide-long RNAs produced by the Piwi-type of Argonaute proteins, have a specific germline function in repressing transposable elements. This repression is thought to involve heterochromatin formation and transcriptional and post-transcriptional silencing. The piRNA pathway has other essential functions in germline stem cell maintenance and in maintaining germline DNA integrity. Here we uncover an unexpected function of the piRNA pathway in the decay of maternal messenger RNAs and in translational repression in the early embryo. A subset of maternal mRNAs is degraded in the embryo at the maternal-to-zygotic transition. In Drosophila, maternal mRNA degradation depends on the RNA-binding protein Smaug and the deadenylase CCR4, as well as the zygotic expression of a microRNA cluster. Using mRNA encoding the embryonic posterior morphogen Nanos (Nos) as a paradigm to study maternal mRNA decay, we found that CCR4-mediated deadenylation of nos depends on components of the piRNA pathway including piRNAs complementary to a specific region in the nos 3' untranslated region. Reduced deadenylation when piRNA-induced regulation is impaired correlates with nos mRNA stabilization and translational derepression in the embryo, resulting in head development defects. Aubergine, one of the Argonaute proteins in the piRNA pathway, is present in a complex with Smaug, CCR4, nos mRNA and piRNAs that target the nos 3' untranslated region, in the bulk of the embryo. We propose that piRNAs and their associated proteins act together with Smaug to recruit the CCR4 deadenylation complex to specific mRNAs, thus promoting their decay. Because the piRNAs involved in this regulation are produced from transposable elements, this identifies a direct developmental function for transposable elements in the regulation of gene expression.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Poliadenilação/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas , Citoplasma/genética , Citoplasma/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Mães , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Zigoto/metabolismoRESUMO
Cytoplasmic polyadenylation has an essential role in activating maternal mRNA translation during early development. In vertebrates, the reaction requires CPEB, an RNA-binding protein and the poly(A) polymerase GLD-2. GLD-2-type poly(A) polymerases form a family clearly distinguishable from canonical poly(A) polymerases (PAPs). In Drosophila, canonical PAP is involved in cytoplasmic polyadenylation with Orb, the Drosophila CPEB, during mid-oogenesis. We show that the female germline GLD-2 is encoded by wispy. Wispy acts as a poly(A) polymerase in a tethering assay and in vivo for cytoplasmic polyadenylation of specific mRNA targets during late oogenesis and early embryogenesis. wispy function is required at the final stage of oogenesis for metaphase of meiosis I arrest and for progression beyond this stage. By contrast, canonical PAP acts with Orb for the earliest steps of oogenesis. Both Wispy and PAP interact with Orb genetically and physically in an ovarian complex. We conclude that two distinct poly(A) polymerases have a role in cytoplasmic polyadenylation in the female germline, each of them being specifically required for different steps of oogenesis.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Proteínas do Olho/genética , Oogênese/genética , Poliadenilação/genética , Polinucleotídeo Adenililtransferase/genética , Animais , Western Blotting , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citoplasma/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imunoprecipitação , Meiose/genética , Metáfase/genética , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
During early development, control of the poly(A) tail length by cytoplasmic polyadenylation is critical for the regulation of specific mRNA expression. Gld2, an atypical poly(A) polymerase, is involved in cytoplasmic polyadenylation in Xenopus oocytes. In this study, a new XGld2-interacting protein was identified: Xenopus RNA-binding motif protein 9 (XRbm9). This RNA-binding protein is exclusively expressed in the cytoplasm of Xenopus oocytes and interacts directly with XGld2. It is shown that XRbm9 belongs to the cytoplasmic polyadenylation complex, together with cytoplasmic polyadenylation element-binding protein (CPEB), cleavage and polyadenylation specificity factor (CPSF) and XGld2. In addition, tethered XRbm9 stimulates the translation of a reporter mRNA. The function of XGld2 in stage VI oocytes was also analysed. The injection of XGld2 antibody into oocytes inhibited polyadenylation, showing that endogenous XGld2 is required for cytoplasmic polyadenylation. Unexpectedly, XGld2 and CPEB antibody injections also led to an acceleration of meiotic maturation, suggesting that XGld2 is part of a masking complex with CPEB and is associated with repressed mRNAs in oocytes.
Assuntos
Citoplasma/metabolismo , Poliadenilação , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Clonagem Molecular , Feminino , Fase G2/genética , Fase G2/fisiologia , Immunoblotting , Imunoprecipitação , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/metabolismo , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/imunologia , Ligação Proteica , Biossíntese de Proteínas , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologia , Xenopus laevisRESUMO
Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.
Assuntos
Fator Estimulador de Clivagem/fisiologia , Citoplasma/metabolismo , Oócitos/metabolismo , Proteínas de Xenopus/fisiologia , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Fator Estimulador de Clivagem/química , Fator Estimulador de Clivagem/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , RNA Mensageiro/metabolismo , Coelhos , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The molecular events triggered during progesterone-induced oocyte maturation in Xenopus are not well understood. One of the first events is the activation of the MAPK cascade and the maturation-promoting factor (MPF). The latter triggers meiosis I resumption and meiosis II progression until the metaphase II arrest. The release of the metaphase II is mediated by the anaphase-promoting complex (APC)-dependent degradation of cyclin B. This degradation activity requires the APC activator Cdc20 that activates ubiquitination reactions by recruiting substrates to the APC. However, recent reports in different organisms involve other APC regulators during different phases of the meiotic cycle. Therefore, we investigated the role of another APC regulator, XCdh1 during the G2/M transition in meiosis I in the Xenopus oocyte. Here, we report that XCdh1 protein is expressed in oocytes. Besides, injection of specific XCdh1 antisense inhibits progesterone-induced G2/M transition that can be rescued by adding back the purified human Cdh1 protein. On the other hand, ectopic expression of low levels of XCdh1 protein has a positive effect on the G2/M transition by facilitating this process. Moreover, the sole injection of XCdh1 mRNA triggers Mos protein synthesis and biphosphorylation of MAPK in absence of progesterone. Altogether, these data show that XCdh1 has a positive role during the G2/M transition in the oocyte. According to our results, its role could be independent of the APC.
Assuntos
Oócitos/fisiologia , Progesterona/fisiologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Ciclossomo-Complexo Promotor de Anáfase , Animais , Células Cultivadas , Embrião não Mamífero , Feminino , Fase G2/efeitos dos fármacos , Fase G2/genética , Regulação da Expressão Gênica no Desenvolvimento , Meiose/genética , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitose/efeitos dos fármacos , Mitose/genética , Oligonucleotídeos Antissenso/farmacologia , Oócitos/efeitos dos fármacos , Fosforilação , Progesterona/farmacologia , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Complexos Ubiquitina-Proteína Ligase/fisiologiaRESUMO
Members of the Bix family of homeobox-containing genes are expressed in the vegetal hemisphere of the Xenopus embryo at the early gastrula stage. Misexpression of at least some of the family members causes activation of mesoderm- and endoderm-specific genes and it is known that some of the proteins, including Bix2 and Bix3, interact with Smad proteins via a motif that is also present in the related protein Mixer. In this paper we study the function of Bix3. Misexpression of Bix3, similar to misexpression of other members of the Bix family, causes the activation of a range of mesendodermal genes, but the spectrum of genes induced by Bix3 differs from that induced by Bix1. More significantly, we find that overexpression of Bix3 also causes apoptosis, as does depletion of Bix3 by use of antisense morpholino oligonucleotides. The ability of Bix3 to causes apoptosis is not associated with its ability to activate transcription and nor with its possession of a Smad interaction motif. Rather, Bix3 lacks a C-terminal motif, which, in Bix1, acts in cis to inhibit apoptosis. Mutation of this sequence in Bix1 causes the protein to acquire apoptosis-inducing activity.
Assuntos
Apoptose/fisiologia , Embrião não Mamífero/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Xenopus , Xenopus laevis/embriologia , Animais , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Oligonucleotídeos Antissenso/metabolismo , Fragmentos de Peptídeos/metabolismo , Fenótipo , Alinhamento de Sequência , Treonina/metabolismo , Transcrição Gênica , Xenopus laevis/fisiologiaRESUMO
Xenopus Brachyury (Xbra) plays a key role in mesoderm formation during early development. One factor thought to be involved in the regulation of Xbra is XSIP1, a zinc finger/homeodomain-like DNA-binding protein that belongs to the deltaEF1 family of transcriptional repressors. We show here that Xbra and XSIP1 are co-expressed at the onset of gastrulation, but that expression subsequently refines such that Xbra is expressed in prospective mesoderm and XSIP1 in anterior neurectoderm. This refinement of the expression patterns of the two genes is due in part to the ability of XSIP1 to repress expression of Xbra. This repression is highly specific, in the sense that XSIP1 does not repress the expression of other regionally expressed genes in the early embryo, and that other members of the family to which XSIP1 belongs, such as deltaEF1 and its Xenopus homologue ZEB, cannot regulate Xbra expression. The function of XSIP1 was studied further by making an interfering construct comprising the open reading frame of XSIP1 fused to the VP16 transactivation domain. Experiments using this chimeric protein suggest that XSIP1 is required for normal gastrulation movements to occur and for the development of the anterior neural plate.