RESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.
Asunto(s)
Distrofia Muscular Oculofaríngea , Animales , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patología , Músculo Esquelético/metabolismo , Respuesta de Proteína Desplegada , Núcleo Celular/metabolismo , Estrés del Retículo Endoplásmico , DrosophilaRESUMEN
Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the RING finger protein RNF219 co-purifies with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, which mediates translational repression in both a deadenylase activity-dependent and -independent manner. Strikingly, RNF219 both inhibits the deadenylase activity of CCR4-NOT and enhances its capacity to repress translation of a target mRNA. We propose that the interaction of RNF219 with the CCR4-NOT complex directs the translational repressive activity of CCR4-NOT to a deadenylation-independent mechanism.
Asunto(s)
Biosíntesis de Proteínas , Ribonucleasas , Regulación de la Expresión Génica , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismoRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.
Asunto(s)
Atrofia Muscular/patología , Distrofia Muscular Oculofaríngea/patología , Proteína I de Unión a Poli(A)/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster , Regulación de la Expresión Génica , Pruebas Genéticas , Humanos , Leupeptinas/farmacología , Leupeptinas/uso terapéutico , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/metabolismo , Distrofia Muscular Oculofaríngea/tratamiento farmacológico , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutación , Proteína I de Unión a Poli(A)/química , Prueba de Estudio Conceptual , Agregado de Proteínas/efectos de los fármacosRESUMEN
Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. Here, we report a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Factor 3 de Iniciación Eucariótica/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/metabolismo , Línea Celular , Células Germinativas/citología , Células Germinativas/metabolismo , Estabilidad del ARNRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Polinucleotido Adenililtransferasa/genética , Polinucleotido Adenililtransferasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Drosophila melanogaster/embriología , Células Germinales Embrionarias/metabolismo , Femenino , Hibridación Fluorescente in Situ , Masculino , Metilación , Estabilidad del ARNRESUMEN
PIWI proteins play essential roles in germ cells and stem cell lineages. In Drosophila, Piwi is required in somatic niche cells and germline stem cells (GSCs) to support GSC self-renewal and differentiation. Whether and how other PIWI proteins are involved in GSC biology remains unknown. Here, we show that Aubergine (Aub), another PIWI protein, is intrinsically required in GSCs for their self-renewal and differentiation. Aub needs to be loaded with piRNAs to control GSC self-renewal and acts through direct mRNA regulation. We identify the Cbl proto-oncogene, a regulator of mammalian hematopoietic stem cells, as a novel GSC differentiation factor. Aub stimulates GSC self-renewal by repressing Cbl mRNA translation and does so in part through recruitment of the CCR4-NOT complex. This study reveals the role of piRNAs and PIWI proteins in controlling stem cell homeostasis via translational repression and highlights piRNAs as major post-transcriptional regulators in key developmental decisions.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Germinativas/metabolismo , Factores de Iniciación de Péptidos/genética , Proteínas Proto-Oncogénicas c-cbl/genética , ARN Interferente Pequeño/genética , Células Madre/metabolismo , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular , Linaje de la Célula/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/crecimiento & desarrollo , Factores de Iniciación de Péptidos/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-cbl/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Ribonucleasas/genética , Ribonucleasas/metabolismo , Células Madre/citologíaRESUMEN
mRNA regulation by poly(A) tail length variations plays an important role in many developmental processes. Recent advances have shown that, in particular, deadenylation (the shortening of mRNA poly(A) tails) is essential for germ-line stem cell biology in the Drosophila ovary. Therefore, a rapid and accurate method to analyze poly(A) tail lengths of specific mRNAs in this tissue is valuable. Several methods of poly(A) test (PAT) assays have been reported to measure mRNA poly(A) tail lengths in vivo. Here, we describe two of these methods (PAT and ePAT) that we have adapted for Drosophila ovarian germ cells and germ-line stem cells.
Asunto(s)
Drosophila/genética , Ovario/química , Poli A/análisis , ARN Mensajero/química , Animales , Femenino , Regulación de la Expresión Génica , Ovario/citología , Poliadenilación , Nicho de Células Madre , Células Madre/química , Células Madre/citologíaRESUMEN
Drosophila Orb, the homolog of vertebrate CPEB, is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb also has an essential function during early oogenesis that has not been addressed at the molecular level. Here, we show that orb prevents cell death during early oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. Autophagy and cell death observed in orb mutant ovaries are reduced by decreasing Atg12 or other Atg mRNA levels. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues.
Asunto(s)
Autofagia/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión al ARN/metabolismo , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia , Ciclo Celular , Muerte Celular , Drosophila/metabolismo , Femenino , Células Germinativas/metabolismo , Homeostasis , Inmunoprecipitación , Mutación , Oocitos/metabolismo , Oogénesis , Ovario/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasas/metabolismoRESUMEN
Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.
Asunto(s)
Proteínas Mitocondriales/genética , Distrofia Muscular Oculofaríngea/genética , Proteína I de Unión a Poli(A)/genética , ARN Mensajero/genética , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas Mitocondriales/biosíntesis , Músculo Esquelético/patología , Distrofia Muscular Oculofaríngea/patología , Proteína I de Unión a Poli(A)/biosíntesis , Poliadenilación/genética , ARN Mensajero/biosíntesisRESUMEN
Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. Here, we show that the CCR4 deadenylase is required for GSC self-renewal and that Nos and Pum act through its recruitment onto specific mRNAs. We identify mei-P26 mRNA as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. We show that fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Óvulo/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasas/metabolismo , Células Madre/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/genética , Femenino , Oogénesis , Óvulo/citología , Óvulo/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasas/genética , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease which affects specific muscles. No pharmacological treatments are currently available for OPMD. In recent years, genetically tractable models of OPMD Drosophila and Caenorhabditis elegans have been generated. Although these models have not yet been used for large-scale primary drug screening, they have been very useful in candidate approaches for the identification of potential therapeutic compounds for OPMD. In this brief review, we summarize the data that validated active molecules for OPMD in animal models including Drosophila, C. elegans and mouse.
Asunto(s)
Modelos Animales de Enfermedad , Descubrimiento de Drogas , Distrofia Muscular Oculofaríngea/tratamiento farmacológico , Animales , HumanosRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset progressive muscle disorder caused by a poly-alanine expansion mutation in the Poly(A) Binding Protein Nuclear 1 (PABPN1). The molecular mechanisms that regulate disease onset and progression are largely unknown. In order to identify molecular pathways that are consistently associated with OPMD, we performed an integrated high-throughput transcriptome study in affected muscles of OPMD animal models and patients. The ubiquitin-proteasome system (UPS) was found to be the most consistently and significantly OPMD-deregulated pathway across species. We could correlate the association of the UPS OPMD-deregulated genes with stages of disease progression. The expression trend of a subset of these genes is age-associated and therefore, marks the late onset of the disease, and a second group with expression trends relating to disease-progression. We demonstrate a correlation between expression trends and entrapment into PABPN1 insoluble aggregates of OPMD-deregulated E3 ligases. We also show that manipulations of proteasome and immunoproteasome activity specifically affect the accumulation and aggregation of mutant PABPN1. We suggest that the natural decrease in proteasome expression and its activity during muscle aging contributes to the onset of the disease.
RESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of specific muscles. OPMD is caused by extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Insoluble nuclear inclusions form in diseased muscles. We have generated a Drosophila model of OPMD that recapitulates the features of the disorder. Here, we show that the antiprion drugs 6-aminophenanthridine (6AP) and guanabenz acetate (GA), which prevent formation of amyloid fibers by prion proteins in cell models, alleviate OPMD phenotypes in Drosophila, including muscle degeneration and nuclear inclusion formation. The large ribosomal RNA and its activity in protein folding were recently identified as a specific cellular target of 6AP and GA. We show that deletions of the ribosomal DNA locus reduce OPMD phenotypes and act synergistically with sub-effective doses of 6AP. In a complementary approach, we demonstrate that ribosomal RNA accelerates in vitro fibril formation of PABPN1 N-terminal domain. These results reveal the conserved role of ribosomal RNA in different protein aggregation disorders and identify 6AP and GA as general anti-aggregation molecules.
Asunto(s)
Guanabenzo/uso terapéutico , Distrofia Muscular Oculofaríngea/metabolismo , Fenantridinas/uso terapéutico , Proteína II de Unión a Poli(A)/metabolismo , Animales , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Larva/metabolismo , Distrofia Muscular Oculofaríngea/tratamiento farmacológico , Fenotipo , Enfermedades por Prión/tratamiento farmacológico , Pliegue de Proteína , ARN Ribosómico/metabolismoRESUMEN
The CCR4-NOT complex is the main enzyme catalyzing the deadenylation of mRNA. We have investigated the composition of this complex in Drosophila melanogaster by immunoprecipitation with a monoclonal antibody directed against NOT1. The CCR4, CAF1 (=POP2), NOT1, NOT2, NOT3, and CAF40 subunits were associated in a stable complex, but NOT4 was not. Factors known to be involved in mRNA regulation were prominent among the other proteins coprecipitated with the CCR4-NOT complex, as analyzed by mass spectrometry. The complex was localized mostly in the cytoplasm but did not appear to be a major component of P bodies. Of the known CCR4 paralogs, Nocturnin was found associated with the subunits of the CCR4-NOT complex, whereas Angel and 3635 were not. RNAi experiments in Schneider cells showed that CAF1, NOT1, NOT2, and NOT3 are required for bulk poly(A) shortening and hsp70 mRNA deadenylation, but knock-down of CCR4, CAF40, and NOT4 did not affect these processes. Overexpression of catalytically dead CAF1 had a dominant-negative effect on mRNA decay. In contrast, overexpression of inactive CCR4 had no effect. We conclude that CAF1 is the major catalytically important subunit of the CCR4-NOT complex in Drosophila Schneider cells. Nocturnin may also be involved in mRNA deadenylation, whereas there is no evidence for a similar role of Angel and 3635.
Asunto(s)
Drosophila melanogaster/enzimología , Ribonucleasas/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Línea Celular , Proteínas de Drosophila/análisis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteoma/análisis , ARN Mensajero/metabolismo , Proteína 4 de Unión a Retinoblastoma/metabolismo , Ribonucleasas/químicaRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is a late onset disorder characterized by progressive weakening of specific muscles. It is caused by short expansions of the N-terminal polyalanine tract in the poly(A) binding protein nuclear 1 (PABPN1), and it belongs to the group of protein aggregation diseases, such as Huntington's, Parkinson's and Alzheimer diseases. Mutant PABPN1 forms nuclear aggregates in diseased muscles in both patients and animal models. Intrabodies are antibodies that are modified to be expressed intracellularly and target specific antigens in subcellular locations. They are commonly generated by artificially linking the variable domains of antibody heavy and light chains. However, natural single-chain antibodies are produced in Camelids and, when engineered, combined the advantages of being single-chain, small sized and very stable. Here, we determine the in vivo efficiency of Llama intrabodies against PABPN1, using the established Drosophila model of OPMD. Among six anti-PABPN1 intrabodies expressed in muscle nuclei, we identify one as a strong suppressor of OPMD muscle degeneration in Drosophila, leading to nearly complete rescue. Expression of this intrabody affects PABPN1 aggregation and restores muscle gene expression. This approach promotes the identification of intrabodies with high therapeutic value and highlights the potential of natural single-chain intrabodies in treating protein aggregation diseases.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Modelos Animales de Enfermedad , Drosophila , Distrofia Muscular Oculofaríngea/terapia , Proteína II de Unión a Poli(A)/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Camélidos del Nuevo Mundo , Drosophila/genética , Drosophila/metabolismo , Femenino , Terapia Genética , Humanos , Inmunoterapia , Masculino , Músculo Esquelético/metabolismo , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/prevención & control , Proteína II de Unión a Poli(A)/metabolismo , Transporte de ProteínasRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Strikingly, the polyalanine tract is not absolutely required for muscle degeneration, whereas another domain of PABPN1, the RNA-binding domain and its function in RNA binding are required. This demonstrates that OPMD does not result from polyalanine toxicity, but from an intrinsic property of PABPN1. We also identify several suppressors of the OPMD phenotype. This establishes our OPMD Drosophila model as a powerful in vivo test to understand the disease process and develop novel therapeutic strategies.
Asunto(s)
Drosophila melanogaster/fisiología , Distrofia Muscular Oculofaríngea , Proteína I de Unión a Poli(A)/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila melanogaster/anatomía & histología , Humanos , Chaperonas Moleculares/metabolismo , Músculo Esquelético/anatomía & histología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/patología , Distrofia Muscular Oculofaríngea/fisiopatología , Fenotipo , Proteína I de Unión a Poli(A)/genética , Proteína p53 Supresora de Tumor/metabolismo , Alas de Animales/anatomía & histologíaRESUMEN
Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. Here, we analyze the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. We also demonstrate an unanticipated cytoplasmic function for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control.
Asunto(s)
Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Proteína II de Unión a Poli(A)/metabolismo , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Ciclo Celular/fisiología , Ciclina B/genética , Ciclina B/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/fisiología , Femenino , Masculino , Datos de Secuencia Molecular , Oocitos/fisiología , Proteína II de Unión a Poli(A)/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismoRESUMEN
The Drosophila larval cardiac tube is composed of 104 cardiomyocytes that exhibit genetic and functional diversity. The tube is divided into the aorta and the heart proper that encompass the anterior and posterior parts of the tube, respectively. Differentiation into aorta and heart cardiomyocytes takes place during embryogenesis. We have observed living embryos to correlate morphological changes occurring during the late phases of cardiogenesis with the acquisition of organ function, including functional inlets, or ostiae. Cardiac cells diversity originates in response to two types of spatial information such that cells differentiate according to their position, both within a segment and along the anteroposterior axis. Axial patterning is controlled by homeotic genes of the Bithorax Complex (BXC) which are regionally expressed within the cardiac tube in non-overlapping domains. Ultrabithorax (Ubx) is expressed in the aorta whereas abdominal A (abd-A) is expressed in the heart, with the exception of the four most posterior cardiac cells which express Abdominal B (Abd-B). Ubx and abd-A functions are required to confer an aorta or a heart identity on cardiomyocytes, respectively. The anterior limit of the expression domain of Ubx, abd-A and Abd-B is independent of the function of the other genes. In contrast, abd-A represses Ubx expression in the heart and ectopic overexpression of abd-A transforms aorta cells into heart cardiomyocytes. Taken together, these results support the idea that BXC homeotic genes in the cardiac tube conform to the posterior prevalence rule. The cardiac tube is also segmentally patterned and each metamere contains six pairs of cardioblasts that are genetically diverse. We show that the transcription of seven up (svp), which is expressed in the two most posterior pairs of cardioblasts in each segment, is dependent on hedgehog (hh) signaling from the dorsal ectoderm. In combination with the axial information furnished by abd-A, the segmental hh-dependent information leads to the differentiation of the six pairs of svp-expressing cells into functional ostiae.
Asunto(s)
Tipificación del Cuerpo , Fase de Segmentación del Huevo/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Proteínas de Homeodominio/genética , Proteínas Nucleares , Receptores de Esteroides/genética , Transducción de Señal , Factores de Transcripción , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Expresión Génica , Genes de Insecto/fisiología , Corazón/embriología , Proteínas Hedgehog , Proteínas de Homeodominio/fisiología , Receptores de Esteroides/metabolismoRESUMEN
The steps that lead to the formation of a single primitive heart tube are highly conserved in vertebrate and invertebrate embryos. Concerted migration of the two lateral cardiogenic regions of the mesoderm and endoderm (or ectoderm in invertebrates) is required for their fusion at the midline of the embryo. Morphogenetic signals are involved in this process and the extracellular matrix has been proposed to serve as a link between the two layers of cells. Pericardin (Prc), a novel Drosophila extracellular matrix protein is a good candidate to participate in heart tube formation. The protein has the hallmarks of a type IV collagen alpha-chain and is mainly expressed in the pericardial cells at the onset of dorsal closure. As dorsal closure progresses, Pericardin expression becomes concentrated at the basal surface of the cardioblasts and around the pericardial cells, in close proximity to the dorsal ectoderm. Pericardin is absent from the lumen of the dorsal vessel. Genetic evidence suggests that Prc promotes the proper migration and alignment of heart cells. Df(3)vin6 embryos, as well as embryos in which prc has been silenced via RNAi, exhibit similar and significant defects in the formation of the heart epithelium. In these embryos, the heart epithelium appears disorganized during its migration to the dorsal midline. By the end of embryonic development, cardial and pericardial cells are misaligned such that small clusters of both cell types appear in the heart; these clusters of cells are associated with holes in the walls of the heart. A prc transgene can partially rescue each of these phenotypes, suggesting that prc regulates these events. Our results support, for the first time, the function of a collagen-like protein in the coordinated migration of dorsal ectoderm and heart cells.