RESUMEN
Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31Bâ¢Tral complex may be at the core of repression.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Animales , ARN Helicasas DEAD-box/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero , Regulación de la Expresión Génica , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , ARN Helicasas/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ribonucleoproteínas/genéticaRESUMEN
Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK-ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Filamentos Intermedios/metabolismo , Queratina-8/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Citoesqueleto/efectos de los fármacos , Citoesqueleto/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Filamentos Intermedios/efectos de los fármacos , Filamentos Intermedios/genética , Queratina-8/genética , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
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
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
Deadenylation is the removal of poly(A) tails from mRNA. Here, we present two methods for assaying deadenylation in vivo. The first is a method for measuring bulk poly(A) tail lengths. When combined with a block in transcription, the method can be used for measuring the rate of bulk poly(A) tail shortening. The second is an RT-PCR method to determine the poly(A) tail lengths of individual RNAs. Again in combination with a block of transcription, the method permits the rate of deadenylation of an individual RNA to be measured.
Asunto(s)
Poli A/química , ARN Mensajero/química , Animales , Drosophila , Poliadenilación/fisiologíaRESUMEN
Deadenylation is the removal of poly(A) tails from mRNA. This chapter presents two methods to assay deadenylation in vitro. The first is a quick and quantitative assay for the degradation of radiolabeled poly(A) that can easily be adapted to be used for many different enzymes. The second method uses an extract from Drosophila embryos to catalyze the deadenylation of an RNA dependent on a specific sequence that also directs deadenylation in vivo.
Asunto(s)
Poli A/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Animales , Drosophila/genéticaRESUMEN
Controlled shortening of the poly(A) tail of mRNAs is the first step in eukaryotic mRNA decay and can also be used for translational inactivation of mRNAs. The CCR4-NOT complex is the most important among a small number of deadenylases, enzymes catalyzing poly(A) tail shortening. Rates of poly(A) shortening differ between mRNAs as the CCR4-NOT complex is recruited to specific mRNAs by means of either sequence-specific RNA binding proteins or miRNAs. This review summarizes our current knowledge concerning the subunit composition and deadenylation activity of the Drosophila CCR4-NOT complex and the mechanisms by which the complex is recruited to particular mRNAs. We discuss genetic data implicating the complex in the regulation of specific mRNAs, in particular in the context of development.
RESUMEN
Shortening of the poly(A) tail is the first and often rate-limiting step in mRNA degradation. Three poly(A)-specific 3' exonucleases have been described that can carry out this reaction: PAN, composed of two subunits; PARN, a homodimer; and the CCR4-NOT complex, a heterooligomer that contains two catalytic subunits and may have additional functions in the cell. Current evidence indicates that all three enzymes use a two-metal ion mechanism to release nucleoside monophosphates in a hydrolytic reaction. The CCR4-NOT is the main deadenylase in all organisms examined, and mutations affecting the complex can be lethal. The contribution of PAN, apparently an initial deadenylation preceding the activity of CCR4-NOT, is less important, whereas the activity of PARN seems to be restricted to specific substrates or circumstances, for example, stress conditions. Rapid deadenylation and decay of specific mRNAs can be caused by recruitment of both PAN and the CCR4-NOT complex. This function can be carried out by RNA-binding proteins, for example, members of the PUF family. Alternatively, miRNAs can recruit the deadenylase complexes with the help of their associated GW182 proteins.
RESUMEN
Endonuclease G (EndoG) is a mitochondrial enzyme believed to be released during apoptosis to participate in the degradation of nuclear DNA. This paper describes a Drosophila protein, EndoGI, which inhibits EndoG specifically. EndoG and EndoGI associate with subpicomolar affinity, forming a 2:1 complex in which dimeric EndoG is bound by two tandemly repeated homologous domains of monomeric EndoGI. Binding appears to involve the active site of EndoG. EndoGI is present in the cell nucleus at micromolar concentrations. Upon induction of apoptosis, levels of the inhibitor appear to be reduced, and it is relocalized to the cytoplasm. EndoGI, encoded by the predicted open reading frame cg4930, is expressed throughout Drosophila development. Flies homozygous for a hypomorphic EndoGI mutation have a strongly reduced viability, which is modulated by genetic background and diet. We propose that EndoGI protects the cell against low levels of EndoG outside mitochondria.
Asunto(s)
Proteínas de Drosophila/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Animales , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Two general pathways of mRNA decay have been characterized in yeast. Both start with deadenylation. The major pathway then proceeds via cap hydrolysis and 5'-exonucleolytic degradation whereas the minor pathway consists of 3'-exonucleolytic decay followed by hydrolysis of the remaining cap structure. In higher eukaryotes, these pathways of mRNA decay are believed to be conserved but have not been well characterized. We have investigated the decay of the hsp70 mRNA in Drosophila Schneider cells. As shown by the use of reporter constructs, rapid deadenylation of this mRNA is directed by its 3'-untranslated region. The main deadenylase is the CCR4.NOT complex; the PAN nuclease makes a lesser contribution. Heat shock prevents deadenylation not only of the hsp70 but also of bulk mRNA. A completely deadenylated capped hsp70 mRNA decay intermediate accumulates transiently and is degraded via cap hydrolysis and 5'-decay. Thus, decapping is a slow step in the degradation pathway. Cap hydrolysis is also inhibited during heat shock. Degradation of reporter RNAs from the 3'-end became detectable only upon inhibition of 5'-decay and thus represents a minor decay pathway. Because two reporter RNAs and at least two endogenous mRNAs were degraded primarily from the 5'-end with cap hydrolysis as a slow step, this pathway appears to be of general importance for mRNA decay in Drosophila.
Asunto(s)
Drosophila melanogaster/genética , Proteínas HSP70 de Choque Térmico/genética , ARN Mensajero/genética , Animales , Células Cultivadas , Proteínas de Drosophila/genética , Calor , Cinética , Poli A/análisis , Reacción en Cadena de la Polimerasa , Interferencia de ARN , ARN Mensajero/metabolismo , Ribonucleasa H/metabolismoRESUMEN
Shortening of the poly(A) tail (deadenylation) is the first and often rate-limiting step in the degradation pathway of most eukaryotic mRNAs and is also used as a means of translational repression, in particular in early embryonic development. The nanos mRNA is translationally repressed by the protein Smaug in Drosophila embryos. The RNA has a short poly(A) tail at steady state and decays gradually during the first 2-3 h of development. Smaug has recently also been implicated in mRNA deadenylation. To study the mechanism of sequence-dependent deadenylation, we have developed a cell-free system from Drosophila embryos that displays rapid deadenylation of nanos mRNA. The Smaug response elements contained in the nanos 3'-untranslated region are necessary and sufficient to induce deadenylation; thus, Smaug is likely to be involved. Unexpectedly, deadenylation requires the presence of an ATP regenerating system. The activity can be pelleted by ultracentrifugation, and both the Smaug protein and the CCR4.NOT complex, a known deadenylase, are enriched in the active fraction. The same extracts show pronounced translational repression mediated by the Smaug response elements. RNAs lacking a poly(A) tail are poorly translated in the extract; therefore, SRE-dependent deadenylation contributes to translational repression. However, repression is strong even with RNAs either bearing a poly(A) tract that cannot be removed or lacking poly(A) altogether; thus, an additional aspect of translational repression functions independently of deadenylation.
Asunto(s)
Adenosina Trifosfato/química , Proteínas de Drosophila/fisiología , Drosophila/embriología , Proteínas de Unión al ARN/fisiología , Regiones no Traducidas 3' , Animales , Bioquímica/métodos , Sistema Libre de Células , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The control of mRNA degradation is an important component of the regulation of gene expression since the steady-state concentration of mRNA is determined both by the rates of synthesis and of decay. Two general pathways of mRNA decay have been described in eukaryotes. Both pathways share the exonucleolytic removal of the poly(A) tail (deadenylation) as the first step. In one pathway, deadenylation is followed by the hydrolysis of the cap and processive degradation of the mRNA body by a 5' exonuclease. In the second pathway, the mRNA body is degraded by a complex of 3' exonucleases before the remaining cap structure is hydrolyzed. This review discusses the proteins involved in the catalysis and control of both decay pathways.
Asunto(s)
Células Eucariotas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Células Eucariotas/enzimología , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Regulación de la Expresión Génica , Poli A/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión a Caperuzas de ARN/metabolismo , Caperuzas de ARN/metabolismo , Precursores del ARN/metabolismo , Transducción de SeñalRESUMEN
The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. We have identified homologs for almost all subunits of this complex in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , ARN Mensajero/metabolismo , Ribonucleasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Secuencia Conservada , Citoplasma/metabolismo , Drosophila/citología , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Interferencia de ARN , Proteína 4 de Unión a Retinoblastoma , Ribonucleasas/química , Ribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Homología de Secuencia de AminoácidoRESUMEN
Poly(A) polymerase (PAP) has a role in two processes, polyadenylation of mRNA precursors in the nucleus and translational control of certain mRNAs by cytoplasmic elongation of their poly(A) tails, particularly during early development. It was found recently that at least three different PAP genes exist in mammals, encoding several PAP isoforms. The in vivo specificity of function of each PAP isoform currently is unknown. Here, we analyse PAP function in Drosophila: We show that a single PAP isoform exists in Drosophila that is encoded by the hiiragi gene. This single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo. Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. This demonstrates that regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development.