RESUMEN
Tubulin posttranslational modifications represent an important mechanism involved in the regulation of microtubule functions. The most widespread among them are detyrosination, α∆2-tubulin, and polyglutamylation. Here, we describe a family of tubulin-modifying enzymes composed of two closely related proteins, KIAA0895L and KIAA0895, which have tubulin metallocarboxypeptidase activity and thus were termed TMCP1 and TMCP2, respectively. We show that TMCP1 (also known as MATCAP) acts as α-tubulin detyrosinase that also catalyzes α∆2-tubulin. In contrast, TMCP2 preferentially modifies ßI-tubulin by removing three amino acids from its C terminus, generating previously unknown ßI∆3 modification. We show that ßI∆3-tubulin is mostly found on centrioles and mitotic spindles and in cilia. Moreover, we demonstrate that TMCPs also remove posttranslational polyglutamylation and thus act as tubulin deglutamylases. Together, our study describes the identification and comprehensive biochemical analysis of a previously unknown type of tubulin-modifying enzymes involved in the processing of α- and ß-tubulin C-terminal tails and deglutamylation.
Asunto(s)
Carboxipeptidasas , Tubulina (Proteína) , Microtúbulos , Aminoácidos , CentriolosRESUMEN
The Hippo signaling pathway is a major regulator of organ growth, which controls the activity of the transcription coactivator Yorkie (Yki) in Drosophila and its homolog YAP in mammals. Both Yki and YAP proteins exist as alternatively spliced isoforms containing either one or two WW domains. The biological importance of this conserved alternative splicing event is unknown. Here, we identify the splicing factor B52 as a regulator of yki alternative splicing in Drosophila and show that B52 modulates growth in part through modulation of yki alternative splicing. Yki isoforms differ by their transcriptional activity as well as their ability to bind and bridge PPxY motifs-containing partners, and can compete in vivo. Strikingly, flies in which yki alternative splicing has been abrogated, thus expressing only Yki2 isoform, exhibit fluctuating wing asymmetry, a signal of developmental instability. Our results identify yki alternative splicing as a new level of modulation of the Hippo pathway, that is required for growth equilibration during development. This study provides the first demonstration that the process of alternative splicing contributes to developmental robustness.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Empalme de ARN/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Empalme Alternativo , Animales , Línea Celular , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Nucleares/química , Unión Proteica , Dominios Proteicos , Factores de Empalme de ARN/genética , Análisis de Secuencia de ARN , Transactivadores/química , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Proteínas Señalizadoras YAPRESUMEN
In eukaryotes, trimethylation of lysine 9 on histone H3 (H3K9) is associated with transcriptional silencing of transposable elements (TEs). In drosophila ovaries, this heterochromatic repressive mark is thought to be deposited by SetDB1 on TE genomic loci after the initial recognition of nascent transcripts by PIWI-interacting RNAs (piRNAs) loaded on the Piwi protein. Here, we show that the nucleosome remodeler Mi-2, in complex with its partner MEP-1, forms a subunit that is transiently associated, in a MEP-1 C-terminus-dependent manner, with known Piwi interactors, including a recently reported SUMO ligase, Su(var)2-10. Together with the histone deacetylase Rpd3, this module is involved in the piRNA-dependent TE silencing, correlated with H3K9 deacetylation and trimethylation. Therefore, drosophila piRNA-mediated transcriptional silencing involves three epigenetic effectors, a remodeler, Mi-2, an eraser, Rpd3 and a writer, SetDB1, in addition to the Su(var)2-10 SUMO ligase.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Autoantígenos/metabolismo , Proteínas de Drosophila/metabolismo , Heterocromatina/química , Histona Desacetilasa 1/metabolismo , Nucleosomas/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/metabolismo , Drosophila melanogaster , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Histonas/química , Ovario/metabolismo , Proteínas Inhibidoras de STAT ActivadosRESUMEN
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
TDP-43 is a critical RNA-binding factor associated with RNA metabolism. In the physiological state, maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. As such, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. TDP-43 is a major disease-causing protein in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several studies argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulating the cycle of TDP-43 production might therefore provide a new therapeutic strategy. In this study, we developed a new transgenic Drosophila model mimicking the TDP-43 autoregulatory feedback loop in order to identify genetic modulators of TDP-43 protein steady-state levels in vivo. First, we showed that our TDP-43_TDPBR Drosophila model recapitulates key features of the TDP-43 autoregulatory processes previously described in mammalian and cellular models, namely alternative splicing events, differential usage of polyadenylation sites, nuclear retention of the transcript and a decrease in steady-state mRNA levels. Using this new Drosophila model, we identified several splicing factors, including SF2, Rbp1 and Sf3b1, as genetic modulators of TDP-43 production. Interestingly, our data indicate that these three RNA-binding proteins regulate TDP-43 protein production, at least in part, by controlling mRNA steady-state levels.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Empalme de ARN/metabolismo , Empalme Alternativo , Esclerosis Amiotrófica Lateral/genética , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Degeneración Lobar Frontotemporal/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Factores de Empalme de ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The Spinal Muscular Atrophy disease protein Survival Motor Neuron (SMN) operates as part of a multiprotein complex whose components also include Gemins 2-8 and Unrip. The fruit fly Drosophila melanogaster is thought to have a slightly smaller SMN complex comprised of SMN, Gemin2/3/5 and, possibly, Unrip. Based upon in vivo interaction methods, we have identified novel interacting partners of the Drosophila SMN complex with homologies to Gemin4/6/7/8. The Gemin4 and Gemin8 orthologues are required for neuromuscular function and survival. The Gemin6/7/Unrip module can be recruited via the SMN-associated Gemin8, hence mirroring the human SMN complex architecture. Our findings lead us to propose that an elaborate SMN complex that is typical in metazoans is also present in Drosophila.
Asunto(s)
Proteínas del Complejo SMN/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN/genéticaRESUMEN
Serine-/arginine-rich (SR) proteins are RNA-binding proteins that are primarily involved in alternative splicing. Expression of some SR proteins is frequently upregulated in tumors, and previous reports have demonstrated that these proteins can directly participate in cell transformation. Identifying factors that can rescue the effects of SR overexpression in vivo is, therefore, of potential therapeutic interest. Here, we analyzed phenotypes induced by overexpression of the SR protein B52 during Drosophila development and identified several proteins that can rescue these phenotypes. Using the mechanosensory bristle lineage as a developmental model, we show that B52 expression level influences cell growth, but not differentiation, in this lineage. In particular, B52 overexpression increases cell growth, upregulates myc transcription, and gives rise to flies lacking thoracic bristles. Using a genetic screen, we identified several suppressors of the phenotypes induced by overexpression of B52 in vivo in two different organs. We show that upregulation of brain tumor (brat), a tumor suppressor and post-transcriptional repressor of myc, and downregulation of lilliputian (lilli), a subunit of the superelongation complex involved in transcription elongation, efficiently rescue the phenotypes induced by B52 overexpression. Our results demonstrate a role of this SR protein in cell growth and identify candidate proteins that may overcome the effects of SR protein overexpression in mammals.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Diferenciación Celular , Procesos de Crecimiento Celular , Linaje de la Célula , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Mecanorreceptores/citología , Mecanorreceptores/metabolismo , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Empalme de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
SRm160 is an SR-like protein implicated in multiple steps of RNA processing and nucleocytoplasmic export. Although its biochemical functions have been extensively described, its genetic interactions and potential participation in signaling pathways remain largely unknown, despite the fact that it is highly phosphorylated in both mammalian cells and Drosophila. To begin elucidating the functions of the protein in signaling and its potential role in developmental processes, we characterized mutant and overexpression SRm160 phenotypes in Drosophila and their interactions with the locus encoding the LAMMER protein kinase, Doa. SRm160 mutations are recessive lethal, while its overexpression generates phenotypes including roughened eyes and highly disorganized internal eye structure, which are due at least in part to aberrantly high levels of apoptosis. SRm160 is required for normal somatic sex determination, since its alleles strongly enhance a subtle sex transformation phenotype induced by Doa kinase alleles. Moreover, modification of SRm160 by DOA kinase appears to be necessary for its activity, since Doa alleles suppress phenotypes induced by SRm160 overexpression in the eye and enhance those in genital discs. Modification of SRm160 may occur through direct interaction because DOA kinase phosphorylates it in vitro. Remarkably, SRm160 protein was concentrated in the nuclei of precellular embryos but was very rapidly excluded from nuclei or degraded coincident with cellularization. Also of interest, transcripts are restricted almost exclusively to the developing nervous system in mature embryos.
Asunto(s)
Apoptosis/genética , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/genética , Alelos , Secuencia de Aminoácidos , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Ojo/embriología , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Sitios Genéticos , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Organogénesis/genética , Fenotipo , Fosforilación , Procesamiento Proteico-Postraduccional/genética , Empalme del ARN/genética , ARN Mensajero/metabolismoRESUMEN
Alternative splicing expands the coding capacity of metazoan genes, and it was largely genetic studies in the fruit-fly Drosophila melanogaster that established the principle that regulated alternative splicing results in tissue- and stage-specific protein isoforms with different functions in development. Alternative splicing is particularly prominent in germ cells, muscle and the central nervous system where it modulates the expression of various proteins including cell-surface molecules and transcription factors. Studies in flies have given us numerous insights into alternative splicing in terms of upstream regulation, the exquisite diversity of their forms and the key differential cellular functions of alternatively spliced gene products. The current inundation of transcriptome sequencing data from Drosophila provides an unprecedented opportunity to gain a comprehensive view of alternative splicing.
Asunto(s)
Empalme Alternativo , Drosophila/genética , Animales , Encéfalo/metabolismo , Drosophila/metabolismo , Músculos/metabolismo , Factores Sexuales , Factores de Transcripción/genéticaRESUMEN
Developmental axon pruning is a general mechanism that is required for maturation of neural circuits. During Drosophila metamorphosis, the larval-specific dendrites and axons of early γ neurons of the mushroom bodies are pruned and replaced by adult-specific processes. We found that the nuclear receptor ftz-f1 is required for this pruning, activates expression of the steroid hormone receptor EcR-B1, whose activity is essential for γ remodeling, and represses expression of Hr39, an ftz-f1 homologous gene. If inappropriately expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. EcR-B1 was previously identified as a target of the TGFß signaling pathway. We found that the ftz-f1 and Hr39 pathway apparently acts independently of TGFß signaling, suggesting that EcR-B1 is the target of two parallel molecular pathways that act during γ neuron remodeling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Metamorfosis Biológica/fisiología , Cuerpos Pedunculados/metabolismo , Neuronas/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Esteroides/fisiología , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Metamorfosis Biológica/genética , Cuerpos Pedunculados/crecimiento & desarrollo , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiología , Neuronas/fisiología , Receptores de Esteroides/genética , Factores de Transcripción/genéticaRESUMEN
DNA- and RNA-processing pathways are integrated and interconnected in the eukaryotic nucleus to allow efficient gene expression and to maintain genomic stability. The recruitment of DNA Topoisomerase I (Topo I), an enzyme controlling DNA supercoiling and acting as a specific kinase for the SR-protein family of splicing factors, to highly transcribed loci represents a mechanism by which transcription and processing can be coordinated and genomic instability avoided. Here we show that Drosophila Topo I associates with and phosphorylates the SR protein B52. Surprisingly, expression of a high-affinity binding site for B52 in transgenic flies restricted localization, not only of B52, but also of Topo I to this single transcription site, whereas B52 RNAi knockdown induced mis-localization of Topo I in the nucleolus. Impaired delivery of Topo I to a heat shock gene caused retention of the mRNA at its site of transcription and delayed gene deactivation after heat shock. Our data show that B52 delivers Topo I to RNA polymerase II-active chromatin loci and provide the first evidence that DNA topology and mRNA release can be coordinated to control gene expression.
Asunto(s)
Cromatina/enzimología , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transcripción Genética , Animales , Nucléolo Celular/metabolismo , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/citología , Proteínas HSP70 de Choque Térmico/metabolismo , Modelos Biológicos , Proteínas Nucleares/deficiencia , Fosfoproteínas/deficiencia , Fosforilación , Cromosomas Politénicos/metabolismo , Unión Proteica , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Factores de Empalme de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.
Asunto(s)
Evolución Molecular , Glicina/metabolismo , Péptido Sintasas/genética , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Péptido Sintasas/química , Ácido Poliglutámico/metabolismo , Alineación de SecuenciaRESUMEN
The genetic programs specifying eye development are highly conserved during evolution and involve the vertebrate Pax-6 gene and its Drosophila melanogaster homolog eyeless (ey). Here we report that the SR protein B52/SRp55 controls a novel developmentally regulated splicing event of eyeless that is crucial for eye growth and specification in Drosophila. B52/SRp55 generates two isoforms of eyeless differing by an alternative exon encoding a 60-amino-acid insert at the beginning of the paired domain. The long isoform has impaired ability to trigger formation of ectopic eyes and to bind efficiently Eyeless target DNA sequences in vitro. When over-produced in the eye imaginal disc, this isoform induces a small eye phenotype, whereas the isoform lacking the alternative exon triggers eye over-growth and strong disorganization. Our results suggest that B52/SRp55 splicing activity is used during normal eye development to control eye organogenesis and size through regulation of eyeless alternative splicing.
Asunto(s)
Empalme Alternativo , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ojo Compuesto de los Artrópodos/anomalías , Secuencia de Consenso , Proteínas de Unión al ADN/fisiología , Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Exones/genética , Datos de Secuencia Molecular , Organogénesis/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. We show here that Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and that PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. On the other hand, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Drosophila/embriología , Elementos de Facilitación Genéticos , Silenciador del Gen , Genes de Insecto , Modelos Biológicos , Complejo Represivo Polycomb 1 , Regiones Promotoras GenéticasRESUMEN
Chromatin insulators or boundaries are proposed to structure the chromatin fiber into functionally independent domains by promoting the formation of chromatin loops. These elements can block the communication between an enhancer and a gene when placed between them. Interestingly, it has been previously observed that two tandem copies of the Drosophila Su(Hw) insulator abolish this enhancer-blocking activity, presumably through pairing. This bypass effect has not been described with other insulators, however. In this report, we show that the insertion of binding sites for the GAGA factor (GAF) between an enhancer and the Su(Hw) insulator allows bypassing of the insulator. This bypass relies on the activity of both the GAF protein and the Mod(mdg4)-67.2 protein, a factor required for Su(Hw) insulator activity. We show that these two proteins interact in vitro and in vivo, providing molecular evidence of pairing between the GAF sites and the Su(Hw) insulator. Finally, we show that placing the Mcp boundary together with the Su(Hw) insulator between an enhancer and a promoter leads to bypass, again in a GAF- and Mod(mdg4)-dependent manner. Our data provide direct evidence that heterologous insulators can be bypassed by distal enhancers and identify the interaction between GAF and Mod(mdg4) as a possible means to regulate insulator activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Elementos Aisladores/genética , Proteínas Recombinantes/metabolismo , Retroelementos/genética , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Proteínas de Unión al ADN/genética , Sistema Digestivo , Proteínas de Drosophila/genética , Elementos de Facilitación Genéticos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética , Retroelementos/fisiología , Factores de Transcripción/genéticaRESUMEN
Hepatitis E virus (HEV) is globally distributed, transmitted enterically and between humans and animals. Phylogenetic analysis has identified five distinct HEV genotypes. The first full-length sequence of an African strain (Chad) is presented and compared to 31 complete HEV genomes available, including the fulminant hepatitis strain from India, swine strains and a strain from Morocco. The two African strains are more closely related to genotype 1 than to any other genotypes and together they possibly form a sub-genotype or sixth genotype. The first evidence for recombination between divergent HEV strains is presented.
Asunto(s)
Genoma Viral , Virus de la Hepatitis E/clasificación , Virus de la Hepatitis E/genética , Hepatitis E/virología , Filogenia , Recombinación Genética , Chad , Heces/virología , Genotipo , Virus de la Hepatitis E/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Citoplasma/metabolismo , Drosophila/fisiología , Poliadenilación/fisiología , Polinucleotido Adenililtransferasa/metabolismo , Animales , Núcleo Celular/metabolismo , Drosophila/embriología , Proteínas de Drosophila/metabolismo , Perfilación de la Expresión Génica , Polinucleotido Adenililtransferasa/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The Suppressor of forked [Su(f)] protein is the Drosophila homologue of CstF-77, a subunit of human cleavage stimulation factor (CstF) that is required for the first step of the mRNA 3' end processing reaction in vitro. We have addressed directly the role of su(f) in the mRNA 3' end processing reaction in vivo. We show that su(f) is required for the cleavage of pre-mRNA during mRNA 3' end formation. Analysis of the functional complementation between Su(f) and CstF-77 shows that most of the Drosophila protein (85%) can be exchanged for the human protein to produce chimeric CstF-77/Su(f) proteins that rescue lethality and cleavage defect during mRNA 3' end formation in su(f) mutants. Interestingly, we show that a domain in human CstF-77 is limiting for the rescue and that this domain is not able to reproduce protein interactions with the CstF subunits of Drosophila. We also show that chimeric CstF-77/Su(f) proteins that rescue lethality of su(f) mutants cannot restore utilization of a regulated poly(A) site in Drosophila. Taken together, these results demonstrate that CstF-77 and Su(f) have the same function in mRNA 3' end formation in vivo, but that these two proteins are not interchangeable for regulation of poly(A) site utilization.