RESUMEN
Protein synthesis is energetically expensive and its rate is influenced by factors such as cell type and environment. Suppression of translation is a canonical response to stressful changes in the cellular environment. In particular, inhibition of the initiation step of translation has been highlighted as the key control step in stress-induced translational suppression as mechanisms that quickly suppress initiation are well-conserved. However, cells have evolved complex regulatory means to control translation apart from initiation. Here, we examine the role of the elongation step of translation in yeast subjected to acute glucose deprivation. The use of ribosome profiling and in vivo reporter assays demonstrated elongation rates slow progressively following glucose removal. We observed that ribosome distribution broadly shifts towards the downstream ends of transcripts after both acute and gradual glucose deprivation but not in response to other stressors. Additionally, on assessed mRNAs, a correlation existed between ribosome occupancy and protein production pre-stress but was lost after stress. These results indicate that stress-induced elongation regulation causes ribosomes to slow down and build up on a considerable proportion of the transcriptome in response to glucose withdrawal. Finally, we report ribosomes that built up along transcripts are competent to resume elongation and complete protein synthesis after readdition of glucose to starved cells. This suggests that yeast has evolved mechanisms to slow translation elongation in response to glucose starvation which do not preclude continuation of protein production from those ribosomes, thereby averting a need for new initiation events to take place to synthesize proteins.Abbreviations: AUG: start codon, bp: base pair(s), CDS: coding sequence, CHX: cycloheximide, eEF2: eukaryotic elongation factor 2, LTM: lactimidomycin, nt: nucleotide, PGK1: 3-phosphoglycerate kinase, ribosomal biogenesis: ribi, RO: ribosome occupancy, RPF: ribosome protected fragment, TE: translational efficiency.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Glucosa , Extensión de la Cadena Peptídica de Translación , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Stress granules (SGs) are condensates of mRNPs that form in response to stress. SGs arise by multivalent protein-protein, protein-RNA, and RNA-RNA interactions. However, the role of RNA-RNA interactions in SG assembly remains understudied. Here, we describe a yeast SG reconstitution system that faithfully recapitulates SG assembly in response to trigger RNAs. SGs assembled by stem-loop RNA triggers are ATP-sensitive, regulated by helicase/chaperone activity, and exhibit the hallmarks of maturation observed for SG proteins that phase-separate in vitro. Additionally, the fraction of total RNA that phase-separates in vitro is sufficient to trigger SG formation. However, condensation of NFT1 mRNA, an enriched transcript in this population, can only assemble an incomplete SG. These results suggest that networks of distinct transcripts are required to form a canonical SG and provide a platform for dissecting the interplay between the transcriptome and ATP-dependent remodeling in SG formation.
Asunto(s)
Gránulos Citoplasmáticos/genética , Ribonucleoproteínas/genética , Estrés Fisiológico/genética , Transcriptoma/genética , Adenosina Trifosfato/genética , Línea Celular , Regulación Fúngica de la Expresión Génica/genética , Humanos , ARN/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress. Furthermore, the product of one of these enzymes, AdoMet, is a regulator of SG assembly and composition. Decreases in AdoMet levels increase SG formation, while chronic elevation of AdoMet produces SG remnants lacking proteins associated with the 5' end of transcripts. Interestingly, acute elevation of AdoMet blocks SG formation in yeast and motor neurons. Treatment of ALS-derived motor neurons with AdoMet also suppresses the formation of TDP-43-positive SGs, a hallmark of ALS. Together, these results argue that AdoMet is an evolutionarily conserved regulator of SG composition and assembly with therapeutic potential in neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Gránulos Citoplasmáticos/metabolismo , Metabolismo Energético , Neuronas Motoras/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , S-Adenosilmetionina/farmacología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: The self-assembly of metabolic enzymes into filaments or foci highlights an intriguing mechanism for the regulation of metabolic activity. Recently, we identified the conserved polymerization of phosphoribosyl pyrophosphate synthetase (PRPS), which catalyzes the first step in purine nucleotide synthesis, in yeast and cultured mammalian cells. While previous work has revealed that loss of PRPS activity regulates retinal development in zebrafish, the extent to which PRPS filament formation affects tissue development remains unknown. RESULTS: By generating novel alleles in the zebrafish PRPS paralogs, prps1a and prps1b, we gained new insight into the role of PRPS filaments during eye development. We found that mutations in prps1a alone are sufficient to generate abnormally small eyes along with defects in head size, pigmentation, and swim bladder inflation. Furthermore, a loss-of-function mutation that truncates the Prps1a protein resulted in the failure of PRPS filament assembly. Lastly, in mutants that fail to assemble PRPS filaments, we observed disorganization of the actin network in the lens fibers. CONCLUSIONS: The truncation of Prps1a blocked PRPS filament formation and resulted in a disorganized lens fiber actin network. Altogether, these findings highlight a potential role for PRPS filaments during lens fiber organization in zebrafish.
Asunto(s)
Cristalino/embriología , Cristalino/crecimiento & desarrollo , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Actinas/metabolismo , Sacos Aéreos/embriología , Alelos , Animales , Ojo/embriología , Ojo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Microscopía Fluorescente , Mutación , Pigmentación , Polimerizacion , Retina/embriología , Epitelio Pigmentado de la Retina/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments.
Asunto(s)
Proteínas Luminiscentes/metabolismo , Redes y Vías Metabólicas , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Proteínas Mitocondriales/genética , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Recent work has found that many metabolic enzymes have the ability to polymerize in response to metabolic changes or environmental stress. This ability to polymerize is well conserved for the few metabolic enzyme paralogs that have been studied in yeast. Here we describe the first set of paralogs, Asn1p and Asn2p, that have differential assembly behavior. Asn1p and Asn2p both co-assemble into filaments in response to nutrient limitation. However, the ability of Asn2p to form filaments is strictly dependent on the presence of Asn1p. Using mutations that block enzyme activity but have differential effects on Asn1p polymerization, we have found that Asn1p polymers are unlikely to have acquired a moonlighting function. Together these results provide a novel system for understanding the regulation and evolution of metabolic enzyme polymerization.
Asunto(s)
Aspartatoamoníaco Ligasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Estrés Fisiológico , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/genética , Mutación , Nutrientes , Polimerizacion , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Oogénesis/genética , Estabilidad del ARN/genética , Alelos , Animales , Drosophila melanogaster/citología , Sitios Genéticos , Mutación/genética , Oocitos/citología , Oocitos/metabolismo , Fenotipo , Subunidades de Proteína/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure-function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Regulación Alostérica , Sitio Alostérico , Ligasas de Carbono-Nitrógeno/química , Dominio Catalítico , Estabilidad de Enzimas , Fosforilación , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its "tail" domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Endosomas/metabolismo , Cinesinas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Secuencia de Aminoácidos , Animales , Biofisica , Línea Celular , Vesículas Citoplasmáticas/metabolismo , Dimerización , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Técnicas de Silenciamiento del Gen , Cinesinas/química , Cinesinas/genética , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Datos de Secuencia Molecular , Peso Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiologíaRESUMEN
Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Genoma , Células Germinativas/crecimiento & desarrollo , Planarias/embriología , Planarias/genética , Animales , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Diferenciación Celular , Embrión no Mamífero/metabolismo , Datos de Secuencia Molecular , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inhibidores Enzimáticos/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/metabolismo , Neuronas/citología , Neuronas/metabolismo , Nucleotidiltransferasas , Priones/biosíntesis , Conformación Proteica , Inhibidores de la Síntesis de la Proteína/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estaurosporina/metabolismoRESUMEN
The Balbiani body or mitochondrial cloud is a large distinctive organelle aggregate found in developing oocytes of many species, but its presence in the mouse has been controversial. Using confocal and electron microscopy, we report that a Balbiani body does arise in mouse neonatal germline cysts and oocytes of primordial follicles but disperses as follicles begin to grow. The mouse Balbiani body contains a core of Golgi elements surrounded by mitochondria and associated endoplasmic reticulum. Because of their stage specificity and perinuclear rather than spherical distribution, these clustered Balbiani body mitochondria may have been missed previously. The Balbiani body also contains Trailer hitch, a widely conserved member of a protein complex that associates with endoplasmic reticulum/Golgi-like vesicles and transports specific RNAs during Drosophila oogenesis. Our results provide evidence that mouse oocytes develop using molecular and developmental mechanisms widely conserved throughout the animal kingdom.
Asunto(s)
Oocitos/ultraestructura , Oogénesis , Folículo Ovárico/ultraestructura , Secuencia de Aminoácidos , Animales , Drosophila , Proteínas de Drosophila/análisis , Femenino , Aparato de Golgi/ultraestructura , Masculino , Ratones , Microscopía Electrónica , Mitocondrias/ultraestructura , Datos de Secuencia Molecular , Estabilidad del ARN , Ribonucleoproteínas/análisisRESUMEN
Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Genes de Insecto/genética , Animales , Secuencia de Bases , Cartilla de ADN , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Ovario/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/metabolismo , Análisis de Secuencia de ADNRESUMEN
Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic "P bodies," including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/trailer hitch) in FMRP-driven, argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons.
Asunto(s)
Proteínas de Drosophila/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/fisiología , Animales , Animales Modificados Genéticamente , Northern Blotting , Western Blotting/métodos , Caspasas/metabolismo , Células Cultivadas , Sistema Nervioso Central/citología , Dendritas/metabolismo , Dendritas/fisiología , Drosophila , Proteínas de Drosophila/metabolismo , Exorribonucleasas/metabolismo , Ojo/metabolismo , Ojo/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica/métodos , Larva , MicroARNs/metabolismo , Microscopía Electrónica de Rastreo/métodos , Neuronas/citología , Biosíntesis de Proteínas/fisiología , Transporte de Proteínas/fisiología , Complejo Silenciador Inducido por ARN/metabolismoRESUMEN
Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. We have identified Drosophila trailer hitch (tral) as a gene that is required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral revealed that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell.
Asunto(s)
Proteínas de Drosophila/fisiología , Retículo Endoplásmico/metabolismo , Ribonucleoproteínas/fisiología , Animales , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Huevo/metabolismo , Retículo Endoplásmico/genética , Transporte de Proteínas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Translational regulation plays an essential role in many phases of the Drosophila life cycle. During embryogenesis, specification of the developing body pattern requires co-ordination of the translation of oskar, gurken and nanos mRNAs with their subcellular localization. In addition, dosage compensation is controlled by Sex-lethal-mediated translational regulation while dFMR1 (the Drosophila homologue of the fragile X mental retardation protein) controls translation of various mRNAs which function in the nervous system. Here we describe some of the mechanisms that are utilized to regulate these various processes. Our review highlights the complexity that can be involved with multiple factors employing different mechanisms to control the translation of a single mRNA.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/fisiología , Factor 4E Eucariótico de Iniciación/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Modelos Genéticos , Factores de Iniciación de Péptidos/fisiología , Interferencia de ARN , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Factor de Crecimiento Transformador alfa/biosíntesis , Factor de Crecimiento Transformador alfa/genéticaRESUMEN
In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end-directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Proteínas de Insectos/genética , Oogénesis/genética , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Polaridad Celular/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Factor 4E Eucariótico de Iniciación/genética , Femenino , Proteínas de Insectos/metabolismo , Sustancias Macromoleculares , Microtúbulos/metabolismo , Oocitos/citología , Oocitos/metabolismo , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
Subcellular localization of mRNAs within the Drosophila oocyte is an essential step in body patterning. Yps, a Drosophila Y-box protein, is a component of an ovarian ribonucleoprotein complex that also contains Exu, a protein that plays an essential role in mRNA localization. Y-box proteins are known translational regulators, suggesting that this complex might regulate translation as well as mRNA localization. Here we examine the role of the yps gene in these events. We show that yps interacts genetically with orb, a positive regulator of oskar mRNA localization and translation. The nature of the genetic interaction indicates that yps acts antagonistically to orb. We demonstrate that Orb protein is physically associated with both the Yps and Exu proteins, and that this interaction is mediated by RNA. We propose a model wherein Yps and Orb bind competitively to oskar mRNA with opposite effects on translation and RNA localization.