RESUMEN
Localization of mRNAs, a process essential for embryonic body patterning in Drosophila, requires recognition of cis-acting signals by cellular components responsible for movement and anchoring. We have purified a large multiprotein complex that binds a minimal form of the bicoid mRNA localization signal in a manner both specific and sensitive to inactivating mutations. Identified complex components include the RNA binding proteins Modulo, PABP, and Smooth, the known localization factor Swallow, and the kinesin family member Nod. We demonstrate that localization of bcd mRNA is defective in modulo mutants. The presence of three required localization components (Swallow, Modulo, and specific RNA binding activity) within the recognition complex strongly implicates it in mRNA localization.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Homeodominio/genética , Proteínas de Microtúbulos/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transactivadores/genética , Animales , Western Blotting , Tipificación del Cuerpo/genética , Diferenciación Celular , Drosophila melanogaster/embriología , Electroforesis en Gel de Poliacrilamida , Femenino , Cinesinas , Sustancias Macromoleculares , Complejos Multiproteicos , Ovario/citología , Ovario/metabolismo , ARN Mensajero/genética , Transducción de SeñalRESUMEN
The appearance of Oskar protein occurs coincident with localization of oskar mRNA to the posterior pole of the Drosophila oocyte, and earlier accumulation of the protein is prevented by translational repression. We find that the nascent polypeptide-associated complex (NAC) is required for correct localization of oskar mRNA. The timing of the defects suggests that, if NAC acts directly via an interaction with nascent Oskar protein, oskar mRNA should be undergoing translation prior to its localization. Polysome analysis confirms that oskar mRNA is associated with polysomes even in the absence of localization of the mRNA or accumulation of Oskar protein. Thus, the mechanisms that prevent accumulation of Oskar protein until it can be secured at the posterior pole of the oocyte include regulated degradation or inhibition of translational elongation.
Asunto(s)
Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila/crecimiento & desarrollo , Factores Eucarióticos de Iniciación/metabolismo , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Biosíntesis de Proteínas/genética , Transactivadores/metabolismo , Animales , Polaridad Celular/genética , Drosophila/citología , Drosophila/metabolismo , Factores Eucarióticos de Iniciación/genética , Femenino , Chaperonas Moleculares , Oocitos/citología , Oocitos/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Transporte de Proteínas/genética , ARN Mensajero/metabolismo , Transactivadores/genéticaRESUMEN
The process of mRNA localization, often used for regulation of gene expression in polarized cells, requires recognition of cis-acting signals by components of the localization machinery. Many known RNA signals are active in the contexts of both the Drosophila ovary and the blastoderm embryo, suggesting a conserved recognition mechanism. We used variants of the bicoid mRNA localization signal to explore recognition requirements in the embryo. We found that bicoid stem-loop IV/V, which is sufficient for ovarian localization, was necessary but not sufficient for full embryonic localization. RNAs containing bicoid stem-loops III/IV/V did localize within the embryo, demonstrating a requirement for dimerization and other activities supplied by stem-loop III. Protein complexes that bound specifically to III/IV/V and fushi tarazu localization signals copurified through multiple fractionation steps, suggesting that they are related. Binding to these two signals was competitive but not equivalent. Thus, the binding complexes are not identical but appear to have some components in common. We have proposed a model for a conserved mechanism of localization signal recognition in multiple contexts.
Asunto(s)
Regiones no Traducidas 3'/genética , Conformación de Ácido Nucleico , Ovario/metabolismo , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Secuencia de Bases , Drosophila melanogaster , Embrión no Mamífero/metabolismo , Femenino , Datos de Secuencia MolecularRESUMEN
Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.