RESUMEN
Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist.
Asunto(s)
Proteínas de Drosophila/metabolismo , Memoria/fisiología , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/fisiología , ARN/metabolismo , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Animales , Animales Modificados Genéticamente , Aminas Biogénicas/administración & dosificación , Encéfalo/metabolismo , Encéfalo/ultraestructura , Línea Celular , Cromatografía Líquida de Alta Presión , Cortejo , Drosophila , Proteínas de Drosophila/clasificación , Proteínas de Drosophila/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Larva , Aprendizaje/fisiología , Masculino , Espectrometría de Masas , Microscopía Inmunoelectrónica , Proteínas Quinasas Activadas por Mitógenos/genética , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/metabolismo , Mutación/genética , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína/fisiología , ARN/genética , ARN Mensajero/metabolismo , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Factores de Escisión y Poliadenilación de ARNm/clasificación , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The chicken anaemia virus-derived protein apoptin is a tumour-specific cell-killing agent. It is biologically active as a highly stable, multimeric complex, consisting of 30-40 monomers. In tumour cells, but negligibly in normal cells, apoptin is imported into the nucleus prior to the induction of apoptosis. Immunoelectron microscopic data we report here indicate that apoptin predominantly co-localises with heterochromatin and nucleoli within tumour cells. Apoptin's preference for these DNA-dense nuclear bodies may be explained by our finding that apoptin cooperatively forms distinct superstructures with DNA in vitro. These superstructures do not grow beyond a diameter of approximately 200 nm, containing up to 20 multimeric apoptin complexes and approximately 3 kb of DNA. Furthermore, we show a single apoptin multimer to have eight independent, non-specific DNA-binding sites which preferentially bind strand ends, but which can also collaborate to bind longer stretches of DNA. Apoptin's high affinity for naked, undecorated double- and single-stranded DNA and for DNA fibre ends suggests that it may also capture such DNA in superstructures in vivo. Since these forms of DNA are predominantly found in transcriptionally active, replicating and damaged DNA, apoptin could be triggering apoptosis by interfering with DNA transcription and synthesis.