RESUMEN
The consolidation of long-term memory for sensitization and synaptic facilitation in Aplysia requires synthesis of new mRNA including the immediate early gene Aplysia CCAAT enhancer-binding protein (ApC/EBP). After the rapid induction of ApC/EBP expression in response to repeated treatments of 5-hydroxytryptamine (5-HT), ApC/EBP mRNA is temporarily expressed in sensory neurons of sensory-to-motor synapses. However, the molecular mechanism underlying the rapid degradation of ApC/EBP transcript is not known. Here, we cloned an AU-rich element (ARE)-binding protein, ApAUF1, which functions as a destabilizing factor for ApC/EBP mRNA. ApAUF1 was found to bind to the 3' UTR of ApC/EBP mRNA that contains AREs and subsequently reduces the expression of ApC/EBP 3' UTR-containing reporter genes. Moreover, overexpression of ApAUF1 inhibited the induction of ApC/EBP mRNA in sensory neurons and also impaired long-term facilitation of sensory-to-motor synapses by repetitive 5-HT treatments. These results provide evidence for a critical role of the posttranscriptional modification of ApC/EBP mRNA during the consolidation of synaptic plasticity.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Transmisión Sináptica , Regiones no Traducidas 3' , Animales , Aplysia , Clonación Molecular , Genes Reporteros , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea D0 , Humanos , Hibridación in Situ , Modelos Biológicos , Modelos Genéticos , Plasticidad Neuronal , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismoRESUMEN
Aplysia CCAAT enhancer-binding protein (ApC/EBP), a key molecular switch in 5-hydroxytryptamine (5-HT)-induced long-term facilitation of Aplysia, is quickly and transiently expressed in response to a 5-HT stimulus, but the mechanism underlying this dynamic expression profile remains obscure. Here, we report that the dynamic expression of ApC/EBP during long-term facilitation is regulated at the post-transcriptional level by AU-rich element (ARE)-binding proteins. We found that the 3'UTR of ApC/EBP mRNA contains putative sequences for ARE, which is a representative post-transcriptional cis-acting regulatory element that modulates the stability and/or the translatability of a distinct subset of labile mRNAs. We cloned the Aplysia homologue of embryonic lethal abnormal visual system homologue (ELAV/Hu) protein, one of the best-studied RNA-binding proteins that associate with ARE, and elucidated the involvement of Aplysia ELAV/Hu protein in ApC/EBP gene expressional regulation. Cloned Aplysia ELAV/Hu protein, Aplysia embryonic lethal abnormal visual system (ApELAV), bound to an AU-rich region within the 3'UTR of ApC/EBP mRNA. Additionally, ApELAV controlled the expression of ApC/EBP 3'UTR-containing reporter gene by functioning as a stability-enhancing factor. In particular, 5-HT-induced long-term facilitation was impaired when the AU-rich region within the 3'UTR of ApC/EBP was over-expressed, which suggests the significance of this region in 5-HT-induced ApC/EBP expression, and in the resultant formation of long-term facilitation. Our results imply that the Aplysia ARE-binding protein, ApELAV, can regulate ApC/EBP gene expression at the mRNA level, and accordingly, ARE-mediated post-transcriptional mechanism may serve a crucial function in regulating the expression of ApC/EBP in response to a 5-HT stimulus.
Asunto(s)
Aplysia/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Serotonina/farmacología , Regiones no Traducidas 3'/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Clonación Molecular , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Ribonucleoproteína Nuclear Heterogénea D0 , Hibridación in Situ , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
cAMP response-element binding protein (CREB), a transcription factor involved in learning, memory and drug addiction, is phosphorylated by calcium-calmodulin-dependent protein kinase IV (CaMKIV). Here, we show that CaMKIV-knockout (KO) mice developed less analgesic tolerance after chronic morphine administration with no alteration in physical dependence or acute morphine-induced analgesia. The increase in phosphorylated CREB expression observed in wild-type mice after chronic morphine was absent in CaMKIV-KO mice, while there was no difference in the expression or phosphorylation of the micro-opioid receptor between groups. Morphine-treated CaMKIV-KO mice showed less G-protein uncoupling from the micro-opioid receptor than did wild-type mice, while uncoupling was similar in control wild-type and KO mice. In addition, morphine reduced inhibitory transmission to a greater degree in CaMKIV-KO mice than in controls after chronic morphine exposure. Our results provide novel evidence for the role of CaMKIV in the development of opioid analgesic tolerance but not physical dependence.