RESUMO
The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.
Assuntos
Proteínas de Transporte/fisiologia , Neuroglia/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Imunoprecipitação da Cromatina , Cromatografia em Gel , Clonagem Molecular , Ciclofilina A/química , Primers do DNA/química , Enzimas Reparadoras do DNA , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Dados de Sequência Molecular , Neurônios/metabolismo , Proteínas Nucleares , Oligonucleotídeos/química , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , Fatores de Processamento de RNA , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Spliceossomos/metabolismo , Fatores de Tempo , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
Trip15/CSN2 is a transcriptional corepressor/a component of COP9 signalosome (CSN) and participates in various signaling pathways. However, participation of Trip15/CSN2 in neural differentiation is still obscure. Here, we show that Trip15/CSN2 plays a critical role in neuronal differentiation. The expression of Trip15/CSN2 mRNA was induced at an early stage of neuronal differentiation in the retinoic acid (RA)-treated P19 cells, but not in the triiodothyronine (T3)-primed cardiac muscular cell differentiation. The expression of Trip15/CSN2 mRNA in the rat brain was detected at E14 and the protein was localized in the nuclei of neonatal rat CNS neurons. Enforced expression of sense rat Trip15/CSN2 mRNA caused the downregulation of Oct-3/4 mRNA expression and was sufficient to convert P19 cells into neurons, but not glial cells, only after the aggregation without RA. In the presence of RA, exogenous expression of the sense mRNA caused the intense and rapid induction of neurogenic Brn-2 and Mash-1 mRNA expressions accompanying the strong downregulation of Oct-3/4 mRNA expression, and stimulated both neuronal and glial cell differentiations of P19 cells. In contrast, enforced expression of the antisense mRNA suppressed the commitment of RA-treated aggregation form of P19 cells to neuronal lineage. These data strongly suggest that Trip15/CSN2 could implicate in the commitment of multipotent embryonal carcinoma (EC) cells to neuronal fate through the downregulation of Oct-3/4 which suppresses neurogenic genes. Moreover, in addition to Trip15/CSN2, RA-regulated other factor(s) may be required for glial cell differentiation.
