RESUMO
Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3'-untranslated region (3'-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3'-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3'-UTR, which prolongs the mRNA lifetime and increases protein level.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Serpinas/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Encéfalo/metabolismo , Proteínas ELAV , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hibridização In Situ , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Células PC12 , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serpinas/genética , Transcrição Gênica , NeuroserpinaRESUMO
Neuroserpin is a serine protease inhibitor expressed in the developing and the adult nervous system. Studies with genetically modified mice indicate a role of neuroserpin in the regulation of anxiety. Mutations in the neuroserpin gene cause protein polymerization and formation of inclusion bodies leading to progressive myoclonic epilepsy and neurodegeneration. Here we demonstrate that neuroserpin expression is regulated by thyroid hormone (T3). Neuroserpin RNA levels are down-regulated in cortical layers II/III and VIa, the hippocampus, the retrosplenial cortex and the medial habenular nucleus, but not in cortical layer V or other areas of the hypothyroid rat brain. Concordantly, neuroserpin RNA and protein expression was induced by T3 in rat PC12 cells containing appropriate thyroid hormone receptor levels. In run-on assays T3 did not affect the transcription rate of the neuroserpin gene, indicating that regulation was post-transcriptional. Moreover, T3 increased in vitro binding of cytoplasmic proteins to neuroserpin 3'-UTR RNA and caused biphasic regulation of the stability of this transcript in PC12 cells. Ectopic neuroserpin expression induced neurite extension in PC12 cells and enhanced neuritogenesis triggered by nerve growth factor. In summary, these results indicate that neuroserpin expression is post-transcriptionally regulated by T3 at the level of RNA stability.
Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , Neuropeptídeos/genética , Estabilidade de RNA/fisiologia , Serpinas/genética , Tri-Iodotironina/metabolismo , Regiões 3' não Traduzidas/efeitos dos fármacos , Regiões 3' não Traduzidas/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas ELAV , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neuropeptídeos/metabolismo , Células PC12 , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/metabolismo , Serpinas/metabolismo , Tri-Iodotironina/farmacologia , NeuroserpinaRESUMO
Many genes governed by thyroid hormone (T3) lack binding sites for its receptor (TR) and are thought to be post-transcriptionally regulated by T3. Here we demonstrate that the HuD gene, which encodes a neurone-specific protein that binds to mRNA and modulates its stability, is regulated by T3. HuD RNA and protein expression were strongly up-regulated in specific areas of the hypothyroid rat brain, and reduced by T3 in rat PC12 and mouse N2a cells containing appropriate TR levels. Furthermore, T3 inhibited the transcription of HuD in run-on assays. Finally, HuD protein bound with high affinity to two sequences in acetylcholinesterase mRNA, and ectopic HuD expression increased its abundance in N2a cells. This is the first report of a gene encoding an mRNA stability regulator that is under T3 control. The results suggest that HuD may mediate some T3 effects by altering the half-life of mRNAs for acetylcholinesterase and other genes.