RESUMO
ZNF265 is a newly identified arginine/serine-rich (SR) protein and has two transcript isoforms (ZNF265-1 and ZNF265-2) that autoregulate between each other. Previous studies have shown that ZNF265 regulates the Tra2 beta isoform splicing. Here, we demonstrate that two ZNF-265 transcript isoforms are expressed in various mouse tissues and that ZNF265-1 is a major isoform. The ZNF265-1 protein level in the cerebral cortex is significantly lower in relative to other tissues. The recombinant proteins of both isoforms are nuclear, in consistent with its functions as pre-mRNA splicing regulators. Splicing analysis with GluR-B and SMN2 minigenes demonstrates that ZNF265-1 inhibits the Flop exon and exon 7 usages in the splicing of two minigenes, respectively. The regulation of GluR-B and SMN2 pre-mRNA splicing by ZNF265 implies this newly identified SR protein may play important roles in maintaining normal neuronal function and SMA pathogenesis.
Assuntos
Proteínas de Ligação a RNA/genética , Animais , Animais Recém-Nascidos , Western Blotting , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Éxons/genética , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/genética , Isomerismo , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/biossíntese , Receptores de AMPA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Complexo SMN , Frações Subcelulares/metabolismoRESUMO
The in vitro studies have shown that Tra2beta1 proteins are important regulators in the alternative pre-mRNA splicing in mammalians. To date, the knowledge regarding the in vivo function of Tra2beta1 proteins, especially the function in regulating the splicing of neural-specific genes and the cell-type specificity of the functions is very limited. In the present study, the cell-type specific splicing of two neural-specific genes (GluR-B and SMN2), and the function of Tra2beta1 proteins in regulation of the splicing were studied in two types of cells (COS-1 and PFSK-1) using in vivo splicing models. The results showed that the splicing of GluR-B and SMN2 minigenes is cell-type specific, while Tra2beta1 proteins regulate the splicing of the minigenes in both cell lines in a similar pattern. It indicates that the Tra2beta1-regulated splicing of GluR-B and SMN2 minigenes regulated by Tra2beta1 is not cell-type specific.
Assuntos
Processamento Alternativo , Antígenos CD/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/genética , Receptores de AMPA/genética , Animais , Neoplasias Encefálicas/patologia , Células COS/metabolismo , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Humanos , Proteína Cofatora de Membrana , Proteínas do Tecido Nervoso/biossíntese , Tumores Neuroectodérmicos Primitivos/patologia , Precursores de RNA/genética , Proteínas de Ligação a RNA , Receptores de AMPA/biossíntese , Proteínas do Complexo SMN , Proteína 2 de Sobrevivência do Neurônio Motor , TransfecçãoRESUMO
We generated the small interference RNAs to specifically silence the expression of neural salient serine/arginine rich protein 1 (NSSR1) and showed that the inhibition of NSSR1 expression in mouse embryonic carcinoma cells (P19) reduces neuronal differentiation. By contrast, its over-expression promotes the differentiation. Neither inhibition nor over-expression shows distinct effect on cell proliferation. The over-expression increases the inclusion of NCAM L1 exon2 while the inhibition reduces the inclusion. The splicing of kinase insert free isoform of TrkC (TrkC-K1) is increased by the over-expression. The results demonstrate that NSSR1 promotes neuronal differentiation and the splicing of NCAML1 exon2 and TrkC-K1.
Assuntos
Proteínas de Transporte/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Neoplasias/fisiologia , Neurônios/patologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/fisiologia , Processamento Alternativo , Animais , Northern Blotting/métodos , Western Blotting/métodos , Bromodesoxiuridina/metabolismo , Carcinoma , Contagem de Células , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Embrião de Mamíferos , Éxons , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica , Camundongos , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Receptor trkC/genética , Receptor trkC/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção/métodos , Tubulina (Proteína)/metabolismoRESUMO
Nestin belongs to the class VI intermediate filament family and it is a marker for neural progenitor cells. In this work, the 3'-terminal coding sequence(396 bp) of human nestin gene was cloned into pGEX-3X plasmid and introduced into BL21 E. coli cells. The GST-nestin protein was purified with an affinity column. Anti-human nestin antiserum was raised by immunizing a rabbit with the fusion protein. The high specificity of the antibody against human nestin was confirmed by western-blot and immunocytochemistry analysis.
Assuntos
Soros Imunes/imunologia , Proteínas de Filamentos Intermediários/imunologia , Proteínas do Tecido Nervoso/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Especificidade de Anticorpos , Clonagem Molecular , Glutationa Transferase/genética , Glutationa Transferase/imunologia , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas do Tecido Nervoso/genética , Nestina , Coelhos , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Na(+)-H(+) exchangers (NHE) are major membrane proteins that have been identified as signal transduction mediators in the regulation of cell differentiation and important membrane ion transporters in the regulation of the intercellular pH and the cell volume. NHE are composed of at least six isoforms and activated in growth factor-regulated cell differentiation. However, little is known about the differential regulation of NHE expression in the development. In the present study, we studied developmental regulation of the expression of NHE isoforms in human fetal tissues by comparing the expression of various isoforms between two developmental stages, i.e., week 11 (11 W) and week 16 (16 W). The results demonstrated that NHE1 transcripts were expressed ubiquitously. In comparison to the expression at 16 W, the level of NHE1 transcripts was low and varied significantly in a tissue-specific pattern at 11 W, suggesting that the house-keeping function of MHE1 occurs at 11 W or earlier and becomes well established at least as early as at 16 W. The tissue-specifically restricted expression of NHE2 and NHE3 was regulated at 11 W and 16 W in an opposite tendency, supporting the overlapping relationship between NHE2 and NHE3 in the tissue distribution as reported in adults. NHE5 expression was relatively ubiquitous at 11 W and became restricted in the cerebellum at 16 W, suggesting that the restrictive expression of NHE5 in the brain occurs later than that of other isoforms. The present study demonstrates a space time-dependent regulation of the tissue-specific expression pattern of NHE isoforms during human development between 11 W and 16 W. The results also suggest that at 16 W or earlier the expression pattern of developing tissues becomes similar to that of adult tissues. The observed developmental regulation of NHE expression provides a molecular basis for further study of the function and regulation of NHE gene during development.
