RESUMO
We have examined the functional significance of the organization of pre-mRNA splicing factors in a speckled distribution in the mammalian cell nucleus. Upon microinjection into living cells of oligonucleotides or antibodies that inhibit pre-mRNA splicing in vitro, we observed major changes in the organization of splicing factors in vivo. Interchromatin granule clusters became uniform in shape, decreased in number, and increased in both size and content of splicing factors, as measured by immunofluorescence. These changes were transient and the organization of splicing factors returned to their normal distribution by 24 h following microinjection. Microinjection of these oligonucleotides or antibodies also resulted in a reduction of transcription in vivo, but the oligonucleotides did not inhibit transcription in vitro. Control oligonucleotides did not disrupt splicing or transcription in vivo. We propose that the reorganization of splicing factors we observed is the result of the inhibition of splicing in vivo.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas , Transcrição Gênica , Autorradiografia , Sequência de Bases , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Células HeLa , Humanos , Microinjeções , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Nucleares/análise , Oligodesoxirribonucleotídeos/farmacologia , Precursores de RNA/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/imunologia , Ribonucleoproteínas Nucleares Pequenas/análise , Fatores de Processamento de Serina-Arginina , Spliceossomos/metabolismo , Spliceossomos/ultraestruturaRESUMO
GH is required for normal postnatal growth and metabolism. GH stimulates postnatal growth through induction of IGF-I gene expression. Although the liver is the major site of GH-regulated IGF-I, recent evidence indicates that GH-regulated IGF-I expression in nonhepatic tissues is sufficient for normal postnatal growth. One potentially important nonhepatic site of GH-stimulated IGF-I expression is skeletal muscle, as injection of GH into animals leads to increased IGF-I mRNA in this tissue. Nevertheless, direct effects of GH in skeletal muscle cells in culture have not been reported. We therefore tested the C2C12 myogenic cell line for its response to GH and demonstrate that C2C12 skeletal muscle cells rapidly respond to physiological levels of GH with increased tyrosine phosphorylation of the GH receptor, Janus kinase 2, signal transducer and activator of transcription-5a and -5b, insulin receptor substrate-1, and activation of MAPKs/ERKs and protein kinase B/Akt. In these cells, GH stimulates the expression of IGF-I and two members of the suppressors of cytokine signaling family, cytokine-inducible SH2-containing protein and suppressor of cytokine signaling-2. Treatment of C2C12 myoblasts with either the MAPK kinase inhibitor PD98059 or the PI3K inhibitor wortmannin results in higher levels of GH-induced IGF-I and suppressor of cytokine signaling-2 mRNA expression, suggesting that activation of MAPK and PI3K pathways has an inhibitory role in IGF-I and suppressor of cytokine signaling-2 gene regulation. Therefore, C2C12 cells provide the first in vitro model system to study various aspects of GH action in skeletal muscle.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento Humano/farmacologia , Fator de Crescimento Insulin-Like I/genética , Proteínas do Leite , Músculo Esquelético/fisiologia , Proteínas/genética , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Animais , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Genes Reporter/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Janus Quinase 2 , Camundongos , Músculo Esquelético/citologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Fator de Transcrição STAT5 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina , Transativadores/metabolismoRESUMO
In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAPc is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/biossíntese , Proteínas , Ribonucleoproteína Nuclear Pequena U4-U6/genética , TATA Box , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Reagentes de Ligações Cruzadas , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Polimerase II/biossíntese , RNA Polimerase III/biossíntese , Relação Estrutura-Atividade , Proteína de Ligação a TATA-Box , Transativadores/metabolismoRESUMO
The human RNA polymerase II and III snRNA promoters share a common basal element, the proximal sequence element (PSE), which is recognized by a complex we refer to as the snRNA-activating protein complex (SNAPc). Biochemical purifications suggest that SNAPc is composed of at least four polypeptides of 43, 45, 50 and 190 kDa, as well as variable amounts of the TATA box binding protein, TBP. cDNAs encoding the 43 and 45 kDa subunits, SNAP43 and SNAP45, have been isolated, but there is no evidence that either of these subunits contacts DNA. Here we report the isolation of cDNAs encoding the 50 kDa subunit of SNAPc, SNAP50. The open reading frame predicts a 411 amino acid protein, which contains two potential zinc finger motifs. Depletions with anti-SNAP50 antibodies inhibit RNA polymerase II and III snRNA gene transcription in vitro. SNAP50 interacts with SNAP43 in co-immunoprecipitation experiments, but not with SNAP45 or TBP. UV cross-linking experiments suggest that SNAP50 contacts DNA in the SNAP complex. These results are consistent with the same core SNAP complex recognizing the PSEs of RNA polymerase II and III snRNA promoters, and provide an initial view of the architecture of the SNAP complex.
Assuntos
Proteínas de Ligação a DNA , Proteínas/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteínas/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , RNA Nuclear Pequeno/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The TATA-box-binding protein TBP exists in the cell complexed with different sets of TBP-associated factors (TAFs). In general, each of these TBP-TAF complexes is dedicated to transcription by a single RNA polymerase. Thus, SL1, TFIID and TFIIIB are required for transcription by polymerases I, II and III, respectively. Here we characterize a fourth TBP-TAF complex called SNAPc. Unlike the other TBP-TAF complexes, SNAPc is implicated in transcription by two types of polymerases; it is required for transcription of both the RNA polymerase II and III small-nuclear RNA genes and binds specifically to the proximal sequence element PSE, a non-TATA-box basal promoter element common to these two types of genes. In addition to TBP, SNAPc is composed of at least three TAFs, SNAP43, SNAP45 and SNAP50. The predicted amino-acid sequence of SNAP43 reveals that it corresponds to a new protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase I/metabolismo , RNA Nuclear Pequeno/genética , TATA Box , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Bacteriófago lambda/genética , Sequência de Bases , Reações Cruzadas , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Nuclear Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box , Fatores de Transcrição/imunologia , Transcrição GênicaRESUMO
The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerase III/metabolismo , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/biossíntese , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , TATA Box , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Clonagem Molecular , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Ligação a TATA-BoxRESUMO
Previously, by a yeast 2-hybrid screen, we identified signal transducer and activator of transcription 5b (Stat5b) as a substrate of the insulin receptor (IR). We demonstrated that refeeding of fasted mice leads to rapid activation of Stat5 proteins in liver, skeletal muscle, and fat, suggesting that Stat5b is a physiological target of insulin. Here, we show that injection of glucose or insulin into fasted mice leads to robust activation of both Stat5a and Stat5b in skeletal muscle. In C2C12 myotubes, we find that insulin stimulates tyrosine phosphorylation of Stat5a and Stat5b by 3-5-fold. This degree of Stat5 activation in vitro is significantly lower than what we observe in vivo and inversely correlates with IRS-1/2 levels. We can recapitulate robust insulin activation of Stat5 in C2C12 cells by stable overexpression of the human IR (hIR). To identify insulin-activated genes that are Stat5 targets, we also overexpressed an IR mutant (LA-hIR) that signals normally for mitogen-activated protein kinase- and phosphatidylinositol 3-kinase-dependent pathways but is deficient in Stat5 signaling in response to insulin. We demonstrate that insulin induces the expression of SOCS-2 mRNA in the wild type hIR but not in the LA-hIR-overexpressing cells. The induction of SOCS-3 by insulin is reduced but not lost in the LA-hIR cells. Therefore, our results suggest that insulin induction of SOCS-2, and in part SOCS-3 mRNA expression, is mediated by Stat5 and can be independent of mitogen-activated protein kinase and phosphatidylinositol 3-kinase-signaling pathways.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Proteínas do Leite , Músculo Esquelético/efeitos dos fármacos , Proteínas/genética , RNA Mensageiro/genética , Proteínas Repressoras , Transativadores/fisiologia , Fatores de Transcrição , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Isoformas de Proteínas/fisiologia , Fator de Transcrição STAT5 , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de CitocinaRESUMO
DNA methylation, by regulating the transcription of genes, is a major modifier of the eukaryotic genome. DNA methyltransferases (DNMTs) are responsible for both maintenance and de novo methylation. We have reported that human immunodeficiency virus type 1 (HIV-1) infection increases DNMT1 expression and de novo methylation of genes such as the gamma interferon gene in CD4(+) cells. Here, we examined the mechanism(s) by which HIV-1 infection increases the cellular capacity to methylate genes. While the RNAs and proteins of all three DNMTs (1, 3a, and 3b) were detected in Hut 78 lymphoid cells, only the expression of DNMT1 was significantly increased 3 to 5 days postinfection. This increase was observed with either wild-type HIV-1 or an integrase (IN) mutant, which renders HIV replication defective, due to the inability of the provirus to integrate into the host genome. Unintegrated viral DNA is a common feature of many retroviral infections and is thought to play a role in pathogenesis. These results indicate another mechanism by which unintegrated viral DNA affects the host. In addition to the increase in overall genomic methylation, hypermethylation and reduced expression of the p16(INK4A) gene, one of the most commonly altered genes in human cancer, were seen in cells infected with both wild-type and IN-defective HIV-1. Thus, infection of lymphoid cells with integration-defective HIV-1 can increase the methylation of CpG islands in the promoters of genes such as the p16(INK4A) gene, silencing their expression.