RESUMO
Regulation of both transcription and RNA splicing requires enhancer elements, that is, cis-acting DNA or RNA sequences that promote the activities of linked promoters or splice sites, respectively. Both types of enhancer associate with regulatory proteins to form multicomponent enhancer complexes that recruit the necessary enzymatic machinery to promoter or splice site recognition sequences. This recruitment occurs as a result of direct interactions between regulatory proteins in the enhancer complexes and components of the basic enzymatic machineries. Recent advances suggest that the high degree of regulatory specificity observed for both transcription and splicing is due, in large part, to the multicomponent nature of enhancer complexes and to their cooperative assembly.
Assuntos
Elementos Facilitadores Genéticos , Splicing de RNA , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Cromatina/fisiologia , Humanos , Precursores de Ácido Nucleico/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction.
Assuntos
Antígenos HLA-DR/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologia , Sequência de Bases , Sítios de Ligação , Primers do DNA/química , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Antígenos HLA-DQ/genética , Cadeias beta de HLA-DQ , Cadeias alfa de HLA-DR , Proteína HMGA1a , Células HeLa , Humanos , Técnicas In Vitro , Interferon gama/farmacologia , Linfócitos/fisiologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Fator 2 de Transcrição de Octâmero , RNA Mensageiro/genética , Transcrição Gênica , Ativação TranscricionalRESUMO
Positive control of the sex-specific alternative splicing of doublesex (dsx) precursor messenger RNA (pre-mRNA) in Drosophila melanogaster involves the activation of a female-specific 3' splice site by the products of the transformer (tra) and transformer-2 (tra-2) genes. The mechanisms of this process were investigated in an in vitro system in which the female-specific 3' splice site could be activated by recombinant Tra or Tra-2 (or both). An exon sequence essential for regulation in vivo was shown to be both necessary and sufficient for activation in vitro. Nuclear proteins in addition to Tra and Tra-2 were found to bind specifically to this exon sequence. Therefore, Tra and Tra-2 may act by promoting the assembly of a multiprotein complex on the exon sequence. This complex may facilitate recognition of the adjacent 3' splice site by the splicing machinery.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas Nucleares/metabolismo , Precursores de RNA/genética , Splicing de RNA , Ribonucleoproteínas/metabolismo , Animais , Baculoviridae/genética , Sítios de Ligação , Núcleo Celular/metabolismo , Clonagem Molecular , Éxons , Feminino , Globinas/genética , Células HeLa , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Caracteres Sexuais , Transcrição GênicaRESUMO
The mammalian splicing factor SC35 is required for the first step in the splicing reaction and for spliceosome assembly. The cloning and characterization of a complementary DNA encoding this protein revealed that it is a member of a family of splicing factors that includes mammalian SF2/ASF. This family of proteins is characterized by the presence of a ribonucleoprotein (RNP)-type RNA binding motif and a carboxyl-terminal serine-arginine-rich (SR) domain. A search of the DNA sequence database revealed that the thymus-specific exon (ET) of the c-myb proto-oncogene is encoded on the antisense strand of the SC35 gene.
Assuntos
DNA/isolamento & purificação , Proteínas Nucleares , Proteínas/genética , Splicing de RNA , Ribonucleoproteínas , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Sítios de Ligação , Northern Blotting , Linhagem Celular , Clonagem Molecular , Códon , DNA/química , Éxons , Humanos , Dados de Sequência Molecular , Proteínas/química , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myb , RNA/metabolismo , Homologia de Sequência do Ácido Nucleico , Fatores de Processamento de Serina-Arginina , TransfecçãoRESUMO
STAT proteins (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that are phosphorylated by Janus kinases in response to cytokines. Phosphorylated STAT proteins translocate to the nucleus, where they transiently turn on specific sets of cytokine-inducible genes. The mechanism that controls the amounts of activated STAT proteins is not understood. STAT1 proteins activated by interferon-gamma treatment in HeLa cells were shown to be stabilized by a proteasome inhibitor and ubiquitinated in vivo. Thus, the amount of activated STAT1 may be negatively regulated by the ubiquitin-proteasome pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interferon gama/farmacologia , Complexos Multienzimáticos/metabolismo , Transativadores/metabolismo , Ubiquitinas/metabolismo , Núcleo Celular/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , DNA/metabolismo , Células HeLa , Humanos , Immunoblotting , Leupeptinas/farmacologia , Mutação , Fosforilação , Complexo de Endopeptidases do Proteassoma , Fator de Transcrição STAT1 , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The effect of hemin on the rate of synthesis and the level of globin messenger RNA's in the human erythroid cell line K562 was examined by means of cloned hybridization probes specific for each of the human embryonic, fetal, and adult globin genes. Hemin increases both the rate of transcription and the level of accumulation of zeta-, epsilon-, gamma-, and alpha-globin messenger RNA's by a factor of 3 to 5. Thus, hemin induction of globin gene expression in K562 cells is at the level of transcription.
Assuntos
Eritroblastos/metabolismo , Eritrócitos/metabolismo , Regulação da Expressão Gênica , Globinas/genética , Transcrição Gênica , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Hemina/farmacologia , Humanos , Leucemia Mieloide/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis. Analysis of every type of mismatch in many different sequence contexts indicates that more than 50 percent of all single base substitutions can be detected. The feasibility of this method for localizing base substitutions directly in genomic DNA samples is demonstrated by the detection of single base mutations in DNA obtained from individuals with beta-thalassemia, a genetic disorder in beta-globin gene expression.
Assuntos
Sequência de Bases , Mutação , Hibridização de Ácido Nucleico , Ribonucleases , Homologia de Sequência do Ácido Nucleico , Talassemia/diagnóstico , Animais , Globinas/genética , Humanos , Camundongos , Talassemia/genéticaRESUMO
A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse beta-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated beta-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription. In contrast, two different mutations in nucleotides immediately upstream from the CCAAT box resulted in a 3- to 3.5-fold increase in transcription. With two minor exceptions, single base substitutions in all other regions of the promoter had no effect on transcription. These results precisely delineate the cis-acting sequences required for accurate and efficient initiation of beta-globin transcription, and they establish a general approach for the fine structure genetic analysis of eukaryotic regulatory sequences.
Assuntos
Globinas/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Genes , Engenharia Genética , Globinas/biossíntese , Células HeLa , Humanos , Camundongos , Mutação , Transcrição GênicaRESUMO
Molecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood. Positive and negative regulatory elements that function only in specific cell types or in response to extracellular inducers have been identified. A number of cases of inducible and tissue-specific gene expression involve the activation of preexisting transcription factors, rather than the synthesis of new proteins. This activation may involve covalent modification of the protein or an allosteric change in its structure. The modification of regulatory proteins may play a central role in the mechanisms of eukaryotic gene regulation.
Assuntos
Regulação da Expressão Gênica , Animais , Elementos Facilitadores Genéticos , Células Eucarióticas , Previsões , Genes Reguladores , Humanos , Modelos Genéticos , Regiões Promotoras Genéticas , Fatores de Transcrição , Vírus/genéticaRESUMO
A new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA. The resulting duplex DNA fragments containing random single-base substitutions are cloned, amplified as a population, and isolated from wild-type DNA by preparative denaturing gradient gel electrophoresis. The physical separation of mutant DNA fragments makes it possible to isolate and characterize large numbers of site-directed single-base substitutions in the absence of a phenotypic selection. This procedure should be generally applicable to the fine-structure genetic analysis of regulatory and protein-coding sequences.
Assuntos
Clonagem Molecular , DNA Recombinante , Mutação , Animais , Sequência de Bases , Enzimas de Restrição do DNA , DNA de Cadeia Simples/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos , Camundongos , Desnaturação de Ácido Nucleico , Plasmídeos , Moldes GenéticosRESUMO
The protein kinase inhibitor 2-aminopurine (2AP) blocks the induction of the human beta-interferon gene by virus or poly(I)-poly(C) at the level of transcription. This inhibition is specific, since 2AP does not inhibit induction of either the hsp70 heat-shock gene by high temperature or the metallothionein gene by cadmium or dexamethasone. However, 2AP does block the induction of the c-fos and c-myc proto-oncogenes by serum growth factors or virus, suggesting that a protein kinase may be involved in the regulation of these genes, as well as of the beta-interferon gene. However, different factors must be required for the induction of these three genes, since they are not coordinately regulated by the same inducers in most of the cell lines examined.
Assuntos
2-Aminopurina/farmacologia , Adenina/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon Tipo I/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Animais , Células Cultivadas , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Inibidores de Proteínas Quinases , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica/efeitos dos fármacosRESUMO
The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.
Assuntos
Genes , Globinas/genética , Hemoglobinas/genética , Transcrição Gênica , Sequência de Bases , Sistema Livre de Células , Hemoglobina Fetal/genética , Genes Reguladores , Ligação Genética , Humanos , Óperon , Capuzes de RNA , RNA Polimerase II/metabolismoRESUMO
Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Corpo Adiposo/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Álcool Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular , DNA/metabolismo , Proteínas de Drosophila , Feminino , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Ovário/metabolismo , Fatores de Transcrição , Dedos de ZincoRESUMO
We have carried out experiments to determine which members of the rel family of transcription factors are involved in virus induction of the beta interferon (IFN-beta) gene. First, we examined the inducibility of artificial DNA binding sites that preferentially interact with different homo- or heterodimeric combinations of rel proteins in vitro. We found that only those sites capable of binding the p50/p65 heterodimer are virus inducible. Second, we analyzed a series of mutant rel DNA-binding sites in the context of the intact IFN-beta promoter. We found a correlation between (i) sites capable of binding both the p50/p65 heterodimer and the high-mobility-group protein HMG I(Y) and (ii) virus inducibility. Third, cotransfection of the IFN-beta gene enhancer/promoter with plasmids capable of expressing several different rel proteins revealed that only the combination of p50 and p65 efficiently activated transcription. Finally, we have used antibodies directed against different rel proteins to show that virus-inducible protein-DNA complexes assembled on the IFN-beta enhancer in vitro contain both p50 and p65. We conclude that the p50/p65 heterodimer is responsible for the NF-kappa B-dependent activation of the IFN-beta gene promoter in response to virus infection.
Assuntos
Regulação Viral da Expressão Gênica , Interferon beta/genética , NF-kappa B/fisiologia , Regiões Promotoras Genéticas , Viroses/genética , Sequência de Bases , Linhagem Celular , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genéticaRESUMO
We have examined the expression of human alpha- and beta-like globin genes in transient heterokaryons formed by fusion of human nonerythroid cells with terminally differentiating mouse erythroleukemia (MEL) cells or with a MEL cell variant (GM979) in which the endogenous mouse embryonic beta-globin genes are activated. In both the parental MEL cells and the heterokaryons, the alpha-globin genes were activated at least 12 h earlier than the embryonic, fetal, and adult beta-globin genes. These results suggest that kinetic differences in the activation of alpha- and beta-like globin genes are not simply the result of different rates of accumulation of erythroid-specific regulatory factors but may reflect differences in the mechanisms governing the transcriptional activation of these genes during erythroid cell differentiation. In mouse GM979 x human nonerythroid heterokaryons, the human embryonic beta-globin gene was activated, consistent with our previous demonstration that erythroid cells contain stage-specific trans-acting regulators of globin gene expression. Moreover, a dramatic increase in the ratio of human fetal to adult beta-globin transcription was observed compared with that seen in MEL-human nonerythroid hybrids. This ratio change may reflect competition between the fetal and adult beta-globin genes for productive interactions with erythroid cell-specific regulatory elements. Finally, we demonstrate that the behavior of naturally occurring mutations that lead to aberrant hemoglobin switching in humans also leads to aberrant expression in transient heterokaryons. Therefore, erythroid cells must contain trans-acting factors that interact with mutated regulatory elements to induce high-level expression of the human fetal globin genes.
Assuntos
Células Precursoras Eritroides/fisiologia , Regulação da Expressão Gênica , Globinas/genética , Fusão Celular , Núcleo Celular/fisiologia , Hemoglobina Fetal/genética , Genes de Troca , Humanos , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
Viral induction of the human beta-interferon (IFN-beta) gene leads to a transient accumulation of high levels of IFN-beta mRNA. Previous studies have shown that the increase in IFN-beta mRNA levels after induction is due to an increase in the rate of IFN-beta gene transcription. In this paper, we show that the rapid postinduction decrease in the level of IFN-beta mRNA is due to a combination of transcriptional repression and rapid turnover of the mRNA. This transcriptional repression can be blocked with cycloheximide, suggesting that the synthesis of a virus-inducible repressor is necessary for the postinduction turnoff of the IFN-beta gene. Analysis of the sequence requirements for IFN-beta mRNA instability revealed two regions capable of destabilizing a heterologous mRNA. One destabilizer is an AU-rich sequence in the 3' untranslated region, and the other is located 5' to the translation stop codon.
Assuntos
Regulação da Expressão Gênica , Interferon Tipo I/genética , Vírus da Parainfluenza 1 Humana/genética , RNA Mensageiro/genética , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Transformação Celular Viral , Células Cultivadas , Cicloeximida/farmacologia , Dados de Sequência Molecular , Plasmídeos , Poli I-C/farmacologia , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.
Assuntos
Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Deleção Cromossômica , Desoxirribonuclease I , Vetores Genéticos , Interferon beta/genética , Íntrons , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Splicing de RNA , Mapeamento por Restrição , Transcrição GênicaRESUMO
PRDI-BFc and PRDI-BFi are proteins that bind specifically to a regulatory element required for virus induction of the human beta interferon (IFN-beta). PRDI-BFc is a constitutive binding activity, while the PRDI-BFi binding activity is observed only after cells are treated with inducers such as virus or poly(I).poly(C) plus cycloheximide or in some cells by cycloheximide alone. In this paper we report that PRDI-BFc is interferon regulatory factor-2 (IRF-2), a known transcriptional repressor. In addition, we find that PRDI-BFi is a truncated form of IRF-2, lacking approximately 185 C-terminal amino acids. Thus, PRDI-BFi appears to be generated by inducible proteolysis. Although the affinity of PRDI-BFc/IRF-2 for the IFN-beta promoter does not appear to be affected by the removal of C-terminal amino acids, the ability of PRDI-BFi to function as a repressor in cotransfection experiments is significantly less than that of intact IRF-2. Studies have shown that IRF-2 can block the activity of the transcriptional activator IRF-1, which also binds specifically to the IFN-beta gene promoter. Thus, the inducible proteolysis of IRF-2 may be involved in the regulation of the IFN-beta gene or of other genes in which the ratio of IRF-1 to IRF-2 can affect the level of transcription.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Repressoras/genética , Fatores de Transcrição , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Cromatografia de Afinidade , Deleção Cromossômica , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Fator Regulador 2 de Interferon , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Processamento de Proteína Pós-Traducional , Transcrição Gênica , TransfecçãoRESUMO
beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells. We conclude that the human cells are deficient in a trans-acting factor required for B-IFN gene activation. The level of induction of the beta-IFN gene in HeLa and 143 tk- cells can also be increased by priming with IFN before induction. If IFN priming is carried out in the presence of cycloheximide, a approximately 200-fold increase in induction is observed. We conclude that activation of the beta-IFN gene requires an IFN-inducible factor that is only expressed at low levels in unprimed HeLa and 143 tk- cells.
Assuntos
Genes/efeitos dos fármacos , Interferon Tipo I/genética , Poli I-C/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Cicloeximida/farmacologia , Genes Reguladores/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos , Interferon Tipo I/biossíntese , Hibridização de Ácido Nucleico , Plasmídeos , Timidina Quinase/genéticaRESUMO
Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.