RESUMO
The RNA-specific adenosine deaminase (ADAR1) is an interferon-inducible editing enzyme that converts adenosine to inosine. ADAR1 contains three distinct domains: a N-terminal Z-DNA binding domain that includes two Z-DNA binding motifs; a central double-stranded RNA binding domain that includes three dsRNA binding motifs (dsRBM); and a C-terminal catalytic domain responsible for A-to-I enzymatic activity. The E3L protein of vaccinia virus mediates interferon resistance. E3L, similar to ADAR1, also contains Z-DNA binding and dsRNA binding motifs. To assess the possible role of E3L in modulating RNA editing by ADAR1, we examined the effect of E3L on ADAR1 deaminase activity. Wild-type E3L protein was a potent inhibitor of ADAR1 deaminase enzymatic activity. Analysis of mutant E3L proteins indicated that the carboxy-proximal dsRBM of E3L was essential for antagonism of ADAR1. Surprisingly, disruption of the Z-DNA binding domain of E3L by double substitutions of two highly conserved residues also abolished its antagonistic activity, whereas deletion of the entire Z domain had little effect on the inhibition. With natural neurotransmitter pre-mRNA substrates, E3L weakly inhibited the site-selective editing activity by ADAR1 at the R/G site of the glutamate receptor B subunit (GluR-B) pre-mRNA and the A site of serotonin 2C receptor (5-HT2CR) pre-mRNA; editing of the intronic hotspot (+)60 site of GluR-B was not affected by E3L. These results demonstrate that the A-to-I RNA editing activity of the IFN-inducible adenosine deaminase is impaired by the product of the vaccinia virus E3L interferon resistance gene.
Assuntos
Adenosina Desaminase/metabolismo , Edição de RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/fisiologia , Vaccinia virus/fisiologia , Proteínas Virais/fisiologia , Adenosina/metabolismo , Adenosina Desaminase/genética , Inibidores de Adenosina Desaminase , Inosina/metabolismo , Interferons/farmacologia , Mutação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2',5'-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2'-5'-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-alpha, IFN-beta, and IFN-gamma, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2',5'-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Interferons/farmacologia , Interferons/uso terapêutico , Apoptose/efeitos dos fármacos , Humanos , Interferons/antagonistas & inibidores , Interferons/metabolismo , Receptores de Interferon/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Viroses/tratamento farmacológico , Viroses/genética , Viroses/virologiaRESUMO
Virus infection triggers innate responses to host cells including production of type I interferon (IFN). Since IFN production is also induced by treatment with poly(I:C), viral double-stranded (ds) RNA has been postulated to play a direct role in the process. In the present study, we investigated the effect of dsRNA binding proteins on virus-induced activation of the IFN-beta gene. We found that PACT, originally identified as protein activator for dsRNA-dependent protein kinase (PKR) and implicated in the regulation of translation, augmented IFN-beta gene activation induced by Newcastle disease virus. Concomitantly with the augmented activity of IFN-beta enhancer, increased activity of NF-kappaB and IRF-3 and IRF-7 was observed. For the observed effect, the dsRNA-binding activity of PACT was essential. We identified residues of PACT that interact with a presumptive target molecule to exert its function. Furthermore, PACT colocalized with viral replication complex in the infected cells. Thus the observed effect of PACT is novel and PACT is involved in the regulation of viral replication and results in a marked increase of cellular IFN-beta gene expression.
Assuntos
Proteínas de Transporte/metabolismo , Interferon beta/genética , Vírus da Doença de Newcastle/patogenicidade , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Células HeLa , Humanos , Fator Regulador 3 de Interferon , Fator Regulador 7 de Interferon , Camundongos , Dados de Sequência Molecular , Mutação , NF-kappa B/metabolismo , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Replicação ViralRESUMO
Gamma interferon (IFN-gamma) is an important cytokine in the host defense against infection by viral and microbial pathogens. IFN-gamma induces a variety of physiologically significant responses that contribute to immunity. Treatment of animal cells with IFN-gamma or infection with viral or microbial pathogens leads to changes in the level of expression of several target genes as revealed by DNA microarray analyses. The signaling pathways leading to the induction of IFN-gamma-regulated gene products and, in some cases, their biochemical functions have been defined in exquisite detail. Studies of transgenic mutant mice deficient in proteins of the IFN-gamma response pathway firmly establish the importance of IFN-gamma in immunity.
Assuntos
Infecções Bacterianas/imunologia , Interferon gama/imunologia , Viroses/imunologia , Animais , Expressão Gênica , Imunidade Celular , Interferon gama/genética , Receptores de Interferon/imunologia , Transdução de SinaisRESUMO
The human ADAR1 gene specifies two size forms of RNA-specific adenosine deaminase, an interferon (IFN) inducible approximately 150 kDa protein and a constitutively expressed N-terminally truncated approximately 110 kDa protein, encoded by transcripts with alternative exon 1 structures that initiate from different promoters. We have now identified a new class of ADAR1 transcripts, with alternative 5'-structures and a deduced coding capacity for the approximately 110 kDa protein. Nuclease protection and 5'-rapid amplification of cDNA ends (5'-RACE) revealed five major ADAR1 transcriptional start sites that mapped within the previously identified and unusually large (approximately 1.6 kb) exon 2. These transcripts were observed with RNA from human amnion U cells and placenta tissue. Their abundance was not affected by IFN-alpha treatment of U cells in culture. Transfection analysis identified a functional promoter within human genomic DNA that mapped to the proximal exon 2 region of the ADAR1 gene. Promoter activity was not affected by IFN. These results suggest that transcripts encoding the constitutively expressed approximately 110 kDa form of the ADAR1 editing enzyme are initiated from multiple promoters, including one within exon 2, that collectively contribute to the high basal level of deaminase activity observed in nuclei of mammalian cells.
Assuntos
Adenosina Desaminase/genética , Processamento Alternativo , Regiões Promotoras Genéticas/genética , Regiões 5' não Traduzidas/genética , Células Cultivadas , DNA Complementar/genética , Éxons/genética , Feminino , Regulação da Expressão Gênica , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Ensaios de Proteção de Nucleases , Mapeamento Físico do Cromossomo , Gravidez , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
The RNA-specific adenosine deaminase (ADAR1) and the RNA-dependent protein kinase (PKR) are both interferon-inducible double-stranded (ds) RNA-binding proteins. ADAR1, an RNA editing enzyme that converts adenosine to inosine, possesses three copies of a dsRNA-binding motif (dsRBM). PKR, a regulator of translation, has two copies of the highly conserved dsRBM motif. To assess the functional selectivity of the dsRBM motifs in ADAR1, we constructed and characterized chimeric proteins in which the dsRBMs of ADAR1 were substituted with those of PKR. Recombinant PKR-ADAR1 chimeras retained significant RNA adenosine deaminase activity measured with a synthetic dsRNA substrate when the spacer region between the RNA-binding and catalytic domains of the deaminase was exactly preserved. However, with natural substrates, substitution of the first two dsRBMs of ADAR1 with those from PKR dramatically reduced site-selective editing activity at the R/G and (+)60 sites of the glutamate receptor B subunit pre-RNA and completely abolished editing of the serotonin 2C receptor (5-HT(2C)R) pre-RNA at the A site. Chimeric deaminases possessing only the two dsRBMs from PKR were incapable of editing either glutamate receptor B subunit or 5-HT(2C)R natural sites but edited synthetic dsRNA. Finally, RNA antagonists of PKR significantly inhibited the activity of chimeric PKR-ADAR1 proteins relative to wild-type ADAR1, further demonstrating the functional selectivity of the dsRBM motifs.
Assuntos
Adenosina Desaminase/fisiologia , Edição de RNA , Precursores de RNA , RNA de Cadeia Dupla/metabolismo , Receptores de Glutamato/genética , Receptores de Serotonina/genética , eIF-2 Quinase/fisiologia , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sítios de Ligação , Desaminação , Humanos , Estrutura Terciária de Proteína , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA , Receptor 5-HT2C de Serotonina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Recent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear lambda-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled L(alpha)C with DNA sandwiched between cationic bilayer membranes. The membrane charge density is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled H(II)C) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, phiDOPE = 0.72) and a low transfectant complex (DOTAP/DOPC, (phiDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.
Assuntos
Terapia Genética/métodos , Lipossomos , Plasmídeos , Transfecção/métodos , Animais , Células Cultivadas , DNA Super-Helicoidal/química , Vetores Genéticos/química , Bicamadas Lipídicas/química , Lipossomos/química , Camundongos , Microscopia , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon (IFN). As an extension of our structural characterization of the exon-intron organization of the mouse Pkr gene, we now have isolated and characterized the mouse Pkr promoter region required for IFN-inducible transcription. Transient transfection analyses, using reporter constructs possessing various 5'-flanking fragments of the Pkr gene, led to the identification of a functional IFN-inducible promoter. A single IFN-stimulated response element (ISRE) was present in a minimal 44-nt TATA-less promoter identified by deletion analysis; the 13-nt ISRE differed from previously described ISRE elements in that the 3'-nt was a purine instead of a pyrimidine. The sequence immediately upstream of the ISRE possessed the 15-nt KCS element that was exactly conserved in sequence and position between the mouse and human Pkr promoters. A single gamma IFN-activated sequence (GAS)-like element and multiple recognition sites for factors including NF-kappaB and NF-IL6 involved in responses to various cytokine and hormone signals in inflammatory responses were also present in the 5'-flanking region. Northern blot analysis showed efficient IFN-alpha induced accumulation of 2.4kb, 4.5kb and approx. 6kb Pkr transcripts, but neither IFN-gamma nor IL-6 induced detectable Pkr mRNA accumulation in L cells.
Assuntos
Interferons/farmacologia , eIF-2 Quinase/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA/química , DNA/genética , DNA/isolamento & purificação , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Células L , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The double-stranded RNA-dependent protein kinase PKR is an interferon-inducible enzyme that possesses antiviral and antiproliferative activities. We examined expression of PKR transcripts in human placenta tissue and cultured human amnion U cells. Alternative exon 2 structures were identified and characterized that possess different functional activities. Cloning and sequence analyses of 5'-RACE cDNAs from human placenta established a linkage between exon 1 and three alternative exon 2 structures that constitute, together with part of exon 3, the 5'-untranslated region of the PKR mRNA. The alternative splice variants of exon 2 were designated Ex2alpha (83 nucleotides), Ex2beta (167 nucleotides), and Ex2gamma (401 nucleotides). All three exon 2 variants were present in placenta tissue. However, only the Ex2alpha and Ex2beta forms were detectable in the amnion U cell line. Nuclease protection analysis revealed that the Ex2beta form was slightly more abundant than the Ex2alpha form, in both placenta tissue and U cells. Interferon treatment of U cells increased the level of both Ex2alpha and Ex2beta RNA by approximately 5-fold. The translational activities, measured in a luciferase reporter assay, of RNA transcripts possessing the Ex2alpha and Ex2beta forms of the PKR 5'-UTR were comparable to each other and more efficient than those with the Ex2gamma form.
Assuntos
Regiões 5' não Traduzidas/genética , Processamento Alternativo , Regulação Enzimológica da Expressão Gênica , Variação Genética , Interferon-alfa/farmacologia , eIF-2 Quinase/genética , Âmnio/citologia , Âmnio/enzimologia , Sequência de Bases , Linhagem Celular , Éxons , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Genes Reporter , Humanos , Íntrons , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Placenta/enzimologia , Gravidez , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , eIF-2 Quinase/biossínteseRESUMO
The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine to inosine in cellular pre-mRNAs. The pre-mRNA for the rat serotonin-2C receptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the encoded G-protein-coupled receptor. Measurements of 5-HT2CR pre-mRNA editing in vitro revealed site-specific deamination catalyzed by ADAR1. Three splice site variants, ADAR1-a, -b, and -c, all efficiently edited the A site of 5-HT2CR pre-mRNA, but the D site did not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-stranded (ds) RNA binding motifs present in ADAR1 revealed a different relative importance of the individual dsRNA binding motifs for deamination of the A site of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the 5-HT2CR pre-mRNA was most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 showed significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bulb, and striatum, where the 5-HT2CR pre-mRNA was extensively edited.
Assuntos
Adenosina Desaminase/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interferons/farmacologia , Edição de RNA , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Cadeia Dupla/metabolismo , Receptores de Serotonina/genética , Adenosina Desaminase/biossíntese , Animais , Células COS , Indução Enzimática , Proteínas de Ligação a RNA , Ratos , Receptor 5-HT2C de Serotonina , Transcrição GênicaRESUMO
RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. We have now identified alternative exon 1 structures of human ADAR1 transcripts that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5'-rapid amplification of cDNA ends (RACE) cDNAs from human placenta established a linkage between exon 2 of ADAR1 and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrated that exon 1B-exon 2 transcripts were synthesized in the absence of IFN and were not significantly altered in amount by IFN treatment. By contrast, exon 1A-exon 2 transcripts were IFN inducible. Transient transfection analysis with reporter constructs led to the identification of two functional promoters, designated PC and PI. Exon 1B transcripts were initiated from the PC promoter whose activity in transient transfection reporter assays was not increased by IFN treatment. The 107-nt exon 1B mapped 14.5 kb upstream of exon 2. The 201-nt exon 1A that mapped 5.4 kb upstream of exon 2 was initiated from the interferon-inducible PI promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the ADAR1 gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed approximately 110-kDa ADAR1 protein (exon 1B) or the interferon-induced approximately 150-kDa ADAR1 protein (exon 1A).
Assuntos
Adenosina Desaminase/genética , Processamento Alternativo , Interferons/farmacologia , Transcrição Gênica , Adenosina Desaminase/biossíntese , Âmnio/citologia , Âmnio/metabolismo , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/biossíntese , Indução Enzimática , Éxons , Feminino , Biblioteca Genômica , Humanos , Íntrons , Dados de Sequência Molecular , Placenta/enzimologia , Gravidez , Edição de RNA , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
The double-stranded RNA-specific adenosine deaminase (ADAR1) is inducible by interferon (IFN) and is implicated in the editing of viral RNAs during lytic and persistent infection. We have now isolated and characterized human genomic clones that contain the promoter region required for transcription of the ADAR1 gene. Rapid amplification of cDNA 5'-ends (5'-RACE) identified additional upstream exon 1 sequence that was localized on P1-phage and lambda-phage genomic clones by Southern gel-blot analysis and sequence analysis. A Northern gel-blot analysis using a probe corresponding to the 5'-RACE exon 1 sequence and adjacent exon 2 sequence detected a major RNA transcript of approximately 6.7kb that was IFN-inducible in human amnion U cells. Transient transfection assays, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the ADAR1 gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed a consensus copy of the IFN-Stimulated Response Element (ISRE) involved in IFN inducibility that was flanked by a Kinase Conserved Sequence (KCS)-like element previously found to be unique to the human and mouse PKR gene promoters. A 63-bp minimal promoter fragment possessing the KCS-like and ISRE elements was sufficient to drive IFN-inducible transcription.
Assuntos
Adenosina Desaminase/genética , Interferons/farmacologia , Regiões Promotoras Genéticas/genética , Bacteriófago P1/genética , Bacteriófago lambda/genética , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Humanos , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Deleção de Sequência/genética , TransfecçãoRESUMO
The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA-editing enzyme that catalyzes the deamination of adenosine in double-stranded RNA structures. Three alternative splice-site variants of ADAR1 (ADAR1-a, -b, and -c) occur that possess functionally distinct double-stranded RNA-binding motifs as measured with synthetic double-stranded RNA substrates. The pre-mRNA transcript encoding the B subunit of glutamate receptor (GluR-B) has two functionally important editing sites (Q/R and R/G sites) that undergo selective A-to-I conversions. We have examined the ability of the three ADAR1 splice-site variants to catalyze the editing of GluR-B pre-mRNA at the Q/R and R/G sites as well as an intron hotspot (+60) of unknown function. Measurement of GluR-B pre-mRNA editing in vitro revealed different site-specific deamination catalyzed by the three ADAR1 variants. The ADAR1-a, -b, and -c splice variants all efficiently edited the R/G site and the intron +60 hotspot but exhibited little editing activity at the Q/R site. ADAR1-b and -c showed higher editing activity than ADAR1-a for the R/G site, whereas the intron +60 site was edited with comparable efficiency by all three ADAR1 splice variants. Mutational analysis revealed that the functional importance of each of the three RNA-binding motifs of ADAR1 varied with the specific target editing site in GluR-B RNA. Quantitative reverse transcription-polymerase chain reaction analyses of GluR-B RNA from dissected regions of rat brain showed significant expression and editing at the R/G site in all brain regions examined except the choroid plexus. The relative levels of the alternatively spliced flip and flop isoforms of GluR-B RNA varied among the choroid plexus, cortex, hippocampus, olfactory bulb, and striatum, but in all regions of rat brain the editing of the flip isoform was greater than that of the flop isoform.
Assuntos
Adenosina Desaminase/genética , Interferons/fisiologia , Edição de RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Receptores de Glutamato/genética , Adenosina Desaminase/biossíntese , Processamento Alternativo , Animais , Sequência de Bases , Encéfalo/metabolismo , Células COS , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The PKR protein kinase is an important regulator of viral mRNA translation. A approximately 50-kb gene (Pkr) encodes the human PKR protein that is inducible by interferon (IFN). The Pkr promoter region has a novel 15-bp DNA element designated as KCS required for transcriptional activity that is located 4 bp upstream of a 13-bp IFN-stimulated response element (ISRE) that confers inducibility by type I IFN. We have carried out a systematic analysis of the 5' flanking region of the human Pkr gene to define how the novel KCS element acts to affect basal as well as IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) revealed that nuclear proteins bound selectively to the KCS element in a manner that was not dependent upon either IFN treatment or protein binding at the adjacent ISRE element. KCS protein binding activity in vitro correlated with activation of transcription in vivo in transient transfection assays. Competitionsupershift EMSA assays revealed that multiple proteins were involved in bandshift complex formation with KCS, one of which was identified as factor Sp1. In addition to the positive regulatory domain containing the KCSISRE elements, a negative regulatory domain (NRD) was identified within a 40-bp region positioned approximately 400-bp upstream of the KCSISRE elements. Deletion and substitution mutations indicated that the NRD negatively affected Pkr transcription by a mechanism dependent upon the KCS element. These results define novel positivenegative regulatory domains within the Pkr promoter that function through the KCS element to affect basalIFN-inducible transcription of Pkr.
Assuntos
Interferon-alfa/farmacologia , Regiões Promotoras Genéticas , Ativação Transcricional , eIF-2 Quinase/genética , Sequência de Bases , Mapeamento Cromossômico , Regulação Enzimológica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
RNA-dependent protein kinase PKR is an important regulator of gene expression in interferon (IFN)-treated and virus-infected cells. The 50-kb gene encoding human PKR kinase (pkr) is inducible by IFN. Transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the human pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription. Sequence determination and mutational analysis of the pkr promoter region revealed, in addition to a functional copy of the IFN-stimulated response element (ISRE) responsible for inducibility by type I IFN, a novel 15-bp element required for optimal promoter activity mediated by the ISRE. This element (5' GGGAAGGCGGAGTCC 3'), designated KCS for kinase-conserved sequence, is exactly conserved between the human and mouse pkr promoters in sequence and position relative to the ISRE. We have now carried out an extensive mutational analysis of the 15-bp KCS element. Site-directed mutagenesis was performed, whereby every base pair position within the KCS element was replaced by each of the other three alternatives. Forty-five substitution mutants were analyzed for promoter activity by transient transfection analysis of untreated and IFN-treated human cells. The results establish 5' NNRRRGG(C,A,T)GGRGYYN 3', where R stands for purine and Y stands for pyrimidine, as the consensus sequence for the KCS element, both for basal and for IFN-inducible promoter activity. KCS-binding proteins were detected by electrophoretic mobility shift analysis (EMSA). Competition EMSA established that constitutively expressed nuclear proteins bound the KCS element selectively; KCS protein binding activity correlated with promoter activity in the transient transfection reporter assay.
Assuntos
Interferon-alfa/farmacologia , Ativação Transcricional/efeitos dos fármacos , eIF-2 Quinase/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Sequência Consenso , Primers do DNA/genética , Genes Reporter , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , TransfecçãoRESUMO
The RNA-specific adenosine deaminase (ADAR1, herein referred to as ADAR) is an interferon-inducible RNA-editing enzyme. ADAR catalyzes the C-6 deamination of adenosine in double-stranded (ds) structures present in viral RNAs and cellular pre-mRNAs as well as synthetic dsRNA substrates. ADAR possesses three functionally distinct copies of the highly conserved double-stranded RNA binding R motif (RI, RII, RIII) implicated in the recognition of dsRNA structures within the substrate RNAs. ADAR is also a Z-DNA-binding protein. Two Z-DNA binding motifs (Zalpha and Zbeta) present in ADAR correspond to repeated regions homologous to the N-terminal region of the vaccinia virus E3L protein. Here we describe assay methods for measurement of ADAR enzymatic activity, dsRNA binding activity, and Z-DNA binding activity.
Assuntos
Adenosina Desaminase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Adenosina Desaminase/genética , Animais , Células COS , Proteínas de Ligação a DNA/genética , Vetores Genéticos , Ligação Proteica , Proteínas de Ligação a RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA. PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD. RNA ligands were selected by binding to His6-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches (5'AG3'/3'GA5'), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)2 reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch.
Assuntos
Conformação de Ácido Nucleico , RNA/metabolismo , eIF-2 Quinase/metabolismo , Sequência de Bases , Cromatografia de Afinidade , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA de Cadeia Dupla/metabolismo , Alinhamento de SequênciaRESUMO
The virus-associated VAI RNA of adenovirus is a small highly structured RNA that is required for the efficient translation of cellular and viral mRNAs at late times after infection. VAI RNA antagonizes the activation of the interferon-inducible RNA-dependent protein kinase, PKR, an important regulator of translation. The RNA-specific adenosine deaminase, ADAR, is an interferon-inducible RNA-editing enzyme that catalyzes the site-selective C-6 deamination of adenosine to inosine. ADAR possesses three copies of the highly conserved RNA-binding motif (dsRBM) that are similar to the two copies found in PKR, the enzyme in which the prototype dsRBM motif was discovered. We have examined the effect of VAI RNA on ADAR function. VAI RNA impairs the activity of ADAR deaminase. This inhibition can be observed in extracts prepared from interferon-treated human cells and from monkey COS cells in which wild-type recombinant ADAR was expressed. Analysis of wild-type and mutant forms of VA RNA suggests that the central domain is important in the antagonism of ADAR activity. These results suggest that VAI RNA may modulate viral and cellular gene expression by modulating RNA editing as well as mRNA translation.