RESUMO
RNA molecules play an essential role in many cellular processes, often as components of ribonucleoprotein complexes. Like proteins, RNA molecules adopt sequence-specific secondary and tertiary structures that are essential for function; alteration of these structures therefore provides a means of regulating RNA function. The discovery of DEAD box proteins, a large family of proteins that share several highly conserved motifs and have known or putative ATP-dependent RNA helicase activity, has provoked growing interest in the concept that regulation of RNA function may occur through local unwinding of complex RNA structures.
RESUMO
The nuclear protein p68 (also known as Ddx5) is a prototypic member of the 'DEAD box' family of RNA helicases, which has been shown to be abnormally expressed and modified in colorectal tumors and to function as an important transcriptional regulator. Here, we show that p68 is modified in vivo on a single site (K53) by the small ubiquitin-like modifier-2 (SUMO-2). We demonstrate that the SUMO E3 ligase PIAS1 interacts with p68 and enhances its SUMO modification in vivo. To determine the functional consequences of SUMO modification, we compared the transcriptional activity of p68 and a K53R mutant that could not be SUMO-modified. Our data show that SUMO modification enhances p68 transcriptional repression activity and inhibits the ability of p68 to function as a coactivator of p53. These findings may be explained by the ability of wild type, but not K53R p68, to alter the modification state of chromatin by recruitment of histone deacetylase 1 (HDAC1).
Assuntos
RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Células HeLa , Histona Desacetilase 1 , Humanos , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
The RNA helicases p68 and p72 are highly related members of the DEAD box family of proteins, sharing 90% identity across the conserved core, and have been shown to be involved in both transcription and mRNA processing. We previously showed that these proteins co-localise in the nucleus of interphase cells. In this study we show that p68 and p72 can interact with each other and self-associate in the yeast two-hybrid system. Co-immunoprecipitation experiments confirmed that p68 and p72 can interact in the cell and indicated that these proteins preferentially exist as hetero-dimers. In addition, we show that p68 can interact with NFAR-2, a protein that is also thought to function in mRNA processing. Moreover, gel filtration analysis suggests that p68 and p72 can exist in a variety of complexes in the cell (ranging from approximately 150 to approximately 400 kDa in size), with a subset of p68 molecules being in very large complexes (>2 MDa). The potential to exist in different complexes that may contain p68 and/or p72, together with a range of other factors, would provide the potential for these proteins to interact with different RNA substrates and would be consistent with recent reports implying a wide range of functions for p68/p72.
Assuntos
Adenosina Trifosfatases/metabolismo , Fosfoproteínas , Proteínas Quinases/metabolismo , RNA Helicases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Ligação Competitiva , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , RNA Helicases DEAD-box , Dimerização , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas do Fator Nuclear 90 , Testes de Precipitina , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , RNA Helicases/química , RNA Helicases/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.
Assuntos
DNA Glicosilases , N-Glicosil Hidrolases/metabolismo , Proteínas Quinases , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Clonagem Molecular , RNA Helicases DEAD-box , Primers do DNA , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , RNA Helicases/química , RNA Helicases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The DEAD box RNA helicase, p68, is upregulated in exponentially growing cells and shows cell cycle-dependent changes in nuclear localization. Although some other DEAD box proteins have been implicated in cancer, there have been no reports of any link between p68 status and carcinogenesis. In the present study we have analysed specimens from 50 patients with colorectal adenocarcinomas, including cases in which an adenomatous polyp was also present, by immunohistochemistry and Western blotting. Our data indicate that p68 protein is consistently overexpressed in tumours as compared with matched normal tissue. Examination of the levels of p68 mRNA from both normal and tumour tissue showed no obvious specific increase in p68 mRNA levels in tumours nor any evidence of underlying mutations in the p68 coding region. Interestingly, however, the accumulated p68 appears to be poly-ubiquitylated, suggesting a possible defect in proteasome-mediated degradation in these tumours. This overexpression/ubiquitylation is observed in both pre-invasive and invasive lesions suggesting that the dysregulation of p68 expression occurs early during tumour development. Finally, we demonstrate that ubiquitylation of p68 occurs in cultured cells, thereby providing a model for the molecular analysis of this process and its potential role in tumorigenesis.
Assuntos
Neoplasias Colorretais/enzimologia , Poliubiquitina/metabolismo , Proteínas Quinases/metabolismo , RNA Helicases/metabolismo , Animais , Western Blotting , Células COS , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , RNA Helicases DEAD-box , Células HeLa , Humanos , Imuno-Histoquímica , Peso Molecular , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Proteínas Quinases/genética , RNA Helicases/biossíntese , RNA Helicases/química , RNA Helicases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA. We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity. Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted. These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA. We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , RNA Helicases/metabolismo , RNA Ribossômico 23S/metabolismo , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , RNA Helicases DEAD-box , Escherichia coli/genética , Hidrólise , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , Proteínas de Ligação a RNA/metabolismo , Especificidade por SubstratoRESUMO
The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective.
Assuntos
Enzimas de Restrição do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo I , Escherichia coli/enzimologia , Autorradiografia , Bacteriófago lambda/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Genes Bacterianos , Mutação , Plasmídeos , Biossíntese de Proteínas , Transcrição GênicaRESUMO
The RNA helicase p68 (DDX5) is an established co-activator of the p53 tumour suppressor that itself has a pivotal role in orchestrating the cellular response to DNA damage. Although several factors influence the biological outcome of p53 activation, the mechanisms governing the choice between cell-cycle arrest and apoptosis remain to be elucidated. In the present study, we show that, while p68 is critical for p53-mediated transactivation of the cell-cycle arrest gene p21(WAF1/CIP1), it is dispensable for induction of several pro-apoptotic genes in response to DNA damage. Moreover, p68 depletion results in a striking inhibition of recruitment of p53 and RNA Pol II to the p21 promoter but not to the Bax or PUMA promoters, providing an explanation for the selective effect on p21 induction. Importantly, these findings are mirrored in a novel inducible p68 knockout mouse model in which p68 depletion results in a selective inhibition of p21 induction in several tissues. Moreover, in the bone marrow, p68 depletion results in an increased sensitivity to γ-irradiation, consistent with an increased level of apoptosis. These data highlight a novel function of p68 as a modulator of the decision between p53-mediated growth arrest and apoptosis in vitro and in vivo.
Assuntos
Apoptose/fisiologia , Pontos de Checagem do Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , RNA Helicases DEAD-box/metabolismo , Dano ao DNA/fisiologia , Animais , Western Blotting , Imunoprecipitação da Cromatina , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
The RNA helicase p68 is a potent co-activator of p53-dependent transcription in response to DNA damage. Previous independent studies have indicated that p68 and the Δ133p53 isoforms, which modulate the function of full-length p53, are aberrantly expressed in breast cancers. Here we identify a striking inverse association of p68 and Δ133p53 expression in primary breast cancers. Consistent with these findings, small interfering RNA depletion of p68 in cell lines results in a p53-dependant increase of Δ133p53 in response to DNA damage, suggesting that increased Δ133p53 expression could result from downregulation of p68 and provide a potential mechanistic explanation for our observations in breast cancer. Δ133p53α, which has been shown to negatively regulate the function of full-length p53, reciprocally inhibits the ability of p68 to stimulate p53-dependent transcription from the p21 promoter, suggesting that Δ133p53α may be competing with p68 to regulate p53 function. This hypothesis is underscored by our observations that p68 interacts with the C-terminal domain of p53, co-immunoprecipitates 133p53α from cell extracts and interacts only with p53 molecules that are able to form tetramers. These data suggest that p68, p53 and 133p53α may form part of a complex feedback mechanism to regulate the expression of Δ133p53, with consequent modification of p53-mediated transcription, and may modulate the function of p53 in breast and other cancers that harbour wild-type p53.
Assuntos
Neoplasias da Mama/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
The DEAD-box RNA helicases p68 (DDX5) and p72 (DDX17) have been shown to act as transcriptional co-activators for a diverse range of transcription factors, including oestrogen receptor-alpha (ERalpha). Here, we show that, although both proteins interact with and co-activate ERalpha in reporter gene assays, small interfering RNA-mediated knockdown of p72, but not p68, results in a significant inhibition of oestrogen-dependent transcription of endogenous ERalpha-responsive genes and oestrogen-dependent growth of MCF-7 and ZR75-1 breast cancer cells. Furthermore, immunohistochemical staining of ERalpha-positive primary breast cancers for p68 and p72 indicate that p72 expression is associated with an increased period of relapse-free and overall survival (P=0.006 and 0.016, respectively), as well as being inversely associated with Her2 expression (P=0.008). Conversely, p68 shows no association with relapse-free period, or overall survival, but it is associated with an increased expression of Her2 (P=0.001), AIB-1 (P<0.001) and higher tumour grade (P=0.044). Our data thus highlight a crucial role for p72 in ERalpha co-activation and oestrogen-dependent cell growth and provide evidence in support of distinct but important roles for both p68 and p72 in regulating ERalpha activity in breast cancer.
Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , RNA Helicases DEAD-box/fisiologia , Receptor alfa de Estrogênio/fisiologia , Estrogênios/farmacologia , Transcrição Gênica , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Células COS , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Chlorocebus aethiops , RNA Helicases DEAD-box/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 1 de Receptor Nuclear/fisiologia , Ligação Proteica , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Ativação Transcricional , Células Tumorais CultivadasRESUMO
SUMO (small ubiquitin-related modifier) modification is known to have diverse effects on the activity of transcriptional regulators, often through alterations in their localization or interactions with other factors, and in most of the cases is associated with transcriptional repression. The DEAD-box family of RNA helicases includes a large number of proteins that are involved in various cellular processes. Several members are now known to be multifunctional and their activities are thought to be governed by interactions with other partners, which may be regulated by post-translational modifications. In the present paper, we shall briefly review recent evidence indicating that SUMO modification is important in modulating the activity of two DEAD-box proteins, p68 (Ddx5) and DP103 (Ddx20), which are known to be important transcriptional regulators.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Gênica/fisiologiaRESUMO
We have analyzed the accumulation of viral genomic and messenger RNAs in tissue culture cells during the first 24 hr of acute infection with lymphocytic choriomeningitis virus (LCMV). This has allowed comparison of the relative amounts of the genomic L and S RNAs (both genomic sense and genomic complementary sense) and of nucleoprotein (NP) and glycoprotein precursor (GP-C) mRNAs. Using these techniques NP mRNA was detected simultaneously with genomic S RNA, but the amount of NP mRNA accumulating during this period of infection was higher than that of GP-C mRNA. This is consistent with a model for ambisense RNA transcription and replication proposed by D.D. Auperin, V. Romanowski, M. Galinski and D. H. L. Bishop (J. Virol. 52: 897-904, 1984). The accumulation of S RNA exceeded that of L RNA and, for both L and S RNAs, the amount of genomic sense RNA was higher than that of genomic complementary RNA.
Assuntos
Vírus da Coriomeningite Linfocítica/genética , Transcrição Gênica , Replicação Viral , RNA Viral/genética , Fatores de TempoRESUMO
The hsd genes of Salmonella typhimurium and Salmonella potsdam encode related type I restriction and modification systems designated SB and SP, respectively; the polypeptide encoded by the hsdS gene dictates the DNA sequence recognized. The hsdS genes of the SB and SP systems have a conserved sequence of around 100 base pairs flanked by two nonhomologous (variable) regions of around 500 base pairs. Recombination between the hsdS genes of SB and SP generated a system (SQ) with a different recognition specificity. We have localized the position of the crossover in the central conserved region by analysis of nucleotide sequences. Concomitant with the generation of a new combination of flanking variable regions is the recombination of minor differences in the central conserved region. A polypeptide domain encoded on the 5' side of the crossover dictates recognition of the trinucleotide component of the target sequence, and a second domain, encoded on the 3' side of the crossover, similarly governs recognition of the tetra- or penta-nucleotide component. Our analysis implicates at least parts of the variable regions in the determination of the specificity of interaction between protein and DNA. Furthermore, the trinucleotide components of the recognition sequences of S. typhimurium and Escherichia coli K-12 are identical, and the 5' segments of their hsdS genes are strikingly homologous rather than variable.
Assuntos
Enzimas de Restrição do DNA/genética , Genes Bacterianos , Salmonella/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Genes , Recombinação Genética , Salmonella/enzimologia , Especificidade por SubstratoRESUMO
We have developed an in vitro assay for the lymphocytic choriomeningitis virus (LCMV) RNA-dependent RNA polymerase with ribonucleoprotein complexes extracted from acutely infected tissue culture cells. The RNA products synthesized in vitro corresponded in size to the full-length genomic L and S RNAs and subgenomic NP and GP mRNAs normally produced in vivo during acute LCMV infection. In a temporal analysis spanning the first 72 h of acute infection, the in vitro polymerase activity of ribonucleoprotein complexes was maximal at 16 h and declined significantly at later times. In contrast, the intracellular levels of the viral L protein (the putative polymerase protein) appeared to be maximal at 48 to 72 h postinfection. Our results suggest that the accumulation of L protein correlates with reduced viral replication and transcription at later times in acute infection and may be involved in the transition from acute to persistent LCMV infection.
Assuntos
Vírus da Coriomeningite Linfocítica/genética , RNA Nucleotidiltransferases/metabolismo , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Animais , Western Blotting , Linhagem Celular , Sistema Livre de Células , Cricetinae , Coriomeningite Linfocítica/enzimologia , Coriomeningite Linfocítica/microbiologia , Ribonucleoproteínas/metabolismo , Fatores de Tempo , Proteínas Virais/metabolismoRESUMO
The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Nucleotidiltransferases/metabolismo , Peptidil Transferases/metabolismo , RNA Helicases , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , RNA Helicases DEAD-box , Análise Mutacional de DNA , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Peptidil Transferases/isolamento & purificação , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
We screened Salmonella typhimurium, Citrobacter freundii, Klebsiella pneumoniae, Shigella boydii, and many isolates of Escherichia coli for DNA sequences homologous to those encoding each of two unrelated type I restriction and modification systems (EcoK and EcoA). Both K- and A-related hsd genes were identified, but never both in the same strain. S. typhimurium encodes three restriction and modification systems, but its DNA hybridized only to the K-specific probe which we know to identify the StySB system. No homology to either probe was detected in the majority of E. coli strains, but in C. freundii, we identified homology to the A-specific probe. We cloned this region of the C. freundii genome and showed that it encoded a functional, A-related restriction system whose specificity differs from those of known type I enzymes. Sequences immediately flanking the hsd K genes of E. coli K-12 and the hsd A genes of E. coli 15T- were shown to be homologous, indicating similar or even identical positions in their respective chromosomes. E. coli C has no known restriction system, and the organization of its chromosome is consistent with deletion of the three hsd genes and their neighbor, mcrB.
Assuntos
Enzimas de Restrição do DNA/genética , DNA Bacteriano/genética , Enterobacteriaceae/genética , Escherichia coli/genética , Genes Bacterianos , Citrobacter/genética , Clonagem Molecular , Desoxirribonuclease EcoRI , Enterobacteriaceae/enzimologia , Escherichia coli/enzimologia , Klebsiella pneumoniae/genética , Hibridização de Ácido Nucleico , Salmonella typhimurium/genética , Homologia de Sequência do Ácido Nucleico , Shigella boydii/genética , Especificidade da EspécieRESUMO
The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , RNA Helicases , RNA Ribossômico 23S/metabolismo , Proteínas de Ligação a RNA , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Cromatografia em Gel , RNA Helicases DEAD-box , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Nucleotidiltransferases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The arenavirus genomic L RNA segment represents approximately 70% of the viral genetic material but current understanding of the organization, regulation, and functioning of the viral L products remains limited. Biological studies with reassortant viruses have implicated the L RNA segment in the lethal infection of adult guinea pigs produced by LCMV-WE but no further explanation of the pathogenic process is presently available. We have initiated a detailed molecular analysis of LCMV L products based on construction and characterization of L-specific cDNA clones and synthesis of L-specific hybridization probes. Nucleotide sequencing studies have allowed the derivation of a partial amino acid sequence for a predicted L protein and antisera raised against synthetic peptides have demonstrated an L protein in Western blotting experiments. Using this approach we have identified a single high molecular weight protein (approximately 200,000 Da) in purified virions and in viral ribonucleoprotein complexes extracted from acutely infected tissue culture cells. This L protein is translated from a nonpolyadenylated, genomic complementary L mRNA and potentially represents part or all of the viral RNA-dependent RNA polymerase.
Assuntos
Vírus da Coriomeningite Linfocítica/genética , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA/genética , Genes Virais , Técnicas de Imunoadsorção , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/imunologiaRESUMO
P72, a novel human member of the DEAD box family of putative RNA-dependent ATPases and ATP-dependent RNA helicases was isolated from a HeLa cDNA library. The predicted amino acid sequence of p72 is highly homologous to that of the prototypic DEAD box protein p68. In addition to the conserved core domains characteristic of DEAD box proteins, p72 contains several N-terminal RGG RNA-binding domains and a serine/glycine rich C-terminus likely involved in mediating protein-protein interactions. A p72-specific probe detects two mRNAs of approximately 5300 and 9300 bases which, although ubiquitously expressed, show variability in their expression levels in different tissues. Purified recombinant p72 exhibits ATPase activity in the presence of a range of RNA moieties. Immunocytochemical studies of p68 and p72 show that these proteins localise to similar locations in the nucleus of HeLa cells, suggesting their involvement in a nuclear process.
Assuntos
Adenosina Trifosfatases/genética , Proteínas Nucleares/genética , Proteínas Quinases , RNA Helicases , Proteínas de Ligação a RNA/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , RNA Helicases DEAD-box , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismoRESUMO
The DEAD box protein, p68, is an established RNA-dependent ATPase and RNA helicase in vitro, but neither the physiological function of this protein nor the macromolecules with which it interacts are known. Using a yeast two-hybrid screen, we identified the nucleolar protein, fibrillarin, as a protein that interacts with p68. Coimmunoprecipitation experiments confirmed that p68 and fibrillarin can form complexes in cellular extracts, and deletion analysis identified regions in each protein responsible for mediating the interaction. Immunofluorescence studies using confocal microscopy revealed that, in interphase cells, while fibrillarin is predominantly nucleolar, p68 shows a diffuse granular nuclear staining but is largely excluded from the nucleoli. Strikingly, both proteins colocalize in nascent nucleoli during late telophase. These data are consistent with a role for p68 either in postmitotic nucleolar reassembly or in the activation of ribosomal DNA transcription/preribosomal RNA processing during telophase and suggest that differential subnuclear compartmentalization may be a mechanism by which interaction of p68 with fibrillarin is regulated in the cell.