RESUMO
The SR (Ser-Arg) proteins are a family of nuclear factors that play multiple important roles in splicing of mRNA precursors in metazoan organisms, functioning in both constitutive and regulated splicing. Certain of these functions are redundant, such that any single SR proteins will suffice, but other functions are unique and are specific to a given family member. A number of studies during the past year have investigated the basis for SR protein specificity.
Assuntos
Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Humanos , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de Serina-ArgininaRESUMO
Polyadenylation of messenger RNA precursors requires a complex protein machinery that is closely integrated with the even more complex transcriptional apparatus. Here a polyadenylation factor, CstF-50 (cleavage stimulation factor), is shown to interact in vitro and in intact cells with a nuclear protein of previously unknown function, BRCA1-associated RING domain protein (BARD1). The BARD1-CstF-50 interaction inhibits polyadenylation in vitro. BARD1, like CstF-50, also interacts with RNA polymerase II. These results indicate that BARD1-mediated inhibition of polyadenylation may prevent inappropriate RNA processing during transcription, perhaps at sites of DNA repair, and they reveal an unanticipated integration of diverse nuclear events.
Assuntos
Proteínas de Transporte/metabolismo , Poli A/metabolismo , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Anticorpos Monoclonais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Núcleo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Rad51 Recombinase , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos , Dedos de Zinco , Fatores de Poliadenilação e Clivagem de mRNARESUMO
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
Nearly every eukaryotic mRNA contains a poly(A) tail at its 3' end. Unlike the factors that catalyze other RNA processing reactions, the basal polyadenylation machinery consists entirely of proteins. As with other steps in gene expression, however, this machinery is complex, consisting of multiple separable factors. Two multisubunit factors are required for specification of the poly(A) site and formation of a stable protein-RNA complex. Two additional proteins, as well as poly(A) polymerase, join the complex and are required for cleavage of the pre-mRNA and synthesis of the poly(A) tail, respectively. These and other properties suggest at least a superficial similarity between transcription initiation and polyadenylation.
Assuntos
Poli A/metabolismo , Proteínas/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Animais , Catálise , Humanos , HidróliseRESUMO
Recent studies have identified additional cis-acting sequence elements that can either positively or negatively regulate pre-mRNA splicing. Trans-acting protein factors involved in tissue-specific splicing regulation have been characterized. The functions of splicing factors have been explored in vivo and certain splicing factors have been demonstrated to be essential for proper development and cell viability. Kinases that can specifically phosphorylate splicing factors have been identified and phosphorylation has been shown to influence the activity of splicing factors, consistent with a role for phosphorylation in splicing regulation.
Assuntos
Precursores de RNA , Splicing de RNA , Animais , Humanos , FosforilaçãoRESUMO
Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery. We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the polyadenylation machinery.
Assuntos
Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Fatores de Poliadenilação e Clivagem de mRNARESUMO
We have employed gene targeting coupled with conditional expression to construct a chicken DT40 cell line in which a tetracycline (Tet)-repressible promoter is exclusively responsible for expression of cTAF(II)31, a histone-like TAF(II) residing in both the transcription factor TFIID and the histone acetylase complex PCAF/SAGA. Tet addition resulted in rapid loss of cTAF(II)31 mRNA and protein, eventually leading to apoptotic cell death. Significantly, five of six other TAF(II)s tested were also rapidly depleted, but levels of the TATA binding protein and subunits of PCAF/SAGA were at most modestly compromised. Strikingly, pulse-labeling experiments indicate that total poly(A)(+) mRNA transcription was not significantly reduced after cTAF(II)31 depletion, and steady-state levels of several specific transcripts remained the same or decreased only mildly. Moreover, activation of c-fos transcription following serum starvation occurred efficiently in the absence of cTAF(II)31. These data, which contrast with comparable studies in yeast, strongly suggest that cTAF(II)31 and perhaps other TAF(II)s are not essential for general mRNA transcription in DT40 cells. We propose that this is due to extensive functional degeneracy in the highly complex metazoan transcriptional machinery.
Assuntos
Evolução Molecular , Proteínas de Saccharomyces cerevisiae , Fatores Associados à Proteína de Ligação a TATA , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição TFIID , Transcrição Gênica , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular/efeitos dos fármacos , Galinhas , Clonagem Molecular , Meios de Cultura , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-Box , Tetraciclina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.
Assuntos
Poli A/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Sítios de Ligação , Sequência Consenso , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Saccharomyces cerevisiae , Fatores de Poliadenilação e Clivagem de mRNARESUMO
To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.
Assuntos
Splicing de RNA , RNA Mensageiro/genética , Vírus 40 dos Símios/genética , Antígenos Virais de Tumores/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Íntrons , Mutação , RNA Viral/genéticaRESUMO
To determine whether a specific nucleotide sequence is required to direct polyadenylation of a simian virus 40 early pre-mRNA in a soluble HeLa whole-cell lysate, we constructed a series of rearranged and deleted DNA templates, transcribed them in vitro, and determined whether the resultant RNAs could be polyadenylated when incubated in whole-cell lysate. When a 237-base-pair DNA fragment encoding the 3' end of the simian virus 40 early pre-mRNA was transferred to recombinant plasmids encoding RNAs that were not substrates for polyadenylation, the resultant RNAs could now be polyadenylated efficiently. In one case, the chimeric RNA was polyadenylated even more efficiently than was the original simian virus 40 early transcript. Analysis of the RNAs produced from the deletion mutant templates revealed that only RNAs containing at least one copy of the AAUAAA sequence situated near the 3' end and implicated in 3'-end formation and polyadenylation in vivo could be polyadenylated in vitro. Surprisingly, this sequence directed polyadenylation of pre-mRNAs not only when near the RNA 3' end, i.e., 50 nucleotides or less away, but also when the 3' end was situated over 400 nucleotides downstream. Thus, our results show that a polyadenylic acid polymerase activity in HeLa lysates can recognize a specific nucleotide sequence in pre-mRNA and then, in the absence of the nucleolytic cleavage that presumably occurs in vivo, locate the RNA 3' end and use it as a primer for polyadenylic acid synthesis.
Assuntos
Precursores de Ácido Nucleico/análise , Poli A/metabolismo , RNA Mensageiro/análise , Sequência de Bases , Deleção Cromossômica , Enzimas de Restrição do DNA/metabolismo , Células HeLa , Humanos , Poli A/análise , RNA/análise , Precursores de RNA , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have previously been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, we isolated the mouse PAP gene and an intronless pseudogene from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which we call PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive. We discuss the possible significance of this complex expression pattern.
Assuntos
Processamento Alternativo , Isoenzimas/biossíntese , Polinucleotídeo Adenililtransferase/biossíntese , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae , Sequência de Bases , Sítios de Ligação , Encéfalo/enzimologia , Bovinos , Primers do DNA , Éxons , Biblioteca Gênica , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Serina , Spodoptera , Treonina , Transfecção , XenopusRESUMO
We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region 1a gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region 1a protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region 1a activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.
Assuntos
Adenovírus Humanos/genética , Regulação da Expressão Gênica , Genes Virais , Proteínas Oncogênicas Virais/fisiologia , Regiões Promotoras Genéticas , Proteínas Precoces de Adenovirus , Sequência de Bases , Linhagem Celular , Elementos Facilitadores Genéticos , Células HeLa , Humanos , Transcrição GênicaRESUMO
To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Proteínas de Homeodomínio , Regiões Promotoras Genéticas , Fatores de Transcrição , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Feminino , Fertilização , Masculino , Dados de Sequência Molecular , Mapeamento de NucleotídeosRESUMO
A region in the adenovirus major late promoter (MLP) containing a CCAAT consensus sequence can direct transcription termination of RNA polymerase II, a mechanism that possibly prevents transcriptional interference from upstream genes. Using a chimeric plasmid template that contains the MLP directing expression of the simian virus 40 early region, we showed that an inserted oligonucleotide containing only 13 base pairs of MLP sequences, including the CCAAT box, is capable of inducing transcription termination in an orientation-dependent, position-independent manner. Point mutations within the CCAAT-specific protein-binding site abolished this effect, while a base substitution outside of this region did not affect termination. These data suggest that termination is mediated by a CCAAT box-binding protein. Several other transcription factor-binding sites do not, however, cause termination, suggesting that this may be a relatively specific property of a CCAAT-binding protein.
Assuntos
Adenovírus Humanos/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transcrição Gênica , Sequência de Bases , Quimera , Dados de Sequência Molecular , Mutação , Plasmídeos , Sequências Reguladoras de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.
Assuntos
Precursores de Ácido Nucleico/metabolismo , Poli A/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vírus 40 dos Símios/genética , Núcleo Celular/metabolismo , Células HeLa/metabolismo , Magnésio/farmacologia , Capuzes de RNA/metabolismo , Precursores de RNA , Espermidina/farmacologia , Moldes GenéticosRESUMO
We showed previously that a branch site mutation in simian virus 40 early pre-mRNA that prevented small t antigen mRNA splicing could be efficiently suppressed by a compensatory mutation in a coexpressed U2 small nuclear (sn) RNA gene. We have now generated second-site mutations in this suppressor gene to investigate regions of U2 RNA required for function. A number of mutations in a putative stem at the 5' end of the molecule inhibited splicing, indicating that bases in this region are important for activity. However, several lines of evidence suggested that formation of the entire stem is not essential for splicing. Indeed, mutations that strengthen the stem actually inhibited splicing, and evidence that this prevents a required base-pairing interaction with U6 snRNA is presented. These results suggest that the relative stabilities of competing intra- and intermolecular base-pairing interactions play an important role in the splicing reaction. Mutations in a conserved single-stranded region immediately 3' to the branch site recognition sequence all inhibited splicing, indicating that this region is required for U2 function, although its exact role remains unknown. Finally, two mutations in the loop of stem IV at the 3' end of the molecule, which destroy the binding site of U2 sn ribonucleoprotein B", prevented small t splicing; this finding contrasts with previous studies which utilized different assay systems. Analysis of the accumulation and subcellular localization of all of the mutant RNAs showed that they were similar to those of the parental suppressor U2 RNA, indicating that the effects observed indeed reflect defects in splicing.
Assuntos
Splicing de RNA , RNA Nuclear Pequeno/genética , Sequência de Bases , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Saccharomyces cerevisiae/genética , TransfecçãoRESUMO
Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.
Assuntos
Fatores Biológicos/metabolismo , Splicing de RNA , Proteínas Virais/metabolismo , Proteínas Precoces de Adenovirus , Adenovírus Humanos/genética , Ligação Competitiva , Células HeLa , Humanos , Proteínas Oncogênicas Virais/genética , Cloreto de Potássio/farmacologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Vírus 40 dos Símios/genéticaRESUMO
The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery. Here we describe experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter. We first compared Eve activities in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II. In each case, equivalent and very efficient levels of repression were observed, indicating that no factors other than those in the minimal system are required for repression. We then show that Eve can function efficiently when its recognition sites are far from the promoter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain. Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to facilitate interaction with TBP. Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, we show that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide. We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , TATA Box , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Drosophila , Proteínas de Homeodomínio/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteína de Ligação a TATA-Box , Moldes Genéticos , Fator de Transcrição TFIIDRESUMO
Vertebrate poly(A) polymerase (PAP) contains a catalytic domain and a C-terminal Ser-Thr-rich regulatory region. Consensus and nonconsensus cyclin-dependent kinase (cdk) sites are conserved in the Ser-Thr-rich region in vertebrate PAPs. PAP is phosphorylated by cdc2-cyclin B on these sites in vitro and in vivo and is inactivated by hyperphosphorylation in M-phase cells, when cdc2-cyclin B is active. In the experiments described here, we undertook a genetic approach in chicken DT40 cells to study the function of PAP phosphorylation. We found that PAP is highly conserved in chicken and is essential in DT40 cells. While cells could tolerate reduced levels of PAP, even modest overexpression of either wild-type PAP or a mutant PAP with two consensus cdk sites mutated (cdk- PAP) was highly deleterious and at a minimum resulted in reduced growth rates. Importantly, cells that expressed cdk- PAP had a significantly lower growth rate than did cells that expressed similar levels of wild-type PAP, which was reflected in increased accumulation of cells in the G0-G1 phase of the cell cycle. We propose that the lower growth rate is due to the failure of hyperphosphorylation and thus M-phase inactivation of cdk- PAP.
Assuntos
Quinases Ciclina-Dependentes , Polinucleotídeo Adenililtransferase/biossíntese , Polinucleotídeo Adenililtransferase/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Bovinos , Divisão Celular , Linhagem Celular , Galinhas , Sequência Conservada , Ciclina B/metabolismo , Quinase 5 Dependente de Ciclina , Éxons , Heterozigoto , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Polinucleotídeo Adenililtransferase/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ranidae , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transfecção , VertebradosRESUMO
The Drosophila homeodomain protein Even-skipped (Eve) has previously been shown to function as a sequence-specific transcriptional repressor, and in vitro and in vivo experiments have shown that the protein can actively block basal transcription. However, the mechanism of repression is not known. Here, we present evidence establishing a direct interaction between Eve and the TATA-binding protein (TBP). Using cotransfection assays with minimal basal promoters whose activity can be enhanced by coexpression of TBP, we found that Eve could efficiently block, or squelch, this enhancement. Squelching did not require Eve DNA-binding sites on the reporter plasmids but was dependent on the presence of the Eve repression domain. Further support for an in vivo interaction between the Eve repression domain and TBP was derived from a two-hybrid-type assay with transfected cells. Evidence that Eve and TBP interact directly was provided by in vitro binding assays, which revealed a specific protein-protein interaction that required an intact Eve repression domain and the conserved C terminus of TBP. The Eve homeodomain was also required for these associations, suggesting that it may function in protein-protein interactions. We also show that a previously characterized artificial repression region behaves in a manner similar to that of the Eve repression domain, including its ability to squelch TBP-enhanced expression in vivo and to bind TBP specifically in vitro. Our results suggest a model for transcriptional repression that involves an interaction between Eve and TBP.