RESUMO
Forkhead-box domain (Fox) containing family members are known to play a role in neocorticogenesis and have also been associated with disorders on the autism spectrum. Here we show that a single RNA-binding protein, Hu antigen R (HuR), dictates translation specificity of bound mRNAs and is sufficient to define distinct Foxp-characterized subpopulations of neocortical projection neurons. Furthermore, distinct phosphorylation states of HuR differentially regulate translation of Foxp mRNAs in vitro. This demonstrates the importance of RNA binding proteins within the framework of the developing brain and further confirms the role of mRNA translation in autism pathogenesis.
Assuntos
Proteína Semelhante a ELAV 1/fisiologia , Fatores de Transcrição Forkhead/biossíntese , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neurogênese/genética , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Transtorno do Espectro Autista/genética , Imunoprecipitação da Cromatina , Feminino , Fatores de Transcrição Forkhead/genética , Idade Gestacional , Masculino , Camundongos , Neocórtex/embriologia , Proteínas do Tecido Nervoso/genética , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genéticaRESUMO
Removal of the mRNA 5' cap is an important step in the regulation of mRNA stability. mRNAs are degraded by at least two distinct exonucleolytic decay pathways, one from the 5' end, and the second from the 3' end. Two major cellular decapping enzymes have been identified, and each primarily functions in one of the two decay pathways. The Dcp2 decapping enzyme utilizes capped mRNA as substrate and hydrolyses the cap to release m(7)GDP (N7-methyl GDP), while a scavenger decapping enzyme, DcpS, utilizes cap dinucleotides or capped oligonucleotides as substrate and releases m(7)GMP (N7-methyl GMP). In this review, we will highlight the function of different decapping enzymes and their role in mRNA turnover.
Assuntos
Capuzes de RNA/metabolismo , Estabilidade de RNA , Animais , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Capuzes de RNA/químicaRESUMO
Mechanistic understanding of mammalian mRNA turnover remains incomplete. We demonstrate that the 3' to 5' exoribonuclease decay pathway is a major contributor to mRNA decay both in cells and in cell extract. An exoribonuclease-dependent scavenger decapping activity was identified that follows decay of the mRNA and hydrolyzes the residual cap. The decapping activity is associated with a subset of the exosome proteins in vivo, implying a higher-order degradation complex consisting of exoribonucleases and a decapping activity, which together coordinate the decay of an mRNA. These findings indicate that following deadenylation of mammal mRNA, degradation proceeds by a coupled 3' to 5' exoribonucleolytic activity and subsequent hydrolysis of the cap structure by a scavenger decapping activity.
Assuntos
Endorribonucleases , Exorribonucleases/metabolismo , Capuzes de RNA/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Animais , Fracionamento Celular , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Células K562 , Mamíferos , Complexos Multienzimáticos , Proteínas de Ligação ao Cap de RNA , Estabilidade de RNA , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoAssuntos
Células Eucarióticas/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Artefatos , Sítios de Ligação/fisiologia , Genes , Vetores Genéticos , Humanos , Fatores de Iniciação de Peptídeos/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Reprodutibilidade dos Testes , Projetos de Pesquisa , Ribossomos/metabolismoRESUMO
CD154 expression is regulated throughout a time course of CD3-dependent T cell activation by differential mRNA decay. To understand the molecular basis of the "stability" phase of this pathway, experiments were conducted to identify sequences and specific complexes important in this regulation. Gel retardation assays using extracts from both Jurkat T cells and CD3-activated CD4(+) T cells revealed a major complex (complex I) that bound a 65-bp highly CU-rich region of the CD154 3' untranslated region. The specificity of the CU-rich element for complex-I formation was confirmed by disruption of this complex by oligo(dCT) competition. Formation of complex I strongly correlated with CD154 mRNA stability across a time course of T cell activation. UV cross-linking identified a major oligo(dCT)-sensitive species at approximately 90 kDa that showed induced and increased expression in extracts from 24- and 48-hr anti-CD3-activated T cells, respectively. This protein was absent in equivalent extracts from resting or 2-h-activated T cells. Using an in vitro decay assay, we found that a CD154-specific transcript was more rapidly degraded in 2-h-activated extract and stabilized in the 24- and 48-h extracts compared to extracts from resting T cells. Disruption of complex I resulted in the rapid decay of a CD154-specific transcript demonstrating a functional role for complex I in mRNA stabilization in vitro. These studies support a model of posttranscriptional regulation of CD154 expression being controlled in part by the interaction of a poly(CU)-binding complex with a specific sequence in the 3' untranslated region.
Assuntos
Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Ligante de CD40/genética , Ativação Linfocitária/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Bases , Ligação Competitiva/genética , Ligação Competitiva/imunologia , Complexo CD3/farmacologia , Ligante de CD40/metabolismo , Células Clonais , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Desnaturação de Ácido Nucleico , Sondas de Oligonucleotídeos/metabolismo , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Mensageiro/isolamento & purificação , Proteínas de Ligação a RNA/metabolismoRESUMO
We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable alpha-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated alpha-globin 3' untranslated region (3'UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3'UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated alpha-globin 3'UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the alphaCP mRNA stability protein. Sequestration of alphaCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of alphaCP to the alpha-globin 3'UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by alphaCP, implying that a stable higher-order structural network is involved in stabilization of the alpha-globin mRNA. Upon deadenylation, the interaction of PABP with alphaCP would be disrupted, rendering the alpha-globin 3'UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.
Assuntos
Endorribonucleases/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Citosina/metabolismo , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Células Precursoras Eritroides/enzimologia , Globinas/metabolismo , Humanos , Camundongos , Plasmídeos/metabolismo , Poli A/metabolismo , Proteínas de Ligação a Poli(A) , Ligação Proteica , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
Stability of the human alpha-globin mRNA is conferred by a ribonucleoprotein complex termed the alpha-complex, which acts by impeding deadenylation. Using our recently devised in vitro decay assay, we demonstrate that the alpha-complex also functions by protecting the 3'-untranslated region (3'-UTR) from an erythroid-enriched, sequence-specific endoribonuclease activity. The cleavage site was mapped to a region protected by the alpha-complex and is regulated by the presence of the alpha-complex. Similar endoribonuclease cleavage products were also detected in erythroid cells expressing an exogenous alpha-globin gene. Nucleotide substitution of the target sequence renders the RNA refractory to the endoribonuclease activity. Insertion of the target sequence onto a heterologous RNA confers sequence-specific cleavage on the chimeric RNA, demonstrating the sequence specificity of this activity. We conclude that the alpha-complex stabilizes the alpha-globin mRNA in erythroid cells by a multifaceted approach, one aspect of which is to protect the 3'-UTR from specific endoribonuclease cleavage.
Assuntos
Endorribonucleases/metabolismo , Globinas/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas/genética , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Quimera , Códon de Terminação , Humanos , Leucemia Eritroblástica Aguda , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Especificidade por Substrato , Transfecção , Células Tumorais CultivadasRESUMO
Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions.
Assuntos
Northern Blotting/métodos , Técnicas Genéticas , Ribonucleoproteínas Nucleares Heterogêneas , Proteínas de Membrana , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glutationa Transferase/metabolismo , Hemoglobinas/genética , Humanos , Células K562 , Modelos Genéticos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , RNA Mensageiro/isolamento & purificação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Tetraspanina 28RESUMO
The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. alpha-Globin mRNA stability is dictated by sequences in the 3' untranslated region (3'UTR) which form a specific ribonucleoprotein complex (alpha-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, alphaCP1 and alphaCP2. Using an in vitro-transcribed and polyadenylated alpha-globin 3'UTR, we have devised an in vitro mRNA decay assay which reproduces the alpha-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the alpha-complex by sequestration of alphaCP1 and alphaCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the alpha-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between alphaCP1 and alphaCP2 with PABP suggests that the alpha-complex can directly interact with PABP. Therefore, the alpha-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.
Assuntos
Proteínas de Ligação a DNA , Globinas/genética , Ribonucleoproteínas Nucleares Heterogêneas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição , Regiões 3' não Traduzidas , Animais , Bovinos , Células HeLa , Humanos , Células K562 , Proteínas de Ligação a Poli(A) , Proteínas de Ligação a RNA/genéticaRESUMO
Regulation of mRNA turnover is a critical control mechanism of gene expression and is influenced by ribonucleoprotein (RNP) complexes that form on cis elements. All mRNAs have an intrinsic half-life and in many cases these half-lives can be altered by a variety of stimuli that are manifested through the formation or disruption of an RNP structure. The stability of alpha-globin mRNA is determined by elements in the 3' untranslated region that are bound by an RNP complex (alpha-complex) which appears to control the erythroid-specific accumulation of alpha-globin mRNA. The alpha-complex could consist of up to six distinct proteins or protein families. One of these families is a prominent polycytidylate binding activity which consists of two highly homologous proteins, alpha-complex proteins 1 and 2 (alphaCP1 and alphaCP2). This article focuses on various methodologies for the detection and manipulation of alphaCP1 and alphaCP2 binding to RNA and details means of isolating and characterizing mRNA bound by these proteins to study mRNA turnover and its regulation.
Assuntos
Proteínas de Ligação a DNA , Globinas/genética , Ribonucleoproteínas Nucleares Heterogêneas , Poli C/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Ribonucleoproteínas/metabolismo , Fatores de Transcrição , Regiões 3' não Traduzidas , Meia-Vida , Humanos , Células K562 , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2. Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex. With the yeast two-hybrid screen, a second alpha-complex protein was identified. This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. We refer to these proteins as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex. The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA.
Assuntos
Proteínas de Ligação a DNA , Globinas/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Leucemia Eritroblástica Aguda , Substâncias Macromoleculares , Complexos Multiproteicos , Poli C/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Accumulation of globin mRNAs during erythroid differentiation is dependent on their extraordinary stability. The longevity of human alpha-globin mRNA is associated with a ribonucleoprotein complex (alpha-complex) formed on the 3' untranslated region (3'UTR). One or more of the proteins within this alpha-complex contain strong polycytosine [poly(C)] binding (alpha PCB) activity. In the present report we purify alpha PCB activity from human erythroid K562 cells. Although not able to bind the alpha-globin 3'UTR directly, alpha PCB activity is sufficient to complement alpha-complex formation in a cytosolic extract depleted of poly(C) binding activity. Peptide microsequencing demonstrates that alpha PCB activity contains two structurally related poly(C) binding proteins. These two proteins, alpha-complex protein (alpha CP)-1 and -2, have an overall structural identity of 80% and contain three repeats of the K homology (KH) domain which is found in a subset of RNA binding proteins. Epitope-tagged recombinant alpha CP-1 and alpha CP-2 expressed in cells are each incorporated into the alpha-complex. We conclude that alpha CP-1 and alpha CP-2, members of the KH domain RNA binding protein family, are involved in formation of a sequence-specific alpha-globin mRNP complex associated with alpha-globin mRNA stability. As such this represents the first example of a specific function for this class of proteins and suggests potential roles for other members of this protein family.
Assuntos
Globinas/biossíntese , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Leucemia Eritroblástica Aguda , Dados de Sequência Molecular , Fases de Leitura Aberta , Poli C/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificação , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.
Assuntos
Globinas/biossíntese , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Cromatografia de Afinidade , Primers do DNA , Fibroblastos , Globinas/genética , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/genética , Humanos , Leucemia Eritroblástica Aguda , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Mutação Puntual , Poli C/metabolismo , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , RNA Mensageiro/isolamento & purificação , Proteínas de Ligação a RNA/isolamento & purificação , Ribonucleoproteínas/isolamento & purificação , Moldes Genéticos , TransfecçãoRESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.
Assuntos
RNA Nuclear Heterogêneo/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , DNA , Eletroforese em Gel de Poliacrilamida , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo U , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Plasmídeos , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Transcrição GênicaRESUMO
The immortalized rat calvarial bone cell line RCT-1 responds to treatment with retinoic acid (RA) by increased expression of osteoblast phenotype-related features, including the induction of liver/bone/kidney alkaline phosphatase (ALP) activity. ALP mRNA could not be demonstrated in unstimulated cells, but was first detected in cells treated for 6 h with 1 microM RA. Cycloheximide failed to block the RA induction of ALP mRNA, indicating that de novo protein synthesis was not a requirement for the RA effect and that the ALP gene may be a direct target for RA action. This was confirmed by nuclear run-on assays, which demonstrated a 2.5-fold increase in the abundance of ALP transcripts after 6 h of RA treatment. To determine whether the RA responsiveness was mediated by a specific segment of the ALP promoter, RCT-1 cells were transfected with a series of plasmids containing deletions of the 5'-flanking sequence of the human ALP gene fused to the chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured in cells cultured in the presence of RA or vehicle. All but the smallest construct, which contained 44 basepairs up-stream of the initiation of transcription, were found to mediate a 2- to 3-fold increase in the expression of CAT activity in response to RA. Furthermore, when the region -108 to -45 of the human ALP gene was inserted into the expression vector pBLcat2, in a position immediately up-stream of the herpes simplex virus thymidine kinase promoter, the construct was found to mediate a 2-fold enhancement of CAT activity in response to RA. In gel retardation assays, a major band was present corresponding to the formation of a complex between the 32P-labeled probe containing the -108 to -45 sequence and proteins present in nuclear extracts of RCT-1 cells stimulated for 3 h with RA. These data suggest that the sequence of 64 basepairs (-108 to -45) 5' to the transcription start site is involved in the RA inducibility of the human ALP gene.
Assuntos
Fosfatase Alcalina/genética , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Sequência de Bases , Osso e Ossos , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cicloeximida/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteonectina/genética , Pró-Colágeno/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , TATA Box , TransfecçãoRESUMO
It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.
Assuntos
Linfócitos B/citologia , Proteínas de Ligação a DNA/fisiologia , Elementos Facilitadores Genéticos , Cadeias Pesadas de Imunoglobulinas/genética , Proteínas Repressoras , Fatores de Transcrição , Células 3T3 , Animais , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica , Proteína 1 Inibidora de Diferenciação , Camundongos , Conformação Proteica , Células-Tronco/citologiaRESUMO
Osteoblasts express high levels of liver/bone/kidney alkaline phosphatase (LBK AP), an enzyme critical for bone formation. Other tissues and cell types generally express much lower levels of LBK AP and correspondingly lower levels of mRNA. In light of our early observations that the human LBK AP promoter is expressed equally when transfected into a variety of different cells, we have carried out a detailed study of LBK AP gene expression in Saos-2 cells which are osteoblast-derived and express high levels of LBK AP mRNA, and in HepG2 hepatoblastoma cells which express LBK AP mRNA at levels which are approximately 1000-fold lower. Our results indicate that both of these cells utilize the same promoter sequences to initiate transcription of their LBK AP genes at roughly the same rates. Moreover, the stability of cytoplasmic LBK AP mRNA is equal in both cell types. The lack of any apparent buildup of unspliced precursor mRNA in the nucleus of HepG2 cells leads us to the conclusion that splicing (and nuclear export) is equivalent. It is therefore likely that differential expression is controlled at a very early step post-transcription, possibly by sequences that destabilize the nascent RNA in HepG2 cells. We reason that these destabilizing sequences are located in the gene's introns because a transfected LBK AP minigene, comprised of the full length cDNA and flanking sequences, is expressed efficiently in both cell types.
Assuntos
Fosfatase Alcalina/genética , Regulação Enzimológica da Expressão Gênica , Northern Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Humanos , Técnicas In Vitro , Osteoblastos/fisiologia , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
We have carried out an analysis of the promoter for the human liver/bone/kidney alkaline phosphatase (LBK AP) gene. Using transient transfection assays, the intact promoter directs equal expression of a linked cat gene in Saos-2 cells (osteoblast-derived cells which express very high levels of endogenous LBK AP mRNA) and in HeLa and HepG2 cells (which express low levels of endogenous message). The activity of the transfected promoter apparently mimics the true in vivo situation since nuclear run-on assays employing Saos-2 and HeLa cells indicate that the endogenous gene is transcribed at approximately the same rate in these two cell types. Transfections of a series of 5' deletion mutants indicate that promoter activity is dependent on multiple motifs, which possibly include several putative Sp1 binding sites and a TATA box. The LBK AP promoter also directs accurate transcription initiation in HeLa whole cell extracts and in vitro activities of the 5' deletion mutants also suggest that the promoter utilizes multiple motifs.
Assuntos
Fosfatase Alcalina/genética , Osso e Ossos/enzimologia , Isoenzimas/genética , Rim/enzimologia , Fígado/enzimologia , Regiões Promotoras Genéticas , Carcinoma Hepatocelular , Linhagem Celular , Núcleo Celular/metabolismo , Células HeLa/enzimologia , Humanos , Neoplasias Hepáticas , Osteossarcoma , Plasmídeos , Mapeamento por Restrição , Transcrição Gênica , Células Tumorais Cultivadas/enzimologiaRESUMO
Activity of the immunoglobulin heavy and kappa light chain gene enhancers depends on a complex interplay of ubiquitous and developmentally regulated proteins. Two complementary DNAs were isolated that encode proteins, denoted ITF-1 and ITF-2, that are expressed in a variety of cell types and bind the microE5/kappa 2 motif found in both heavy and kappa light chain enhancers. The complementary DNAs are the products of distinct genes, yet both ITF-1 and ITF-2 are structurally and functionally similar. The two proteins interact with one another through their putative helix-loop-helix motifs and each possesses a distinct domain that dictates transcription activation.
Assuntos
DNA/metabolismo , Elementos Facilitadores Genéticos , Genes de Imunoglobulinas , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , DNA/genética , Humanos , Cadeias kappa de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Plasmídeos , Conformação Proteica , Saccharomyces cerevisiae/genética , Transcrição Gênica , Transfecção , Transformação GenéticaRESUMO
We have investigated the effect of polymerizing defined segments of the immunoglobulin heavy-chain enhancer on the activity of a single, linked transcription unit. Transient assays in lymphoid cells have led to the following observations. First, polymerizing the entire enhancer led to an increase in overall transcription. Second, polymerizing defined DNA segments revealed two distinct functional domains within the enhancer. Although each domain alone possessed only partial enhancer activity, greater than wild-type levels of activity could be obtained upon polymerization. One of these domains contains three regions thought to be involved in protein binding in vivo and in vitro (E motifs E1, E2, and E3). The other domain contains the fourth E motif (E4) and the conserved octanucleotide, ATTTGCAT. We have tested the functional importance of these motifs by determining the effect of mutating these elements singly or in combination in the context of the isolated domains. Although E2, E3, E4, and the octanucleotide are clearly important for enhancer function, mutation of the E1 motif did not appear to have an effect on enhancer activity in our assay. Transient assays in mouse L cells indicate that nonlymphoid cells are able to use a distinct subset of these motifs.