RESUMO
Plasminogen activator inhibitor type 2 (PAI-2; SERPINB2) is a highly-regulated gene that is subject to both transcriptional and post-transcriptional control. For the latter case, inherent PAI-2 mRNA instability was previously shown to require a nonameric adenylate-uridylate element in the 3' UTR. However, mutation of this site was only partially effective at restoring complete mRNA stabilization. In the present study, we have identified additional regulatory motifs within the 3' UTR that cooperate with the nonameric adenylate-uridylate element to promote mRNA destabilization. These elements are located within a 74 nucleotide U-rich stretch (58%) of the 3' UTR that flanks the nonameric motif; deletion or substitution of this entire region results in complete mRNA stabilization. These new elements are conserved between species and optimize the destabilizing capacity with the nonameric element to ensure complete mRNA instability in a manner analogous to some class I and II adenylate-uridylate elements present in transcripts encoding oncogenes and cytokines. Hence, post-transcriptional regulation of the PAI-2 mRNA transcript involves an interaction between closely spaced adenylate-uridylate elements in a manner analogous to the post-transcriptional regulation of oncogenes and cytokines.
Assuntos
Regiões 3' não Traduzidas , Citocinas/metabolismo , Elementos Facilitadores Genéticos , Oncogenes/genética , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Regiões 3' não Traduzidas/fisiologia , Animais , Sequência de Bases , Citocinas/genética , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RatosAssuntos
Peptídeo Hidrolases/genética , Ativadores de Plasminogênio/genética , Inibidores de Proteases/metabolismo , Regiões 3' não Traduzidas , Animais , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Biossíntese de Proteínas , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transdução de SinaisRESUMO
Plasminogen activator inhibitor type-2 (PAI-2) is a nonconventional serine protease inhibitor (serpin) with unique and tantalizing properties that is generally considered to be an authentic and physiological inhibitor of urokinase. However, the fact that only a small percentage of PAI-2 is secreted has been a long-standing argument for alternative roles for this serpin. Indeed, PAI-2 has been shown to have a number of intracellular roles: it can alter gene expression, influence the rate of cell proliferation and differentiation, and inhibit apoptosis in a manner independent of urokinase inhibition. Despite these recent advances in defining the intracellular function of PAI-2, it still remains one of the most mysterious and enigmatic members of the serpin superfamily.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/metabolismo , Inibidores de Serina Proteinase/metabolismo , Animais , Apoptose , Regulação da Expressão Gênica , Humanos , Metástase Neoplásica/patologia , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genéticaRESUMO
The human prothrombin G20210A polymorphism located at the 3' cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous thrombosis. This polymorphism has been shown to directly influence a variety of processes related to prothrombin mRNA metabolism. We have constructed plasmids that express the full-length prothrombin mRNA that is polyadenylated at its natural site. The A allele prothrombin variant was more efficient than the G allele at promoting cleavage at this site in the presence of a competing poly (A) sequence. In the absence of competition, both allelic variants give rise to a similar level of cleavage site heterogeneity. An upstream sequence element (USE) was also identified within the prothrombin 3'-UTR. When placed upstream of two competing poly (A) sites, the USE directed cleavage preferentially to the proximal poly (A) site. In the absence of competition, the USE had no effect on cleavage site selection. This study suggests that the basis for the increase in prothrombin expression in A allele carriers is not due to allelic changes in cleavage site selection per se. In addition, the functionality of USEs needs to be considered within the context of endogenous sequence architecture.
Assuntos
Regiões 3' não Traduzidas , Polimorfismo de Nucleotídeo Único , Protrombina/genética , Processamento de Terminações 3' de RNA , RNA Mensageiro/metabolismo , Sequência de Bases , Sequência Conservada , Humanos , Dados de Sequência Molecular , Poliadenilação , Protrombina/biossíntese , RNA Mensageiro/química , Sequências Reguladoras de Ácido RibonucleicoRESUMO
Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.
Assuntos
Antígenos de Superfície , Proteínas de Ligação a DNA , Proteínas Imediatamente Precoces/farmacologia , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Northern Blotting , Western Blotting , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Proteínas Fúngicas/metabolismo , Biblioteca Gênica , Globinas/química , Globinas/metabolismo , Humanos , Modelos Genéticos , Testes de Precipitina , Ligação Proteica , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Transfecção , Tristetraprolina , Técnicas do Sistema de Duplo-Híbrido , Raios UltravioletaRESUMO
Cells respond to a wide variety of stresses through the transcriptional activation of genes that harbour stress elements within their promoters. While many of these elements are shared by genes encoding proteins representative of all subcellular compartments, cells can also respond to stresses that are specific to individual organelles, such as the endoplasmic reticulum un folded protein response. Here we report on the discovery and characterization of a mitochondrial stress response in mammalian cells. We find that the accumulation of unfolded protein within the mitochondrial matrix results in the transcriptional upregulation of nuclear genes encoding mitochondrial stress proteins such as chaperonin 60, chaperonin 10, mtDnaJ and ClpP, but not those encoding stress proteins of the endoplasmic reticulum. Analysis of the chaperonin 60/10 bidirectional promoter identified a CHOP element as the mitochondrial stress response element. Dominant-negative mutant forms of CHOP and overexpression of CHOP revealed that this transcription factor, in association with C/EBPbeta, regulates expression of mitochondrial stress genes in response to the accumulation of unfolded proteins.
Assuntos
Proteínas de Choque Térmico/biossíntese , Mitocôndrias/fisiologia , Estresse Fisiológico/fisiopatologia , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Células COS , Núcleo Celular/metabolismo , Chaperonina 10/biossíntese , Chaperonina 10/genética , Chaperonina 60/biossíntese , Chaperonina 60/genética , Chlorocebus aethiops , Endopeptidase Clp , Regulação da Expressão Gênica , Genes Dominantes , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico/genética , Ornitina Carbamoiltransferase/química , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Regiões Promotoras Genéticas , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/fisiologia , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Transdução de Sinais , Estresse Fisiológico/genética , Fator de Transcrição CHOP , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
The G20210A polymorphism has been shown to alter the efficiency of prothrombin mRNA processing. Here we show that the G20210A mutation also alters prothrombin mRNA stability. Three-fold more prothrombin protein and mRNA were produced in NIH-3T3 cells transfected with the prothrombin cDNAs containing the 20210A variant compared to cells expressing the 20210G variant. mRNA stability assays using chimeric globin transcripts harboring the G or A variant of the 97 nt prothrombin 3'-UTR indicated that the 20210G variant conferred greater instability to the globin reporter transcript than the A variant in transfected HepG2 cells. Both variants of the prothrombin 3'-UTR were shown to provide binding sites for a number of cellular proteins including HuR, an RNA binding protein associated with mRNA stability. Our results indicate that the G20210A is a bifunctional polymorphism, as it not only alters the efficiency of mRNA processing, but also the decay rate of prothrombin mRNA.
