RESUMEN
As the mechanisms underlying neuronal development and degeneration become clarified, a number of common effectors and signaling pathways are becoming apparent. Here we describe the identification of Abeta, long considered a pathologic mediator of Alzheimers Disease and Down Syndrome, as similarly over-expressed in the neurodevelopmental disease, Fragile X Syndrome. We also show that mGluR5 inhibitors, currently employed for the treatment of Fragile X, reduce Abeta production in rodent models of Fragile X and AD as well as reduce disease phenotypes including seizures. Thus seemingly disparate neurologic diseases may share a common pathologic instigator and be treatable with a common, currently available class of therapeutics.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/fisiopatología , Ácido Glutámico/metabolismo , Humanos , Fenotipo , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/antagonistas & inhibidoresRESUMEN
The ability to control gene expression is central to normal development and function. For a growing number of genes in the central nervous system and peripheral tissues, expression is determined by changes in the rate of mRNA decay. At a molecular level, regulated interactions between the mRNA target and sequence-specific binding proteins either inhibit or accelerate decay, affording tight control over gene expression. This review discusses several examples of such posttranscriptional gene regulation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Sistema Nervioso/metabolismo , Biosíntesis de Proteínas/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Genes Reguladores/fisiología , Humanos , Proteína Básica de Mielina/biosíntesis , Proteína Básica de Mielina/genética , Sistema Nervioso/crecimiento & desarrollo , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Short-lived peripheral blood eosinophils are recruited to the lungs of asthmatics after allergen challenge, where they become long-lived effector cells central to disease pathophysiology. GM-CSF is an important cytokine which promotes eosinophil differentiation, function, and survival after transit into the lung. In human eosinophils, GM-CSF production is controlled by regulated mRNA stability mediated by the 3' untranslated region, AU-rich elements (ARE). We identified human Y box-binding factor 1 (YB-1) as a GM-CSF mRNA ARE-specific binding protein that is capable of enhancing GM-CSF-dependent survival of eosinophils. Using a transfection system that mimics GM-CSF metabolism in eosinophils, we have shown that transduced YB-1 stabilized GM-CSF mRNA in an ARE-dependent mechanism, causing increased GM-CSF production and enhanced in vitro survival. RNA EMSAs indicate that YB-1 interacts with the GM-CSF mRNA through its 3' untranslated region ARE. In addition, endogenous GM-CSF mRNA coimmunoprecipitates with endogenous YB-1 protein in activated eosinophils but not resting cells. Thus, we propose a model whereby activation of eosinophils leads to YB-1 binding to and stabilization of GM-CSF mRNA, ultimately resulting in GM-CSF release and prolonged eosinophil survival.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/fisiología , Proteínas de Unión al ADN , Eosinófilos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Estabilidad del ARN , Factores de Transcripción , Regiones no Traducidas 3' , Asma/inmunología , Proteínas Potenciadoras de Unión a CCAAT/genética , Supervivencia Celular , Productos del Gen tat/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Hipersensibilidad Inmediata/inmunología , Factores de Transcripción NFI , Proteínas Nucleares , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta , Transducción Genética , Proteína 1 de Unión a la Caja YRESUMEN
The precise signaling pathways which contribute to amyloid precursor protein (APP) gene expression remain incompletely characterized. We evaluated the role of protein kinases, calcium and phospholipase C (PLC) in modulating APP mRNA levels. There was a rapid 35-40% reduction in the steady state level of APP mRNA upon stimulation of peripheral blood mononuclear cells (PBMC) with phorbol 12-myristate 13-acetate (PMA), A23187 or ionomycin. However the protein kinase C (PKC), protein kinase A (PKA) or PLC pathways did not mediate these changes in APP mRNA levels. Rather, PMA or ionophore caused a rapid activation of extracellular-regulated kinase (ERK). This effect was independent of PKC and sensitive to U0126. After 4 h of PMA treatment, the remaining APP mRNA became indefinitely stable. We propose a model for the biphasic decay of APP mRNA in which ERK activation by PMA causes sequential upregulation of two APP mRNA binding proteins, nucleolin and hnRNP C. We attribute the initial rapid loss of APP mRNA to the helicase activity associated with nucleolin and later stabilization to hnRNP C binding to the 29 base instability element in the 3'-UTR of APP mRNA.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN Mensajero/metabolismo , Butadienos/farmacología , Calcimicina/farmacología , Calcio/metabolismo , Carcinógenos/farmacología , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Ionomicina/farmacología , Ionóforos/farmacología , Leucocitos Mononucleares/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Nitrilos/farmacología , Proteína Quinasa C/metabolismo , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/fisiología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
In these studies, we examined signaling through the transcription factor STAT5 in human peripheral blood eosinophils after treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-5. In response to either cytokine, STAT5 was rapidly tyrosine phosphorylated and acquired interferon gamma activation site (GAS) DNA binding activity. Tyrosine-phosphorylated STAT5 was associated with both cytosolic and nuclear cell fractions. Consistent with activation, the transcription of a STAT5-dependent gene, cytokine inducible, SH2-containing protein (CIS1), was enhanced after cytokine stimulation. This is the first report of IL-5 regulation of CIS1 gene expression in any cell type. Given its role in cytokine signaling, CIS1 upregulation may serve to attenuate IL-5 and GM-CSF modulation of eosinophil function. These data suggest that active nuclear STAT5 participates in the regulation of IL-5 and GM-CSF--inducible genes in stimulated human peripheral blood eosinophils.
Asunto(s)
Proteínas de Unión al ADN/sangre , Eosinófilos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Proteínas Inmediatas-Precoces/genética , Interleucina-5/farmacología , Proteínas de la Leche , Transactivadores/sangre , Núcleo Celular/metabolismo , Citocinas/sangre , Citocinas/genética , Citosol/metabolismo , Eosinófilos/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/sangre , Técnicas In Vitro , Cinética , Fosforilación , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5 , Proteínas Supresoras de la Señalización de CitocinasRESUMEN
Airway eosinophils show prolonged in vitro survival compared with peripheral blood eosinophils (PBEos). Recent studies have shown that autocrine production and release of GM-CSF is responsible for enhanced survival, but the mechanisms controlling cytokine production remain obscure. We compared GM-CSF mRNA decay in eosinophils from bronchoalveolar lavage (BALEos) after allergen challenge or from PBEos. BALEos showed prolonged survival in vitro (60% at 4 days) and expressed GM-CSF mRNA. The enhanced survival of BALEos was 75% inhibited at 6 days by neutralizing anti-GM-CSF Ab. Based on transfection studies, GM-CSF mRNA was 2.5 times more stable in BALEos than in control PBEos. Treatment of PBEos with fibronectin and TNF-alpha increased their in vitro survival, GM-CSF mRNA expression, and GM-CSF mRNA stability to a comparable level as seen in BALEos. These data suggest that TNF-alpha plus fibronectin may increase eosinophil survival in vivo by controlling GM-CSF production at a posttranscriptional level.
Asunto(s)
Bronquios/metabolismo , Eosinófilos/metabolismo , Fibronectinas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Bronquios/citología , Bronquios/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Movimiento Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Combinación de Medicamentos , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Humanos , ARN Mensajero/sangre , Regulación hacia Arriba/inmunologíaRESUMEN
Allergic asthma is characterized by pulmonary infiltration and accumulation of eosinophils, which is enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). T cells, fibroblasts, and eosinophils themselves produce GM-CSF, suggesting it functions in the lung microenvironment as a survival factor. However, the amounts and the mechanism by which GM-CSF supports eosinophil survival remain poorly understood. We have previously reported that human peripheral blood eosinophils (PBEo) can be transfected with GM-CSF mRNA using particle-mediated gene transfer (PMGT). Using this technology, GM-CSF mRNA was introduced into resting PBEo, and GM-CSF production and cell survival were assessed. GM-CSF protein was undetectable (< 1 pg/ml) in the supernatant but present intracellularly at very low levels. Unexpectedly, the in vitro survival of transfected PBEo was 4-fold greater than that of controls. Neutralizing anti-GM-CSF but not anti-interleukin-5 (anti-IL-5) antibody added up to 24 h after transfection abolished enhanced survival, demonstrating that the continuous presence of GM-CSF was required. Conditioned medium prepared from transfected PBEo prolonged the survival of naive cells. Comparable survival activity was mimicked by a single dose of 100-500 pg/ml or multiple administrations of 0.1 pg/ml recombinant human GM-CSF (rHuGM-CSF). Survival was completely inhibited by a Jak2 inhibitor, suggesting that GM-CSF-mediated survival involved signaling through the Jak-Stat pathway. Thus, autocrine production of low levels of GM-CSF by a minority of PBEo can block apoptosis of the entire culture by a minute but sustained GM-CSF release.
Asunto(s)
Eosinófilos/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Proteínas Proto-Oncogénicas , Apoptosis/fisiología , Asma/etiología , Asma/patología , Asma/fisiopatología , Supervivencia Celular/fisiología , Medios de Cultivo Condicionados , Eosinófilos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Técnicas In Vitro , Janus Quinasa 2 , Pruebas de Neutralización , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes/farmacología , Transducción de Señal , TransfecciónRESUMEN
The signal transduction pathways regulating nucleolin mRNA and protein production have yet to be elucidated. Peripheral blood mononuclear cells treated with phorbol 12-myristate 13-acetate showed steady state levels of nucleolin mRNA that were 2-2.5-fold greater than untreated control cells. The up-regulation of nucleolin mRNA was substantially repressed by U0126, a specific inhibitor that blocks phosphorylation of extracellular-regulated kinase (ERK). Calcium ionophores and ionomycin also activated ERK and substantially elevated nucleolin mRNA levels, demonstrating phorbol 12-myristate 13-acetate and calcium signaling converge on ERK. Drugs that affected protein kinase C, protein kinase A, and phospholipase C signal transduction pathways did not alter nucleolin mRNA levels significantly. The half-life of nucleolin mRNA increased from 1.8 h in resting cells to 3.2 h with phorbol ester activation, suggesting ERK-mediated posttranscriptional regulation. Concomitantly, full-length nucleolin protein was increased. The higher levels of nucleolin protein were accompanied by increased binding of a 70-kDa nucleolin fragment to the 29-base instability element in the 3'-untranslated region of amyloid precursor protein (APP) mRNA in gel mobility shift assays. Supplementation of rabbit reticulocyte lysate with nucleolin decreased APP mRNA stability and protein production. These data suggest ERK up-regulates nucleolin posttranscriptionally thereby controlling APP production.
Asunto(s)
Regulación de la Expresión Génica , Leucocitos Mononucleares/metabolismo , Proteínas Quinasas Activadas por Mitógenos/sangre , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Transcripción Genética/efectos de los fármacos , Regiones no Traducidas 3'/genética , Adenilil Ciclasas/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Calcimicina/farmacología , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Cinética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Nucleares/sangre , Fosfoproteínas/sangre , Biosíntesis de Proteínas , Proteína Quinasa C/sangre , ARN Mensajero/sangre , Proteínas de Unión al ARN/sangre , Conejos , Reticulocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/sangre , NucleolinaRESUMEN
Using a cell-free translation system, we previously demonstrated that the turnover and translation of amyloid precursor protein (APP) mRNA was regulated by a 29-nucleotide instability element, located 200 nucleotides downstream from the stop codon. Here we have examined the regulatory role of this element in primary human capillary endothelial cells under different nutritional conditions. Optimal proliferation required a growth medium (endothelial cell growth medium) supplemented with epidermal, basic fibroblast, insulin-like, and vascular endothelial growth factors. In vitro transcribed mRNAs with the 5'-untranslated region (UTR) and coding region of beta-globin and the entire 3'-UTR of APP 751 were transfected into cells cultured in endothelial cell growth medium. Wild-type globin-APP mRNA containing an intact APP 3'-UTR and mutant globin-APP mRNA containing a mutated 29-nucleotide element decayed with identical half-lives (t 1/2 = 60 min). Removal of all supplemental growth factors from the culture medium significantly accelerated the decay of transfected wild-type mRNA (t 1/2 = 10 min), but caused only a moderate decrease in the half-life of transfected mutant mRNA (t 1/2 = 40 min). We therefore conclude that the 29-nucleotide 3'-UTR element is an mRNA destabilizer whose function can be inhibited by inclusion of the aforementioned mixture of growth factors in the culture medium.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Precursor de Proteína beta-Amiloide/genética , Secuencia Conservada/fisiología , Sustancias de Crecimiento/fisiología , Estabilidad del ARN , ARN Mensajero/genética , Secuencia de Bases/genética , Fenómenos Fisiológicos Sanguíneos , Células Cultivadas , Medios de Cultivo/química , Sustancias de Crecimiento/farmacología , Semivida , Homeostasis/fisiología , Humanos , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , TransfecciónRESUMEN
Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMV-driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t1/2 in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t1/2 of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded, suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM-CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow detailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.
Asunto(s)
Eosinófilos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , ARN Mensajero/genética , Transfección/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Vectores Genéticos/síntesis química , Vectores Genéticos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Humanos , Ionomicina/farmacología , Ionóforos/farmacología , Biosíntesis de Proteínas/inmunología , ARN Mensajero/sangre , Transfección/efectos de los fármacos , Transfección/métodos , Células Tumorales CultivadasRESUMEN
Eosinophils produce granulocyte macrophage colony-stimulating factor (GM-CSF), which enhances their survival and function. In T cells and fibroblasts, GM-CSF production is controlled predominantly by variable messenger RNA (mRNA) stability involving 3' untranslated region (3' UTR) adenosine-uridine-rich elements (AREs) and sequence-specific mRNA binding proteins. However, the mode of regulation of this critical cytokine remains unknown in eosinophils. Therefore, we measured GM-CSF mRNA decay in an eosinophil-like cell line (AML14.3D10) and, with a radiolabeled GM-CSF RNA probe, asked whether ARE-specific, mRNA binding proteins were present in cytoplasmic lysates of these cells. Human GM-CSF mRNA transfected into unstimulated AML14.3D10 cells decayed with a half-life of 6 min, which increased to 14 min after 1 h, and to 22 min after 2 h, of ionophore-mediated activation. GM-CSF RNA mobility shift assays using cytoplasmic extracts from resting or ionophore-stimulated AML14.3D10 cells revealed multiple RNA-protein complexes of 55, 60, 85, 100, and 125 kD. A 47-kD complex was also detected with an 80-base RNA probe containing four consecutive AUUUA motifs. On the basis of competition studies, all of the observed binding protein activities interacted with the 3' UTR AREs. In addition, binding activity increased 2.5-fold in cytoplasmic lysates from cells stimulated with calcium ionophore for 2 h, contemporaneous with GM-CSF mRNA stabilization. These data provide direct evidence that ionophore stabilizes GM-CSF mRNA in AML14.3D10 cells and simultaneously increases the activity of a series of AUUUA-specific mRNA binding proteins. We conclude that the interaction of AU-specific binding proteins may stabilize GM-CSF mRNA in activated eosinophil-like cell lines.
Asunto(s)
Proteínas Portadoras/metabolismo , Eosinófilos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Ionóforos/farmacología , Estabilidad del ARN/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Unión Competitiva/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Eosinófilos/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Ionomicina/farmacología , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Tiempo , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA levels are controlled post-transcriptionally by the 3'-untranslated region (UTR) adenosine-uridine-rich element (ARE). In untransformed, resting cells, the ARE targets GM-CSF mRNA for rapid degradation, thereby significantly suppressing protein expression. We used a rabbit reticulocyte lysate (RRL) cell-free system to examine translational regulation of GM-CSF expression. We uncoupled decay rates from rates of translation by programming the RRL with an excess of mRNAs. Capped, full-length, polyadenyl-ated human GM-CSF mRNA (full-length 5'-UTR AUUUA+A90) and an ARE-modified version (full-length 5'-UTR AUGUA+A90) produced identical amounts of protein. When the 5'-UTR was replaced with an irrelevant synthetic leader sequence (syn 5'-UTR), translation of syn 5'-UTR AUUUA+A90 mRNA was suppressed by >20-fold. Mutation of the ARE or removal of the poly(A) tail relieved this inhibition. Thus, in the absence of a native 5'-UTR, the ARE and poly(A) tail act in concert to block GM-CSF mRNA translation. Substitutions of different regions of the native 5'-UTR revealed that the entire sequence was essential in maintaining the highest rates of translation. However, shorter 10-12 nt contiguous 5'-UTR regions supported 50-60% of maximum translation. The 5'-UTR is highly conserved, suggesting similar regulation in multiple species and in these studies was the dominant element regulating GM-CSF mRNA translation, overriding the inhibitory effects of the ARE and the poly(A) tail.
Asunto(s)
Regiones no Traducidas 5'/genética , Silenciador del Gen , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Poli A/genética , Biosíntesis de Proteínas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Regiones no Traducidas 5'/química , Regiones no Traducidas 5'/metabolismo , Adenosina/genética , Adenosina/metabolismo , Secuencia de Bases , Sitios de Unión , Secuencia Conservada/genética , Humanos , Cinética , Mutación/genética , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Temperatura , Uridina/genética , Uridina/metabolismoRESUMEN
Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes. Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1. In an effort to identify these mRNAs, we incubated total mRNA from mitogen-activated peripheral blood mono-nuclear cells (PBMCs) with AUF1 in vitro. AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. We have obtained partial nucleotide sequences from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs. As a test of the validity of this method, we also show that a randomly selected, novel mRNA contained in the library is stabilized upon cell activation.
Asunto(s)
Genes Inmediatos-Precoces , Ribonucleoproteína Heterogénea-Nuclear Grupo D , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Cromatografía de Afinidad , ADN Complementario/genética , Biblioteca de Genes , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Ribonucleoproteína Nuclear Heterogénea D0 , Ligandos , Proteínas de Unión al ARN/genética , Proteínas Recombinantes , Análisis de Secuencia de ADNRESUMEN
Human eosinophils activated by calcium ionophore produce granulocyte-macrophage colony-stimulating factor (GM-CSF). In T lymphocytes GM-CSF messenger RNA (mRNA) stability is regulated by 3' untranslated region (UTR) adenosine-uridine-rich elements (AREs). We show endogenous GM-CSF mRNA is rapidly induced in an eosinophil cell-line (AML14.3D10) after activation with ionomycin. To calculate the decay rate of GM-CSF mRNA in activated cells, eosinophils were transfected with wild-type, full-length GM-CSF mRNA or a mutant version lacking the AUUUA motifs. In unstimulated cells, wild-type GM-CSF mRNA decayed with a half-life time (t1/2) of 6+/-2 min while the mutant decayed with a t1/2 of 20+/-4 min, demonstrating the dominant, destabilizing effect of multiple AUUUA motifs. Within 1 hr of activation by ionomycin, the half-life of transfected wild-type mRNA increased by 2.5-fold, which increased up to 4-fold after 2 hr of activation. The half-life of the mutant GM-CSF was unaffected by ionomycin, demonstrating that ionophore-mediated stabilization requires intact AUUUA motifs. Actinomycin D (ActD) stabilized wild-type GM-CSF mRNA as well, causing poly(A) tail elongation and translation inhibition. These data show that in eosinophil-like cell lines, GM-CSF mRNA is exquisitely unstable but can be markedly stabilized by calcium ionophore. Both effects require intact 3' UTR AREs.
Asunto(s)
Eosinófilos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Sitios de Unión , Línea Celular , Dactinomicina/farmacología , Eosinófilos/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , ARN MensajeroRESUMEN
We have previously shown that heterogeneous nuclear ribonucleoprotein C (hnRNP C) and nucleolin bound specifically to a 29 nt sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. Upon activation of peripheral blood mononuclear cells, hnRNP C and nucleolin acquired APP mRNA binding activity, concurrent with APP mRNA stabilization. These data suggested that the regulated interaction of hnRNP C and nucleolin with APP mRNA controlled its stability. Here we have directly examined the role of the cis element and trans factors in the turnover and translation of APP mRNA in vitro . In a rabbit reticulocyte lysate (RRL) translation system, a mutant APP mRNA lacking the 29 nt element was 3-4-fold more stable and synthesized 2-4-fold more APP as wild-type APP mRNA. Therefore, the 29 nt element functioned as an APP mRNA destabilizer. RNA gel mobility shift assays with the RRL suggested the presence of endogenous nucleolin, but failed to show hnRNP C binding activity. However, wild-type APP mRNA was stabilized and coded for 6-fold more APP when translated in an RRL system supplemented with exogenous active hnRNP C. Control mRNAs lacking the 29 nt element were unaffected by hnRNP C supplementation. Therefore, occupancy of the 29 nt element by hnRNP C stabilized APP mRNA and enhanced its translation.
Asunto(s)
Precursor de Proteína beta-Amiloide/biosíntesis , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Precursor de Proteína beta-Amiloide/genética , Secuencia de Bases , Sistema Libre de Células , Ribonucleoproteína Heterogénea-Nuclear Grupo C , Ribonucleoproteínas Nucleares Heterogéneas , Biosíntesis de ProteínasRESUMEN
We have demonstrated the existence of multiple mRNA binding proteins that interact specifically with defined regions in posttranscriptionally regulated mRNAs. These domains appear to be destabilizers whose function can be attenuated by the interaction with the specific binding proteins. Thus, the ability to alter mRNA decay rates on demand, given different environmental or intracellular conditions, appears to be mediated by controlling the localization, activity, and overall function of the cognate binding protein. Based on our limited experience, we predict that most, if not all, of similarly regulated mRNAs will ultimately be found to interact with regulatory mRNA binding proteins. Under conditions whereby the mRNA binding proteins are constitutively active (e.g., tumor cell lines), abnormal mRNA decay will result, with accumulation and overtranslation. Such appears to be the case for cytokines and possibly amyloid protein precursor mRNAs in cancer and Alzheimer's disease, respectively. Conversely, mutagenesis of these critical 3' untranslated region elements will likely have comparable deleterious effects on the regulation of gene expression. To the extent that such derangements exist in human disease, attention to understanding the mechanistic detail at this level may provide insights into the development of appropriate therapeutics or treatment strategies.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Precursor de Proteína beta-Amiloide/genética , Citocinas/genética , Células Eucariotas , Terapia Genética/métodos , Globinas/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Semivida , Humanos , Conformación de Ácido NucleicoAsunto(s)
Citoplasma/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Células Eucariotas , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Poli A/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismoRESUMEN
We have developed a novel system to examine intracellular mRNA decay pathways in the absence of transcriptional blockade. In vitro transcribed, capped, and adenylated granulocyte-macrophage colony stimulating factor (GM-CSF) or globin mRNAs were introduced by particle-mediated gene transfer into primary cultures of normal peripheral blood mononuclear cells. Transfected wild-type, human GM-CSF (hGM-AUUUA) mRNA decayed rapidly (t1/2 = 9 min), while a mutated version lacking AUUUA repeats (hGM-AUGUA) was significantly more stable (t1/2 = 30 min). A truncated GM-CSF mRNA lacking the entire 3'-UTR (hGM-Delta3'-UTR) was still more stable (t1/2 = 80 min) demonstrating the existence of non-AUUUA, 3'-UTR destabilizing domains. Transfected beta-globin mRNA was very stable, decaying with a half-life of >360 min. Transfected mRNAs were >90% polysome associated with transgenic protein detectable within 15 min of transfection. The most stable GM-CSF mRNAs were not associated with maximal GM-CSF protein production. Agents known or hypothesized to interfere with mRNA decay, including cycloheximide, phorbol ester, or actinomycin D, stabilized both hGM-AUUUA and hGM-AUGUA mRNAs. These data demonstrate the presence of 3'-UTR, destabilizing, and translational regulatory elements outside of the AUUUA repeats and unambiguously show that actinomycin D at concentrations commonly used to inhibit transcription stabilizes cytokine mRNAs.