RESUMEN
AIM: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is known as a relatively mild leukoencephalopathy. We investigated the occurrence of severe variants of LBSL with extensive brain magnetic resonance imaging (MRI) abnormalities. METHOD: MRIs of approximately 3,000 patients with an unknown leukoencephalopathy were retrospectively reviewed for extensive signal abnormalities of the cerebral and cerebellar white matter, posterior limb of the internal capsule, cerebellar peduncles, pyramids, and medial lemniscus. Clinical data were retrospectively collected. RESULTS: Eleven patients fulfilled the MRI criteria (six males); six had DARS2 mutations. Clinical and laboratory findings did not distinguish between patients with and without DARS2 mutations, but MRI did. Patients with DARS2 mutations more often had involvement of structures typically affected in LBSL, including decussatio of the medial lemniscus, anterior spinocerebellar tracts, and superior and inferior cerebellar peduncles. Also, involvement of the globus pallidus was associated with DARS2 mutations. Earliest disease onset was neonatal; earliest death at 20 months. INTERPRETATION: This study confirms the occurrence of early infantile, severe LBSL, extending the known phenotypic range of LBSL. Abnormality of specific brainstem tracts and cerebellar peduncles are MRI findings that point to the correct diagnosis.
Asunto(s)
Encéfalo/patología , Leucoencefalopatías/patología , Enfermedades Mitocondriales/patología , Fibras Nerviosas Mielínicas/patología , Aspartato-ARNt Ligasa/deficiencia , Aspartato-ARNt Ligasa/genética , Niño , Preescolar , Femenino , Humanos , Lactante , Leucoencefalopatías/genética , Imagen por Resonancia Magnética , Masculino , Enfermedades Mitocondriales/genética , Mutación , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Médula Espinal/patologíaRESUMEN
Megalencephaly is as a rule accompanied by macrocephaly, an occipitofrontal circumference (OFC) greater than the 98th percentile. Megalencephaly is divided into an anatomic type (developmental) and a metabolic type. Metabolic megalencephaly refers to various storage and degenerative encephalopathies. The differential diagnosis includes Alexander's disease, Canavan's disease, glutaric aciduria type 1, GM1 and GM2 gangliosidosis, merosin-deficient variant of congenital muscular dystrophy and megalencephalic leukoencephalopathy with subcortical cysts (MLC). The distinctive features of this syndrome are enlarged cranial circumference, present at birth or starting in the first year of life, and magnetic resonance imaging (MRI) evidence of diffuse with matter abnormalities with subcortical cysts in the tips of the temporal lobes and in frontoparietal subcortical areas. Mutations in the MLC1 gene have been found as causative of MLC in 60-70 % of affected subjects, without genotype-phenotype correlation. The child we describe presented with progressive macrocephaly not associated with dysmorphic features and large abdominoscrotal hydrocele. At the age of 8 months, encephalic MRI showed anomalies suggestive for MLC and brainstem auditory evoked potentials (BAEP) documented alterations of signal conduction in right tracts. At the time, clinical neurologic examination was normal. Extensive metabolic assays were within normal range. Sequence analysis for MLC1 gene revealed a compound heterozygosity for two mutations in MLC1 gene, inherited from healthy non consanguineous parents.
Asunto(s)
Proteínas de la Membrana/genética , Mutación , Quistes/complicaciones , Quistes/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Humanos , Masculino , Megalencefalia/etiologíaRESUMEN
OBJECTIVE: Vanishing white matter (VWM) is an autosomal recessive leukoencephalopathy characterized by slowly progressive ataxia and spasticity with additional stress-provoked episodes of rapid and major deterioration. The disease is caused by mutations in the genes encoding the subunits of eukaryotic initiation factor 2B, which is pivotal in translation of mRNAs into proteins. The disease onset, clinical severity, and disease course of VWM vary greatly. The influence of genotype and gender on the phenotype is unclear. METHODS: From our database of 184 patients with VWM, we selected those with the following mutations in the gene EIF2B5: p.Arg113His in the homozygous state (n = 23), p.Arg113His in the compound-heterozygous state (n = 49), p.Thr91Ala in the homozygous state (n = 8), p.Arg113His/p.Arg339any (n = 9), and p.Thr91Ala/p.Arg339any (n = 7). We performed a cross-sectional observational study. Evaluated clinical characteristics were gender, age at onset, age at loss of walking without support, and age at death. Means, male/female ratios, and Kaplan-Meier curves were compared. RESULTS: Patients homozygous for p.Arg113His had a milder disease than patients compound heterozygous for p.Arg113His and patients homozygous for p.Thr91Ala. Patients with p.Arg113His/p.Arg339any had a milder phenotype than patients with p.Thr91Ala/p.Arg339any. Overall, females tended to have a milder disease than males. CONCLUSIONS: The clinical phenotype in VWM is influenced by the combination of both mutations. Females tend to do better than males.
Asunto(s)
Factor 2B Eucariótico de Iniciación/genética , Estudios de Asociación Genética/métodos , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Leucoencefalopatías , Fibras Nerviosas Mielínicas/patología , Polimorfismo de Nucleótido Simple/genética , Adulto , Estudios Transversales , Femenino , Genotipo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Leucoencefalopatías/complicaciones , Leucoencefalopatías/genética , Leucoencefalopatías/patología , Masculino , Fenotipo , Probabilidad , Factores Sexuales , Análisis de SupervivenciaAsunto(s)
Encefalopatías/complicaciones , Encefalopatías/diagnóstico , Encéfalo/patología , Demencia/diagnóstico , Imagen por Resonancia Magnética , Edad de Inicio , Encefalopatías/patología , Demencia/etiología , Demencia/patología , Femenino , Humanos , Leucoencefalopatía Multifocal Progresiva/complicaciones , Leucoencefalopatía Multifocal Progresiva/diagnóstico , Persona de Mediana EdadRESUMEN
Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a recently described disorder with autosomal recessive mode of inheritance. Lately, mutations in the DARS2 gene, which encodes mitochondrial aspartyl-tRNA synthetase, have been found as the underlying defect. We report a 19-year-old male patient with cerebellar, pyramidal and dorsal column dysfunctions and specific magnetic resonance imaging (MRI) and characteristic magnetic resonance spectroscopy (MRS) abnormalities. The patient was compound-heterozygous for two mutations in DARS2. MRI showed selective involvement of cerebral and cerebellar white matter and superior and inferior cerebellar peduncles, without contrast enhancement. The U-fibers were spared. The sensory and the pyramidal tracts were affected over their entire length. Involvement of the intraparenchymal trajectories of the trigeminal nerves and mesencephalic trigeminal tracts was demonstrated. In the spinal cord, signal abnormalities were identified in the dorsal columns and the lateral corticospinal tracts. Proton-MRS of the frontal and cerebellar white matter showed elevated lactate, reduced N-acetylaspartate, increased myoinositol and mildly elevated choline. In LBSL, distinct MRI findings should lead to the diagnosis, which can be confirmed by the analysis of the disease gene DARS2.
Asunto(s)
Encefalopatías/metabolismo , Encefalopatías/patología , Tronco Encefálico/patología , Ácido Láctico/metabolismo , Médula Espinal/patología , Adulto , Aspartato-ARNt Ligasa/genética , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Encefalopatías/genética , Encefalopatías/fisiopatología , Creatina/metabolismo , Humanos , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Mutación , Conducción Nerviosa/fisiología , ProtonesRESUMEN
Vanishing white matter disease (VWM) is one of the most frequent inherited childhood leukoencephalopathies. Five genes have been implicated in this disease ( EIF2B1-5), which encode the five subunits of translation initiation factor eIF2B. The disease has an autosomal recessive mode of inheritance. The age of onset and clinical severity vary widely. The diagnosis is based on magnetic resonance imaging (MRI) findings and is confirmed by molecular studies. We describe an affected female patient with a common and a novel mutation of the EIF2B5 gene, who demonstrated a progressive neurological and radiological deterioration. An unusual feature was her striking macrocephaly. She had an early clinical onset at two years of age and is currently still alive at 26 years of age.
Asunto(s)
Encéfalo/patología , Factor 2 Eucariótico de Iniciación/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Mutación , Adolescente , Adulto , Ataxia Cerebelosa/etiología , Ataxia Cerebelosa/patología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Humanos , Imagen por Resonancia Magnética , Fenotipo , Pronóstico , Tomografía Computarizada por Rayos XAsunto(s)
Quistes del Sistema Nervioso Central/diagnóstico , Quistes del Sistema Nervioso Central/genética , Aberraciones Cromosómicas , Factor 2B Eucariótico de Iniciación/genética , Genes Recesivos/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Imagen por Resonancia Magnética , Fibras Nerviosas Mielínicas/patología , Encéfalo/patología , Diagnóstico Diferencial , Tamización de Portadores Genéticos , HumanosRESUMEN
Vanishing white matter disease is a newly recognised leukoencephalopathy of identified genetic background, characterised by cystic degeneration and progressive vanishing of white matter. The characteristic clinical symptoms are spasticity and ataxia with relatively preserved cognitive functions. A characteristic feature of the disease is the occurrence of the symptoms after a physical stress situation such as mild head trauma or febrile infection. We would like to present a case of a 6-year-old girl whose first symptoms of the disease occurred after being frightened by a horse.
Asunto(s)
Encefalopatías/genética , Encefalopatías/patología , Miedo/fisiología , Encefalopatías/complicaciones , Niño , ADN/genética , Femenino , Trastornos Neurológicos de la Marcha/etiología , Trastornos Neurológicos de la Marcha/patología , Humanos , Espasticidad Muscular/etiología , Espasticidad Muscular/patología , Mutación/genéticaAsunto(s)
Factores de Iniciación de Péptidos/metabolismo , Animales , Línea Celular , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4F Eucariótico de Iniciación , Proteínas de Choque Térmico/genética , Fosforilación , Unión Proteica , Biosíntesis de Proteínas , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de SeñalRESUMEN
Translation initiation is a key point of regulation in eukaryotic gene expression. 4E-binding proteins (4E-BPs) inhibit initiation by blocking the association of eIF4E with eIF4G, two integral components of the mRNA cap-binding complex. Phosphorylation of 4E-BP1 reduces its ability to bind to eIF4E and thereby to compete with eIF4G. A novel combination of biophysical and biochemical tools was used to measure the impact of phosphorylation and acidic side chain substitution at each potentially modulatory site in 4E-BP1. For each individual site, we have analyzed the effects of modification on eIF4E binding using affinity chromatography and surface plasmon resonance analysis, and on the regulatory function of the 4E-BP1 protein using a yeast in vivo model system and a mammalian in vitro translation assay. We find that modifications at the two sites immediately flanking the eIF4E-binding domain, Thr(46) and Ser(65), consistently have the most significant effects, and that phosphorylation of Ser(65) causes the greatest reduction in binding affinity. These results establish a quantitative framework that should contribute to understanding of the molecular interactions underlying 4E-BP1-mediated translational regulation.
Asunto(s)
Proteínas Portadoras , Modelos Moleculares , Fosfoproteínas/fisiología , Biosíntesis de Proteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Ciclo Celular , Cromatografía de Afinidad , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Mamíferos , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Factor 4E Eucariótico de Iniciación , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Mutación , Factores de Iniciación de Péptidos/química , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Piridinas/farmacología , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Protein synthesis in rat H35 Reuber hepatoma cells is rapidly inhibited on heat shock. At mild heat-shock temperatures the main cause for inhibition is the inactivation of the guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF2B); under more severe heat-shock conditions the activity of several initiation factors is compromised. eIF2B is required for GDP/GTP exchange on eIF2, which delivers methionyl-tRNA to the 40 S ribosomal subunit. We have tried to elucidate the mechanism underlying the inactivation of eIF2B by assays in vitro. Incubation of cell extracts at 41 degreesC or higher led to the inactivation of eIF2B. In agreement with observations in cells exposed to mild heat shocks, the thermal inactivation of eIF2B could be ascribed to neither eIF2alpha phosphorylation nor the induction of another inhibitor. With the use of glycerol gradients we show that eIF2B forms aggregates in heat-treated extracts. Furthermore eIF2B activity is protected against heat shock in thermotolerant cells. Taken together, these results suggest a role for chaperones in the control of eIF2B activity.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas/antagonistas & inhibidores , Animales , Factor 2B Eucariótico de Iniciación , Factores de Intercambio de Guanina Nucleótido , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Calor , Cinética , Fosforilación , Proteínas/química , Proteínas/aislamiento & purificación , Ratas , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Regulation of eukaryotic translation initiation is a process that requires collaboration between multiple proteins. The cap-binding factor eukaryotic initiation factor (eIF)4E, its binding protein 4E-BP1, and the guanine-nucleotide-exchange factor eIF2B play important roles in the regulation of the rate of protein synthesis. This review describes the regulation of the activity of these three proteins and the signal-transduction pathways involved therein.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Proteínas/metabolismo , Transducción de Señal , Animales , Factor 2B Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Factores de Intercambio de Guanina Nucleótido , Humanos , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de ProteínasRESUMEN
The regulation of protein synthesis and of eukaryotic initiation factor eIF2B was studied in PC12 cells. An increase in protein synthesis was observed after nerve growth factor (NGF) and epidermal growth factor (EGF) treatment of PC12 cells, and this increase coincided with activation of eIF2B. Growth factor addition in the presence of the phosphatidylinositol-3'-OH kinase inhibitor wortmannin showed that both NGF- and EGF-induced protein synthesis and eIF2B activation were phosphatidylinositol-3'-OH kinase dependent. The EGF-induced stimulation of protein synthesis and activation of eIF2B was dependent upon FK506-binding protein-rapamycin-associated protein, as shown with the immunosuppressant rapamycin, whereas NGF induction was partially dependent upon FK506-binding protein-rapamycin-associated protein. The activities of two kinases that act on eIF2B, glycogen synthase kinase-3 and casein kinase II, were measured to assess their potential roles in the activation of eIF2B in PC12 cells. Inactivation of glycogen synthase kinase-3 was seen in response to both NGF and EGF and this coincided with activation of eIF2B. However, inactivation of glycogen synthase kinase-3 was not rapamycin sensitive, in contrast to the activation of eIF2B. This indicates the involvement of another protein kinase or regulatory mechanism in the eIF2B activation. Both growth factors activated casein kinase II. However, the time course of its activation and its insensitivity to wortmannin and rapamycin suggest that casein kinase II does not play a major regulatory role in eIF2B activation under these conditions.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Factores de Crecimiento Nervioso/farmacología , Células PC12/fisiología , Biosíntesis de Proteínas , Proteínas/metabolismo , Androstadienos/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Portadoras/fisiología , Quinasa de la Caseína II , Proteínas de Unión al ADN/fisiología , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Factor 2B Eucariótico de Iniciación , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Factores de Intercambio de Guanina Nucleótido , Proteínas de Choque Térmico/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación/efectos de los fármacos , Polienos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Proteína S6 Ribosómica , Proteínas Ribosómicas/metabolismo , Sirolimus , Proteínas de Unión a Tacrolimus , WortmaninaRESUMEN
Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 degrees C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.
Asunto(s)
Proteínas Portadoras , Respuesta al Choque Térmico , Neoplasias Hepáticas Experimentales/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Factores de Intercambio de Guanina Nucleótido , Guanosina Difosfato/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cinética , Células PC12 , Factores de Iniciación de Péptidos/metabolismo , Fosforilación , Biosíntesis de Proteínas , Proteínas/antagonistas & inhibidores , Ratas , Proteínas Represoras/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Células Tumorales CultivadasRESUMEN
Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the insect-cell lysates was measured. The translational properties of the p10 transcripts were very similar in lysates made from either uninfected or baculovirus-infected insect cells. Determination of optimal salt conditions for either uncapped or capped transcripts showed that the p10 5'UTR was used very efficiently for translation initiation in vitro, even in the absence of a cap-structure at its 5' end. Addition of cap-analogue to the in vitro translation assays did not inhibit p10 5'UTR-driven translation, while translation of a cap-dependent mRNA was severely inhibited. These data suggest that the very late mRNAs of baculovirus are translated in a cap-independent manner.
Asunto(s)
Nucleopoliedrovirus/genética , Biosíntesis de Proteínas , ARN Mensajero , ARN Viral , Proteínas Virales/genética , Animales , Línea Celular , Mariposas Nocturnas/virología , Spodoptera/citologíaRESUMEN
In concert with the translation initiation factors 'trans-acting' factors function specifically during internal initiation on picornaviral mRNAs. Of these trans-acting factors, two have been identified as the La-protein and the polypyrimidine tract binding protein. Within the internal ribosomal entry site on the viral RNA, sequences are present that direct the ribosome to the initiation codon. We suggest that selection of the correct AUG initiation codon occurs through basepairing with a part of 18S ribosomal RNA.
Asunto(s)
Biosíntesis de Proteínas , ARN Ribosómico 18S/química , ARN Ribosómico 18S/metabolismo , Composición de Base , Secuencia de Bases , Codón , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Picornaviridae/genética , Picornaviridae/metabolismo , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismo , ARN Viral/química , ARN Viral/metabolismoRESUMEN
The encephalomyocarditis virus 5' untranslated region (EMC 5' UTR) has a binding site for eukaryotic initiation factor eIF-2. Mutations in the 3' end or deletion of the 5' end of the internal ribosomal entry site had a negative effect on the binding of eIF-2 to the EMC 5' UTR. The binding of eIF-2 to the mutant 5' UTRs was completely inhibited by the addition of competitor tRNA. Cross-linking of the EMC 5' UTR with proteins from rabbit reticulocyte lysates showed binding of trans-acting factors p52 and p57. Deletions in the 5' end of the internal ribosomal entry site resulted in a loss of the ability to bind trans-acting factor p57, in accordance with literature data, while p52 binding to these deletion mutants was weak compared to the wildtype EMC 5' UTR. Mutations in the 3' part of the 5' UTR of EMC still resulted in binding of both trans-acting factors, as with wild type RNA, but binding was more sensitive to competitor tRNA when compared to the binding of p52/p57 to the wild type 5' UTR.
Asunto(s)
Virus de la Encefalomiocarditis/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Proteína de Unión al Tracto de Polipirimidina , ARN Viral/metabolismo , Transactivadores/metabolismoRESUMEN
The adenovirus tripartite leader (TPT) 5' untranslated region (5'UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5'UTR. The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct. Surprisingly, the TPT 5'UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap-independent initiation. In vitro protein-synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT-5'UTR-directed translation and poliovirus-induced host cell shut-off is discussed.