RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The average age of onset of both sporadic and familial cases is 50-60 years of age. The presence of cytoplasmic inclusions of the RNA-binding protein TAR DNA-binding protein-43 (TDP-43) in the affected neurons is seen in 95% of the ALS cases, which results in TDP-43 nuclear clearance and loss of function. The Drosophila melanogaster ortholog of TDP-43 (TBPH) shares many characteristics with the human protein. Using a TDP-43 aggregation inducer previously developed in human cells, we created a transgenic fly that shows an adult locomotive defect. Phenotype onset correlates with a physiologically age-related drop of TDP-43/TBPH mRNA and protein levels, seen both in mice and flies. Artificial reduction of mRNA levels, in vivo, anticipates the locomotion defect to the larval stage. Our study links, for the first time, aggregation and the age-related, evolutionary conserved reduction of TDP-43/TBPH levels with the onset of an ALS-like locomotion defect in a Drosophila model. A similar process might trigger the human disease.
Asunto(s)
Envejecimiento/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/deficiencia , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster , Locomoción/fisiología , Ratones , Interferencia de ARN , ARN Mensajero/metabolismo , Especificidad de la EspecieRESUMEN
Two proteins have recently received considerable attention in the neurodegenerative research field: TDP-43 and FUS/TLS. The reason is that both proteins have been found to represent major protein components of the intracellular inclusions occurring in the neuronal tissues of patients affected by Fronto Temporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. One of the most interesting features of this discovery is that both proteins have in common several structural properties. In particular, they are multifunctional RNA-binding proteins (RBPs) already known to play a role in several cellular processes such as transcription, pre-mRNA splicing, and mRNA stability. The potential consequences of changes in their intracellular localization and protein modification status (phosphorylation, ubiquitination, and cleavage) on neuronal metabolism represent one of the major research challenges faced today by researchers. There is hope that a detailed knowledge of the gain- or loss-of-function mechanisms mediated by alterations in these proteins in the neuronal environment may provide novel therapeutic strategies for the treatment of these diseases. Here, we aim to provide an updated review of ways by which TDP-43 and FUS/TLS influence gene expression. In particular, we will focus on the characterized properties of both proteins that involve gene transcription and also RNA splicing, transport and stability processes.
Asunto(s)
Proteínas de Unión al ADN/genética , Degeneración Lobar Frontotemporal/genética , Regulación de la Expresión Génica/genética , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/fisiología , Animales , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , HumanosRESUMEN
Recent studies identified rare missense mutations in amyotrophic lateral sclerosis (ALS) patients in the TARDBP gene encoding TAR DNA binding protein (TDP)-43, the major protein of the ubiquitinated inclusions (UBIs) found in affected motor neurons (MNs). The aim of this study was to further define the spectrum of TARDBP mutations in a large cohort of 666 Italian ALS patients (125 familial and 541 sporadic cases). The entire coding region was sequenced in 281 patients, while in the remaining 385 cases only exon 6 was sequenced. In 18 patients, of which six are familial, we identified 12 different heterozygous missense mutations (nine novel) all locating to exon 6, which were absent in 771 matched controls. The c.1144G>A (p.A382T) variation was observed in seven patients, thus representing the most frequent TARDBP mutation in ALS. Analysis of microsatellites surrounding the TARDBP gene indicated that p.A382T was inherited from a common ancestor in 5 of the 7 patients. Altogether, the frequency of TARDBP gene mutations appears to be particularly high in Italian ALS patients compared to individuals of mainly Northern European origin (2.7% vs. 1%). Western blot analysis of lymphocyte extracts from two patients carrying the p.A382T and p.S393L TARDBP mutations showed the presence of lower molecular weight TDP-43 bands, which were more abundant than observed in healthy controls and patients negative for TARDBP mutations. In conclusion, this report contributes to the demonstration of the causative role of the TARDBP gene in ALS pathogenesis and indicates that mutations may affect the stability of the protein even in nonneuronal tissues.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Mutación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Italia , Linfocitos/metabolismo , Masculino , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Adulto JovenRESUMEN
The extracellular matrix (ECM) is a highly dynamic structure that not only provides a physical framework for cells within connective tissues, but also imparts instructive signals for development, tissue homeostasis and basic cell functions through its composition and ability to exert mechanical forces. The ECM of tissues is composed of, in addition to proteoglycans and hyaluronic acid, a number of proteins, most of which are generated after alternative splicing of their pre-mRNA. However, the precise function of these protein isoforms is still obscure in most cases. Fibronectin (FN), one of the main components of the ECM, is also one of the best-known examples of a family of proteins generated by alternative splicing, having at least 20 different isoforms in humans. Over the last few years, considerable progress on elucidating the functions of the alternatively spliced FN isoforms has been achieved with the essential development of key engineered mouse strains. Here we summarize the phenotypes of the mouse strains having targeted mutations in the FN gene, which may lead to novel insights linking function of alternatively spliced isoforms of fibronectin to human pathologies.
Asunto(s)
Empalme Alternativo , Fibronectinas/genética , Animales , Aterosclerosis/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Fenotipo , Filogenia , Conformación Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fibrosis Pulmonar/genética , Trombosis/genéticaRESUMEN
The hepatitis C virus (HCV) 5'-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation.
Asunto(s)
Hepacivirus/genética , Biosíntesis de Proteínas , ARN Viral/química , Ribosomas/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , MutaciónRESUMEN
Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.
Asunto(s)
Empalme Alternativo , Núcleo Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Unión al ADN/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Unión Competitiva , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN/metabolismo , Exones , Eliminación de Gen , Glutatión Transferasa/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Ácidos Nucleicos/metabolismo , Oligonucleótidos/metabolismo , Fenilalanina/química , Plásmidos/metabolismo , Polimorfismo Genético , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Rayos UltravioletaRESUMEN
Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.
Asunto(s)
Empalme Alternativo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas de Unión al ADN/metabolismo , Exones , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/genética , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Polimorfismo Genético , ARN sin Sentido , ARN Mensajero , Proteínas de Unión al ARN , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Empalme Serina-Arginina , Distribución Tisular , TransfecciónRESUMEN
The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon-intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.
Asunto(s)
Exones/genética , Intrones/genética , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Trombopoyetina/genética , Empalme Alternativo/genética , Composición de Base , Secuencia de Bases , ADN Complementario/genética , Humanos , Cinética , Leucocitos , Datos de Secuencia Molecular , Mutación/genética , Especificidad por Sustrato , Transfección , Células Tumorales CultivadasAsunto(s)
Enfermedades Cardiovasculares/genética , Alelos , Animales , Apolipoproteínas E/genética , Línea Celular , Genoma , Humanos , Mutación , Polimorfismo GenéticoAsunto(s)
Apolipoproteína A-I/genética , Lipoproteínas HDL/sangre , Animales , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Arteriosclerosis/genética , Arteriosclerosis/metabolismo , Humanos , Ratones , Ratones Transgénicos , Estrés Oxidativo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Unión ProteicaRESUMEN
Polymorphic variants of the cytoskeletal protein adducin have been associated with hypertension in humans and rats. However, the direct role of this protein in modulating arterial blood pressure has never been demonstrated. To assess the effect of beta-adducin on blood pressure, a beta-adducin-deficient mouse strain (-/-) was studied and compared with wild-type controls (+/+). Aortic blood pressure was measured in nonanesthetized, freely moving animals with the use of telemetry implants. It is important to note that these mice have at least 98% of C57Bl/6 genetic background, with the only difference from wild-type animals being the beta-adducin mutation. We found statistically significant higher levels of systolic blood pressure (mm Hg) (mean+/-SE values: -/-: 126.94+/-1.14, n=5; +/+: 108.06+/-2. 34, n=6; P:=0.0001), diastolic blood pressure (-/-: 83.54+/-1.07; +/+: 74.87+/-2.23; P:=0.005), and pulse blood pressure (-/-: 43. 32+/-1.10; +/+: 33.19+/-1.96; P:=0.001) in beta-adducin-deficient mice. Western blot analysis showed that as a result of the introduced genetic modification, beta-adducin was not present in heart protein extracts from -/- mice. Consequently, this deficiency produced a sharp decrease of alpha-adducin and a lesser reduction in gamma-adducin levels. However, we found neither cardiac remodeling nor modification of the heart function in these animals. This is the first report showing direct evidence that hypertension is triggered by a mutation in the adducin gene family.
Asunto(s)
Proteínas de Unión a Calmodulina/deficiencia , Proteínas del Citoesqueleto/deficiencia , Hipertensión/genética , Animales , Presión Sanguínea/genética , Proteínas de Unión a Calmodulina/genética , Proteínas del Citoesqueleto/genética , Electrocardiografía , Frecuencia Cardíaca/genética , Frecuencia Cardíaca/fisiología , Ventrículos Cardíacos/patología , Hipertensión/patología , Hipertensión/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patologíaRESUMEN
Gaucher disease, the most common glycolipid storage disease, can be caused by a large variety of mutations. We report here the identification and characterization of a novel mutation in the human glucocerebrosidase gene, IVS 8 (-11delC) (-14T>A), in two siblings with Gaucher disease type I which occurs within the 3' end of intron 8. Both siblings were compound heterozygotes for the IVS 8 (-11delC) (-14T>A) mutation and for the c.626 G>C (R170P) substitution within exon 6. No mRNA species carrying the IVS 8 (-11delC) (-14T>A) mutation were detected by RT-PCR analysis of the RNA extracted from the patients' fibroblasts. To study the possible effects of the IVS 8 (-11delC) (-14T>A) sequence alteration on the splicing of the proximal exon 9, we have established an in vitro system generating a minigene carrying the genomic region of human glucocerebrosidase spanning from exon 8 to exon 10. Transfections into the human Hep3B cell line of the wild-type construct resulted in the expression of mRNA with the glucocerebrosidase exons correctly spliced. On the contrary, transfections of the construct carrying the IVS 8 (-11delC) (-14T>A) mutation resulted in the expression of mRNA with an 11-bp insertion located between the end of exon 8 and the beginning of exon 9. These results indicated that the 5243T>A substitution created a new 3' splice site 11 bp upstream of the wild-type one, leading to the incorporation into the mRNA of these extra 11 bases. Moreover, the new 3' splice site created by this 5243T>A transversion was preferred over the wild-type one in 100% of cases. The in vitro studies suggest that, in the patients, the 11-bp inclusion causes a shift in the reading frame with the generation of a stop codon after codon 388 which undergoes early degradation.
Asunto(s)
Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Eliminación de Secuencia , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Exones , Femenino , Enfermedad de Gaucher/enzimología , Heterocigoto , Humanos , Intrones , Italia , Masculino , Datos de Secuencia Molecular , Núcleo Familiar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales CultivadasRESUMEN
Hereditary eosinophil peroxidase deficiency is a genetic abnormality characterized by a decrease or absence of peroxidase activity and a reduction of the granule matrix volume. Recently, we identified two mutations associated with eosinophil peroxidase deficiency in a subject and his siblings, i.e. a base insertion causing the appearance of a premature stop codon and a base transition causing the replacement of an Arg at codon 286 with a His (R286H). In this article we report the stable expression of both the recombinant wild-type and the R286H eosinophil peroxidase precursor in the K-562 cell line, and the effects of the R286H substitution on the structure and function of the eosinophil peroxidase precursor. Heme group incorporation into both the recombinant wild-type and the recombinant R286H eosinophil peroxidase precursor was comparable, as was the stability of both proteins. Instead, the recombinant R286H eosinophil peroxidase precursor exhibited marked alterations of the catalytic properties and an increased sensitivity to four peroxidase inhibitors with respect to both the recombinant wild-type eosinophil peroxidase precursor and the native enzyme. In addition, the recombinant wild-type, but not the R286H, eosinophil peroxidase precursor was immunoprecipitated by two anti-(eosinophil peroxidase) mAbs. Altogether, our results suggest a protein misfolding of the R286H eosinophil peroxidase precursor which might account for its altered catalytic properties and the absence of expression of some epitopes.
Asunto(s)
Peroxidasas/genética , Peroxidasas/metabolismo , Sustitución de Aminoácidos , Bencidinas/metabolismo , Línea Celular , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Peroxidasa del Eosinófilo , Humanos , Peróxido de Hidrógeno/metabolismo , Peroxidasas/inmunología , Fenilendiaminas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of beta-adducin produces an 80% decrease of alpha-adducin and a fourfold up-regulation of gamma-adducin in erythrocytes. beta-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of -/- erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of -/- mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably alphagamma adducin) could be functional and might account for the mild phenotype. (Blood. 2000;95:3978-3985)
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Eliptocitosis Hereditaria/sangre , Eliptocitosis Hereditaria/genética , Eritrocitos/fisiología , Animales , Proteínas de Unión a Calmodulina/deficiencia , Proteínas de Unión a Calmodulina/genética , Quimera , Cruzamientos Genéticos , Proteínas del Citoesqueleto/metabolismo , Femenino , Hematócrito , Hemoglobinas/análisis , Heterocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragilidad Osmótica , Recuento de ReticulocitosRESUMEN
BACKGROUND: The role of HCV infection in the development of chronic liver disease is still unclear. OBJECTIVES: Assess the presence of HCV infection in patients with liver cirrhosis. STUDY DESIGN: 123 cases of cirrhotic liver randomly selected over a 25 years period (1969-1994) from the autopsy archives of the Pathology Department of the University of Trieste, Italy, were analyzed for the presence of HCV viral genome. METHODS: Total RNA was extracted from formalin-fixed paraffin-embedded tissues of the cirrhotic liver. Genotype analysis for HCV was performed after RT-PCR by dot-blot hybridization with the three major genotype-specific probes (G1, G2 and G3). RESULTS: The overall HCV genome frequency was 50.4% (62/123). The positivity was quite constant in the 1969-1979 period (35-38%), rose to 65% in 1984, peaked to 77% in 1989 (P<0.005 vs. the previous decade), and decreased to 50% in 1994. HCV genotype G1 was found in 89% of the 62 positive samples. The mean age of death of HCV-positive and HCV-negative patients was comparable (69+/-12 vs. 67+/-16 years, NS). CONCLUSIONS: These data show an increasing frequency of HCV infection in cirrhotic liver tissues from 1969 to 1994, which peaked in 1989. The genotype G1 was the almost uniquely associated with cirrhosis. These findings indicate that the HCV infection occurred around the late 1950s-early 1960s, thus supporting the hypothesis of a cohort effect. HCV infection seems not to alter the natural history of liver cirrhosis as indicated by the comparable age at death of HCV positive and HCV negative patients.
Asunto(s)
Hepacivirus/aislamiento & purificación , Hepatitis C/complicaciones , Cirrosis Hepática/complicaciones , Hígado/virología , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Genoma Viral , Genotipo , Hepacivirus/genética , Hepatitis C/epidemiología , Hepatitis C/virología , Humanos , Cirrosis Hepática/epidemiología , Cirrosis Hepática/virología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Prevalencia , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Exones , Intrones , Animales , Secuencia de Bases , Secuencia Conservada , Amplificación de Genes , Genes Sintéticos , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Polimorfismo Genético , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo RestrictivoRESUMEN
Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).
Asunto(s)
Hepacivirus/genética , Conformación de Ácido Nucleico , ARN Viral/química , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Datos de Secuencia MolecularRESUMEN
The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3' splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG)(n)repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3' splice site sequences, the extent of exon 9 skipping in CFTR mRNA.
Asunto(s)
Empalme Alternativo/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Exones , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/análisis , Alineación de Secuencia , Células Tumorales CultivadasRESUMEN
We have investigated the involvement of human apolipoprotein A-IV (apoA-IV) in gastric acid secretion and ulcer formation in recently generated apoA-IV transgenic mice. Compared to control littermates, transgenic animals showed a gastric acid secretion decreased by 43-77% whereas only slight variations were observed in the different cell population densities within the gastric mucosa. In addition, no variation in gastrin levels was observed. Transgenics were protected against indomethacin-induced ulcer formation, with lesions diminishing by 45 to 64% compared to controls. These results indicate that endogenous apoA-IV expression can regulate gastric acid secretion and ulcer development.