RESUMEN
Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.
Asunto(s)
Mutación , Proteínas Nucleares , Precursores del ARN/metabolismo , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Precursores del ARN/genética , ARN Mensajero/genética , ARN Nuclear Pequeño/análisis , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteínas/metabolismo , Factor de Empalme U2AFRESUMEN
The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use.
Asunto(s)
Empalme Alternativo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas Receptoras , Receptores de Factores de Crecimiento de Fibroblastos/genética , Secuencia de Bases , Análisis Mutacional de ADN , Exones , Células HeLa , Humanos , Técnicas In Vitro , Queratinocitos/metabolismo , Datos de Secuencia Molecular , Precursores de Ácido Nucleico/metabolismo , Oligodesoxirribonucleótidos/química , ARN Mensajero/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Relación Estructura-ActividadRESUMEN
We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Exones , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Proteínas de Unión al ADN/aislamiento & purificación , Intrones , Mamíferos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Moldes GenéticosRESUMEN
Transcriptional activation endowed by AP-1 or CREB binding sites can be significantly reduced in transient transfection tests by expression from the corresponding cloned cDNAs of protein tyrosine phosphatases. Both the protein tyrosine phosphatase 1B and the T-cell protein tyrosine phosphatase, as well as a novel form of this latter protein generated by an alternative splicing even show this activity. The effect is specific, as none of the protein tyrosine phosphatases alters transcriptional activation by either the estrogen receptor, GAL4, or a GAL4-VP16 fusion protein. Furthermore, the activities of the SV40 early gene promoter and a Moloney murine leukemia virus long terminal repeat promoter are not reduced by these phosphatases. We conclude that a yet to be identified protein phosphorylated on tyrosine is necessary for a full transcriptional response via AP-1 or CREB binding sites.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fosfoproteínas Fosfatasas/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , ADN/genética , ADN/aislamiento & purificación , Expresión Génica , Genes src , Humanos , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Regiones Promotoras Genéticas , Proteínas Tirosina Fosfatasas , Proteínas Proto-Oncogénicas c-jun , TransfecciónRESUMEN
The BEK transmembrane protein tyrosine kinase is a receptor for both acidic and basic fibroblast growth factors. We identify several different transcripts which code for BEK-related proteins. These proteins differ from BEK in regions expected to control receptor activity. Thus, some of the proteins have altered extracellular, ligand-binding domains, and others an altered carboxy-terminal tail. Still other forms of BEK differ only in their juxtamembrane domains. Sequencing of parts of the BEK gene shows that alternative splicing of the premessenger can account for at least some of this diversity. In particular, an apparently tissue specific, mutually exclusive splicing of two internal exons permits both the previously described K-SAM mRNA and the BEK mRNA to be derived from the same premessenger.
Asunto(s)
ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras , Receptores de Superficie Celular/genética , Transcripción Genética/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/ultraestructura , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/ultraestructura , Línea Celular , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Variación Genética/genética , Humanos , Inmunoglobulinas/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Empalme del ARN/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Receptores Mitogénicos/genética , Receptores Mitogénicos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/ultraestructuraRESUMEN
Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary, this study describes a general method to titrate rAAV, independently of the transgene and its expression, and to measure the level of contamination with adenovirus and rep-positive AAV. Furthermore, we report a new production procedure using adenoviral plasmids instead of virions and resulting in rAAV stocks with undetectable adenovirus contamination.
Asunto(s)
Dependovirus/genética , Vectores Genéticos , Cultivo de Virus , Animales , Dependovirus/crecimiento & desarrollo , Células HeLa , Virus Helper/genética , Humanos , Immunoblotting , Músculo Esquelético/química , Plásmidos , Ratas , Ratas Wistar , Recombinación Genética , Transfección , beta-Galactosidasa/genéticaRESUMEN
The mammalian spliceosome-associated protein, SAP 49, is associated specifically with U2 snRNP and is the most efficiently UV cross-linked protein in the spliceosomal complexes A, B, and C. We show here that SAP 49 cross-links to a region in the pre-mRNA immediately upstream of the branchpoint sequence in the prespliceosomal complex A. In addition to the RNA-binding activity of SAP 49, we show that this protein interacts directly and highly specifically with another U2 snRNP-associated spliceosomal protein, SAP 145. We have isolated a cDNA-encoding SAP 49 and find that it contains two amino-terminal RNA-recognition motifs (RRMs), consistent with the observation that SAP 49 binds directly to pre-mRNA. The remainder of the protein is highly proline-glycine rich (39% proline and 17% glycine). Unexpectedly, the SAP 49-SAP 145 protein-protein interaction requires the amino-terminus of SAP 49 that contains the two RRMs. The observation that SAP 49 and SAP 145 interact directly with both U2 snRNP and the pre-mRNA suggests that this protein complex plays a role in tethering U2 snRNP to the branch site.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Reactivos de Enlaces Cruzados , ADN Complementario/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U2 , Proteínas de Saccharomyces cerevisiae , Empalmosomas/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Genes Fúngicos , Células HeLa , Humanos , Mamíferos , Datos de Secuencia Molecular , Unión Proteica , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/genética , Factores de Empalme de ARN , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Gene therapy is obviously a controversial issue and a wave of suspicion has dampened the initial enthusiasm raised by this new therapeutic approach. It has now become fashionable to downplay the potential for gene therapy in most fields including kidney-related diseases. In our opinion, this is an unfair and unrealistic view of the future. In fact, gene therapy of well-selected kidney diseases will certainly become feasible, but a large data base on vectors and transfer methods both in the normal kidney and in disease models has first to be collected. Any significant progress in the biology of the vectors, in the cellular interactions of the newly introduced DNA, and in the regulation and persistency of the transgene should be rapidly translated to the kidney in relevant experimental models. Herein, we present the use and current limitations of gene transfer to the kidney and the potential therapeutic perspectives.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Riñón/fisiología , Animales , Humanos , Enfermedades Renales/genética , Enfermedades Renales/terapia , Virus/genéticaRESUMEN
We have isolated, from a human tumor cDNA library, a gene encoding a putative receptor-like protein-tyrosine kinase that we call TK14. The amino acid sequence of the TK14 protein is closely related to the available partial sequence of the mouse protein bek, and more distantly related to the sequences of a chicken basic fibroblast growth factor receptor (73% sequence homology) and the apparent human equivalent of this receptor, the FLG protein (encoded by the fms-like tyrosine kinase gene). Overexpression of the TK14 protein by transfection of COS-1 cells with the corresponding cDNA in a simian virus 40-based expression vector leads to the appearance of new cell-surface binding sites for both acidic and basic fibroblast growth factors. This has been demonstrated by specific binding assays and chemical cross-linking experiments using 125I-labeled growth factors. It appears, therefore, that the human genome contains at least two distinct genes, for TK14 and FLG, that code for related fibroblast growth factor receptors.