RESUMEN
The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.
Asunto(s)
Ribonucleoproteína Nuclear Pequeña U2/ultraestructura , Empalmosomas/química , Resinas de Intercambio Aniónico , Secuencia de Bases , Cromatografía Liquida , Células HeLa , Humanos , Nucleasa Microcócica , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Fosfoproteínas/química , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestructura , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Factores de Empalme de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Resinas Sintéticas , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/metabolismo , Empalmosomas/ultraestructuraRESUMEN
Mammalian splicing factor SF1 consists of a single polypeptide of 75 kDa and is required for the formation of the first ATP-dependent spliceosomal complex. Three cDNAs encoding variant forms of SF1 have been isolated and four highly related cDNAs have been found in current databases. Comparison of the cDNA sequences suggests that different SF1 mRNAs are generated by alternative splicing of a common pre-mRNA. In agreement with this idea, at least three mRNAs that are differentially expressed in different cell types have been detected by northern blot analysis. All SF1 cDNAs identified encode proteins with a common N-terminal half that contains two structural motifs implicated in RNA binding (an hnRNP K homology [KH] domain and a zinc knuckle), but the proteins differ in the length of a proline-rich region and have distinct C-termini. Three SF1 isoforms expressed in insect cells via baculovirus transfer vectors show comparable activities in the assembly of a pre-splicing complex. Consistent with the presence of a KH domain and a zinc knuckle, we show that SF1 binds directly to RNA. This interaction appears to be largely sequence-independent with a preference for guanosine- and uridine-rich sequences. The KH domain of SF1 is embedded in a 160-amino acid sequence that is shared with human Sam68, a target of Src during mitosis, as well as Caenorhabditis elegans GLD-1 and mouse Qkl, both of which play roles during cellular differentiation. The presence of this shared region in SF1 suggests functions in addition to its role in pre-spliceosome assembly.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Unión al ADN , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario , Expresión Génica , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Factores de Empalme de ARN , ARN Mensajero , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Homología de Secuencia de Aminoácido , Spodoptera/citologíaRESUMEN
The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs apparently by recruiting host factor(s) required for export of cellular messenger RNAs. Here, we report the identification of TAP as the cellular factor that specifically binds to wild-type CTE but not to export-deficient CTE mutants. Microinjection experiments performed in Xenopus oocytes demonstrate that TAP directly stimulates CTE-dependent export. Furthermore, TAP overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Thus, TAP, like its yeast homolog Mex67p, is a bona fide mRNA nuclear export mediator. TAP is the second cellular RNA binding protein shown to be directly involved in the export of its target RNA.