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
In the assembly of a prespliceosome, U2 small nuclear ribonucleoprotein (snRNP) functions in pre-messenger RNA (mRNA) splicing together with splicing factors (SFs) 3a, SF3b, and several other proteins. The 17S but not the 12S form of U2 snRNP is active in splicing-complex formation. Here it is shown that the SF3a subunits correspond to three of the 17S U2 snRNP-specific polypeptides. SF3a interacts with U2 snRNP in the presence of SF3b to generate a structure similar to 17S U2 snRNP, which suggests a function for SF3a and SF3b in the incorporation of U2 snRNP into the spliceosome. Furthermore, the 60-kilodalton subunit of SF3a is related to the yeast splicing protein PRP9.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Proteínas de Saccharomyces cerevisiae , Empalmosomas/metabolismo , Adenosina Trifosfato/metabolismo , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/inmunología , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/inmunología , Factores de Empalme de ARN , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U2/químicaRESUMEN
Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.
Asunto(s)
Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas/genética , Animales , Secuencia de Bases , Unión Competitiva , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Caperuzas de ARN/metabolismo , ARN Nuclear Pequeño/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares Pequeñas , Especificidad por Sustrato , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
The kill constants beta biol. for cocci are determined in a static aerosol chamber. To do this, the particle diminution (living and dead colony-forming units, KE) is determined with a scattered light photometer and the decrease in reseedability of the colony-forming units is measured by a six-stage Andersen collector. The difference is the kill constant beta biol. which is independent of the physical diminution. For use with model calculations it is recommended that limit values should be assumed which realistically express the wide scatter range of the values.
Asunto(s)
Microbiología del Aire , Fenómenos Fisiológicos Bacterianos , Aerosoles , Micrococcus/fisiología , Microscopía Electrónica de Rastreo , Especificidad de la Especie , Staphylococcus/fisiología , Streptococcaceae/fisiologíaRESUMEN
A cDNA encoding the 60 kDa subunit of mammalian splicing factor SF3a has been isolated. The deduced protein sequence reveals a 30% identity to the PRP9 splicing protein of the yeast S.cerevisiae. The highest homology is present in a zinc finger-like region in the C-terminal domain of both proteins. The PRP9 zinc finger-like motif has been replaced by the equivalent region of mammalian SF3a60. The chimeric protein rescues the temperature-sensitive phenotype of the prp9-1 mutant strain demonstrating that not only the structure but also the function of this domain has been conserved during evolution.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U2 , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Escherichia coli/genética , Genes/genética , Genes Fúngicos/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Peso Molecular , Mutación/fisiología , Fenotipo , Factores de Empalme de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/química , Alineación de Secuencia , Análisis de Secuencia de ADN , Dedos de Zinc/genéticaRESUMEN
Mammalian splicing factor SF3a consists of three subunits of 60, 66, and 120 kDa and functions early during pre-mRNA splicing by converting the U2 snRNP into its active form. A cDNA encoding the 120-kDa subunit of SF3a has been cloned. The SF3a120 gene was localized to human chromosome 22, and three mRNAs of 3.2, 3.8, and 5.7 kb are ubiquitously expressed. The N-terminal half of the deduced SF3a120 amino acid sequence contains a tandemly repeated motif (the SURP module) that has recently been identified in the essential splicing factor PRP21p of Saccharomyces cerevisiae, the Drosophila alternative splicing regulator suppressor-of-white-apricot, and four proteins from nematodes and mammals; the C-terminal half is organized into a proline-rich region and a ubiquitin-like domain. The spacing between the SURP modules and the protein's essential function in constitutive splicing identify SF3a120 as the mammalian homologue of yeast PRP21p. Binding studies with truncated derivatives of SF3a120 revealed that the SURP domains function in binding to SF3a60, whereas a region of 130 amino acids C-terminal to these domains is essential for contacts with SF3a66.
Asunto(s)
Proteínas Fúngicas/genética , Familia de Multigenes , Empalme del ARN , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U2 , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 22 , Clonación Molecular , Cricetinae , ADN Complementario/genética , Femenino , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.