RESUMO
The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5' splice site of both genes revealed that proper transient interaction with the 5' end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5' splice sites and weak branch sequences.
Assuntos
Proteínas Serina-Treonina Quinases/genética , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Íntrons/genética , Mutação , Fatores de Processamento de RNA , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Fatores de Transcrição/genéticaRESUMO
The characterization of alternatively spliced RNA is a frequently performed task in the molecular biology laboratory. Several methods have been established to characterize specific transcripts, of which microarrays, northern analysis, RT-PCR and nuclease protection assays are the most frequently performed methods in the laboratory. Here, we describe the analysis of alternatively spliced RNA by using 5(')-end labelled DNA oligonucleotide probes and S1 nuclease. The method is sensitive, allowing detection of as little as a few hundred femtograms of a specific RNA, and useful for the quantitation of alternatively spliced mRNA isoforms. Because of its insensitivity towards RNA secondary structures and partially degraded RNA, it may perform better in the quantitation of RNA than northern analysis or RT-PCR, especially when long transcripts are studied.
Assuntos
Processamento Alternativo/genética , Oligonucleotídeos/genética , Precursores de RNA/genética , Northern Blotting/métodos , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Técnicas de Sonda Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes.
Assuntos
Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/genética , Spliceossomos/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Deleção de SequênciaRESUMO
Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30-60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing.
Assuntos
Ribonucleoproteína Nuclear Pequena U1/fisiologia , Ribonucleoproteínas Nucleares Pequenas/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Cromatografia de Afinidade , Proteômica , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/isolamento & purificação , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Spliceossomos/química , Espectrometria de Massas em TandemRESUMO
Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-containing RNAs must be exported from the nucleus where they are normally retained, it must be ensured that the unspliced HIV-1 RNA is actively exported from the nucleus and protected from degradation by processes such as nonsense-mediated decay. Here we report the identification of a novel 178-nt-long exon located in the gag-pol gene of HIV-1 and its inclusion in at least two different mRNA species. Although efficiently spliced in vitro, this exon appears to be tightly repressed and infrequently used in vivo. The splicing is activated or repressed in vitro by the splicing factors ASF/SF2 and heterogeneous nuclear ribonucleoprotein A1, respectively, suggesting that splicing is controlled by these factors. Interestingly, mutations in the 5'-splice site resulted in a dramatic reduction in the steady-state level of HIV-1 RNA, and this effect was partially reversed by expression of U1 small nuclear RNA harboring the compensatory mutation. This implies that U1 small nuclear RNA binding to optimal but non-functional splice sites might have a role in protecting unspliced HIV-1 mRNA from degradation.
Assuntos
Éxons , Proteínas de Fusão gag-pol/genética , HIV-1/genética , Splicing de RNA , RNA Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Genoma Viral , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Sítios de Splice de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/metabolismo , Replicação Viral/genéticaRESUMO
U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3'-splice site in a pre-mRNA. The U2AF65 subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF(35) subunit contacts the 3'-splice site AG. Here we show that U2AF35 appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3'-splice sites and require U2AF35 for splicing in nuclear extracts from uninfected cells, are transformed to U2AF35-independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3'-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3'-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.
