RESUMO
Identification of splice sites is essential for the expression of most eukaryotic genes, allowing accurate splicing of pre-mRNAs. The splice sites are recognized by the splicing machinery based on sequences within the pre-mRNA. Here, we show that the exon sequences at the splice junctions play a significant, previously unrecognized role in the selection of 3' splice sites during the second step of splicing. The influence of the exon sequences was enhanced by the Prp18 mutant Prp18DeltaCR, and the strength of an exon sequence in Prp18DeltaCR splicing predicted its effect in wild-type splicing. Analysis of the kinetics of splicing in vitro demonstrated that 3' splice sites were chosen competitively during the second step, likely at the same time as exon ligation. In wild-type yeast, splice site selection for two genes studied was altered by point mutations in their exon bases, affecting splicing fidelity and alternative splicing. Finally, we note that the degeneracy of the genetic code allows competing 3' splice sites to be eliminated from coding regions, and we suggest that the evolution of the splicing signals and the genetic code are connected.
Assuntos
Processamento Alternativo/genética , Éxons/genética , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Actinas/genética , Processamento Alternativo/fisiologia , Sequência de Bases , Cobre , Resistência a Medicamentos , Evolução Molecular , Cinética , Metalotioneína/genética , Dados de Sequência Molecular , Plasmídeos/genética , Mutação Puntual/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Proteínas de Saccharomyces cerevisiae/genética , LevedurasRESUMO
After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37 degrees C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22.
Assuntos
Proteínas Nucleares/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Células Cultivadas , Esterificação , Éxons/genética , Humanos , Proteínas Nucleares/metabolismo , Precursores de RNA/genética , Fatores de Processamento de RNA , RNA Fúngico/química , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Interaction of the ends of the exons with loop 1 of the U5 snRNA aligns the exons for ligation in the second step of pre-mRNA splicing. To study the effect of Prp18 on the exons' interactions, we analyzed the splicing of pre-mRNAs with random sequences in the exon bases at the splice junctions. The exon mutations had large effects on splicing in yeast with a Prp18 protein lacking its most conserved region, but not in wild-type yeast. Analysis of splicing kinetics demonstrated that only the second step was affected in vivo and in vitro, showing that Prp18 - and specifically its conserved region - plays a key role in stabilizing the interaction of the exons with the spliceosome at the time of exon joining. Superior exon sequences defined by the prp18 results accelerated the second step of splicing by wild-type spliceosomes with inefficient AT-AC pre-mRNAs, implying that normal exon interactions follow the rules we discerned for prp18 splicing. Our results show that As are preferred at the ends of both exons and support a revised model of the interactions of the exons with U5 in which the exons are arranged in a continuous double helix that facilitates the second reaction.
Assuntos
Éxons/genética , Proteínas Nucleares/genética , Precursores de RNA/genética , Splicing de RNA/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo , Pareamento de Bases , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Mutação/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U5 , Spliceossomos/genética , LevedurasRESUMO
The kinetochore (centromeric DNA and associated proteins) mediates the attachment of chromosomes to the mitotic spindle apparatus and is required for faithful chromosome transmission. We established that evolutionarily conserved Saccharomyces cerevisiae SPT4, previously identified in genetic screens for defects in chromosome transmission fidelity (ctf), encodes a new structural component of specialized chromatin at kinetochores and heterochromatic loci, with roles in kinetochore function and gene silencing. Using chromatin immunoprecipitation assays (ChIP), we determined that kinetochore proteins Ndc10p, Cac1p, and Hir1p are required for the association of Spt4p to centromeric (CEN) loci. Absence of functional Spt4p leads to altered chromatin structure at the CEN DNA and mislocalization of the mammalian CENP-A homolog Cse4p to noncentromeric loci. Spt4p associates with telomeres (TEL) and HMRa loci in a Sir3p-dependent manner and is required for transcriptional gene silencing. We show that a human homolog of SPT4 (HsSPT4) complements Scspt4-silencing defects and associates with ScCEN DNA in an Ndc10p-dependent manner. Our results highlight the evolutionary conservation of pathways required for genome stability in yeast and humans.
Assuntos
Centrômero/metabolismo , Sequência Conservada , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Centrômero/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Inativação Gênica , Heterocromatina/genética , Humanos , Cinetocoros/metabolismo , Mutação/genética , Fenótipo , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismoRESUMO
Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G(2)/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested a model in which alterations or defects in kinetochore or spindle integrity lead to the distinct enrichment of Bub3p at these structures. In support of our model, kinetochore-associated Bub3-GFP is enriched upon activation of the spindle checkpoint due to nocodazole-induced spindle disassembly, overexpression of the checkpoint kinase Mps1p, or the presence of a defective centromere (CEN). Most importantly, using a novel approach with the chromatin immunoprecipitation (ChIP) technique and genetically engineered defective CEN [CF/CEN6(Delta31)], we determined that Bub3-GFP can associate with a single defective kinetochore. Our studies represent the first comprehensive molecular analysis of spindle checkpoint protein function in the context of a wild-type or defective kinetochore(s) by use of live-cell imaging and the ChIP technique in S. cerevisiae.
