RESUMO
The molecular mechanisms of the second step of pre-mRNA splicing in yeast and higher eukaryotes are reviewed. The important elements in the pre-mRNA, the participating proteins, and the proposed secondary structures and roles of the snRNAs are described. The sequence of events in the second step is presented, focusing on the actions of the proteins in setting up and facilitating the second reaction. Mechanisms for avoiding errors in splicing are discussed.
Assuntos
Precursores de RNA/metabolismo , Splicing de RNA , RNA Helicases DEAD-box/metabolismo , Éxons , Precursores de RNA/química , Sítios de Splice de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismoRESUMO
Two recent papers, one from the Staley laboratory (Koodathingal and colleagues) and the other from the Cheng laboratory (Tseng and colleagues), show that the RNA-dependent ATPase Prp16, which is required for the second step of splicing, acts to reject slowly splicing pre-mRNAs immediately before the first catalytic reaction in pre-mRNA splicing. The results answer long-investigated questions about the actions of Prp16 and provide a wealth of molecular details on the proofreading process in pre-mRNA splicing. The discussion here reviews and integrates the results of the two papers and describes the implications for proofreading in splicing.
Assuntos
Adenosina Trifosfatases/metabolismo , RNA Helicases/metabolismo , Precursores de RNA/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Catálise , RNA Helicases/genética , Fatores de Processamento de RNA , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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
Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly restored the pre-mRNA splicing activity that was lost in the prp18 mutants. The close functional relationship of Prp18 and U5 is emphasized by the finding that two snr7 alleles, U5A and U6A, are dominant synthetic lethal with prp18 alleles. Our results support the idea that Prp18 and the U5 snRNA act in concert during the second step of pre-mRNA splicing and suggest a model in which the conserved loop of Prp18 acts to stabilize the interaction of loop 1 of the U5 snRNA with the splicing intermediates.
Assuntos
Proteínas Nucleares/fisiologia , Splicing de RNA/fisiologia , RNA Nuclear Pequeno/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Supressão Genética/genética , Alelos , Sequência de Aminoácidos , Sequência Conservada/genética , Sequência Conservada/fisiologia , Evolução Molecular , Genes Fúngicos/genética , Genes Fúngicos/fisiologia , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Mutação Puntual/genética , Splicing de RNA/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U5 , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Supressão Genética/fisiologiaRESUMO
The spliceosomal cyclophilin H is a specific component of the human U4/U6 small nuclear ribonucleoprotein particle, interacting with homologous sequences in the proteins U4/U6-60K and hPrp18 during pre-mRNA splicing. We determined the crystal structure of the complex comprising cyclophilin H and the cognate domain of U4/U6-60K. The 31 amino acid fragment of U4/U6-60K is bound to a region remote from the cyclophilin active site. Residues Ile118-Phe121 of U4/U6-60K expand the central beta-sheet of cyclophilin H and the side-chain of Phe121 inserts into a hydrophobic cavity. Concomitantly, in the crystal the cyclophilin H active site is occupied by the N terminus of a neighboring cyclophilin H molecule in a substrate-like manner, indicating the capacity of joint binding to a substrate and to U4/U6-60K. Free and complexed cyclophilin H have virtually identical conformations suggesting that the U4/U6-60K binding site is pre-shaped and the peptidyl-prolyl-cis/trans isomerase activity is unaffected by complex formation. The complex defines a novel protein-protein interaction mode for a cyclophilin, allowing cyclophilin H to mediate interactions between different proteins inside the spliceosome or to initiate from its binding platforms isomerization or chaperoning activities.
Assuntos
Ciclofilinas/química , Peptidilprolil Isomerase/química , Ribonucleoproteína Nuclear Pequena U4-U6/química , Spliceossomos/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Ligação Proteica , Conformação ProteicaRESUMO
Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA. Prp18 interacts with Slu7, and the functions of the two proteins are intertwined. Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing. Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7. Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome. Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7. Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function. Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop.
Assuntos
Proteínas Nucleares , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Análise Mutacional de DNA , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Conformação Proteica , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U5 , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
We report that the cyclophilin USA-CyP is part of distinct complexes with two spliceosomal proteins and is involved in both steps of pre-mRNA splicing. The splicing factors hPrp18 and hPrp4 have a short region of homology that defines a high affinity binding site for USA-CyP in each protein. USA-CyP forms separate, stable complexes with hPrp18 and hPrp4 in which the active site of the cyclophilin is exposed. The cyclophilin inhibitor cyclosporin A slows pre-mRNA splicing in vitro, and we show that its inhibition of the second step of splicing is caused by blocking the action of USA-CyP within its complex with hPrp18. Cyclosporin A also slows splicing in vivo, and we show that this slowing results specifically from inhibition of USA-CyP. Our results lead to a model in which USA-CyP is carried into the spliceosome in complexes with hPrp4 and hPrp18, and USA-CyP acts during splicing within these complexes. These results provide an example of the function of a cyclophilin in a complex process and provide insight into the mechanisms of action of cyclophilins.
