Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 56
Filtrar
1.
Curr Biol ; 4(3): 264-7, 1994 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-7922333

RESUMO

Genetic and biochemical studies of pre-mRNA splicing have recently converged to elucidate an early step in the process: the targeting of the U2 small nuclear ribonucleoprotein particle to the pre-mRNA.


Assuntos
Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Processamento Alternativo , Animais , Humanos , Modelos Biológicos , Precursores de RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo
2.
Mol Cell Biol ; 8(3): 1067-75, 1988 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2835658

RESUMO

Strains of Saccharomyces cerevisiae that bear the temperature-sensitive mutation rna8-1 are defective in nuclear pre-mRNA splicing at the restrictive temperature (36 degrees C), suggesting that the RNA8 gene encodes a component of the splicing machinery. The RNA8 gene was cloned by complementation of the temperature-sensitive growth defect of an rna8-1 mutant strain. Integrative transformation and gene disruption experiments confirmed the identity of the cloned DNA and demonstrated that the RNA8 gene encodes an essential function. The RNA8 gene was shown to be represented once per S. cerevisiae haploid genome and to encode a low-abundance transcript of approximately 7.4 kilobases. By using antisera raised against beta-galactosidase-RNA8 fusion proteins, the RNA8 gene product was identified in S. cerevisiae cell extracts as a low-abundance protein of approximately 260 kilodaltons. Immunodepletion of the RNA8 protein specifically abolished the activity of S. cerevisiae in vitro splicing extracts, confirming that RNA8 plays an essential role in splicing.


Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Precursores de RNA/genética , Splicing de RNA , Saccharomyces cerevisiae/genética , Animais , Clonagem Molecular , Enzimas de Restrição do DNA , Feminino , Proteínas Fúngicas/análise , Teste de Complementação Genética , Soros Imunes , Imunoensaio , Mutação , Hibridização de Ácido Nucleico , Plasmídeos , RNA Fúngico/genética , Coelhos , Temperatura , Transcrição Gênica , Transformação Genética
3.
Mol Cell Biol ; 20(19): 7238-46, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10982841

RESUMO

Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or in vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A(0), A(1), and A(2), while Dhr1p depletion inhibited cleavage at sites A(1) and A(2). No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5' end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot.


Assuntos
RNA Helicases/isolamento & purificação , Precursores de RNA/metabolismo , Splicing de RNA , RNA Fúngico/metabolismo , RNA Ribossômico 18S/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Nucléolo Celular/metabolismo , RNA Helicases DEAD-box , Deleção de Genes , Humanos , Recém-Nascido , Substâncias Macromoleculares , Dados de Sequência Molecular , Família Multigênica , RNA Helicases/genética , RNA Helicases/metabolismo , Sequências Reguladoras de Ácido Nucleico , Esferoplastos/metabolismo , Especificidade por Substrato
4.
Mol Cell Biol ; 9(9): 3698-709, 1989 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2528686

RESUMO

The PRP4 (RNA4) gene product is involved in nuclear mRNA processing in yeast cells; we have previously cloned the gene by complementation of a temperature-sensitive mutation. Sequence and transcript analyses of the cloned gene predicted the gene product to be a 52-kilodalton protein, which was confirmed with antibodies raised against the PRP4 gene product. These antibodies inhibited precursor mRNA splicing in vitro, demonstrating a direct role of PRP4 in splicing. Immunoprecipitations with the antibodies indicated that the PRP4 protein is associated with the U4/U6 small nuclear ribonucleoprotein particle.


Assuntos
Genes Fúngicos , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Anticorpos Antifúngicos , Sequência de Bases , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Dados de Sequência Molecular , Splicing de RNA , Ribonucleoproteínas/imunologia , Ribonucleoproteínas Nucleares Pequenas , Transcrição Gênica
5.
Cancer Res ; 50(24): 7789-92, 1990 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-2123741

RESUMO

Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells. The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reductase. In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism. In order to provide further evidence that the lower reductase activity is a factor in the resistance mechanism, we incorporated NADPH:cytochrome P-450 reductase into cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C. Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed. In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reductase cDNA in a Salmonella typhimurium strain, LR5000. The bacteria expressing the rat NADPH: cytochrome P-450 reductase showed increased sensitivity to mitomycin C when incubated with this compound under aerobic conditions. However, under hypoxic conditions increased sensitivity was not observed. This parallels the previous finding with mitomycin C-resistant Chinese hamster ovary cells. These data provide direct evidence for the role of NADPH:cytochrome P-450 reductase in the cytotoxic action of this mitomycin C under aerobic but not hypoxic conditions and suggest that reduced levels of this enzyme can lead to drug resistance. P-450 reductase expressed in S. typhimurium may provide a valuable tool for evaluating the role of this enzyme in the toxicity of drugs activated through a one electron reduction pathway.


Assuntos
Antineoplásicos/farmacologia , Mitomicinas/farmacologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Neoplasias da Mama , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Escherichia coli/genética , Feminino , Humanos , Cinética , Mitomicina , NADPH-Ferri-Hemoproteína Redutase/genética , Plasmídeos , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética
6.
Genetics ; 157(4): 1451-67, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11290703

RESUMO

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.


Assuntos
Proteínas Fúngicas/fisiologia , RNA Helicases/fisiologia , Splicing de RNA , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae , Spliceossomos/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Fases de Leitura Aberta , Testes de Precipitina , RNA Helicases/genética , RNA Helicases/metabolismo , Fatores de Processamento de RNA , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Técnicas do Sistema de Duplo-Híbrido
7.
Genetics ; 156(4): 1503-17, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11102353

RESUMO

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclo Celular/genética , Proteínas de Ligação a DNA , Proteínas Fúngicas/fisiologia , Genes Fúngicos , RNA Helicases , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fase G2/genética , Humanos , Dados de Sequência Molecular , Fatores de Processamento de RNA , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Spliceossomos/genética
8.
Genetics ; 154(1): 61-71, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10628969

RESUMO

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.


Assuntos
Ciclo Celular/genética , Proteínas de Ligação a DNA , Genes Fúngicos , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Alelos , Sequência de Bases , Proteínas de Ciclo Celular/genética , Mecanismo Genético de Compensação de Dose , Proteínas Fúngicas/genética , Mutagênese , Fenótipo , RNA , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Saccharomyces cerevisiae/genética
9.
Gene ; 110(1): 33-9, 1992 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-1544575

RESUMO

We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Oxigenases/genética , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/biossíntese , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Oxigenases/biossíntese , Ratos , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/química
10.
FEBS Lett ; 318(1): 4-6, 1993 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-8436222

RESUMO

We have investigated whether a spliceosomal protein analogous to the yeast protein, PRP8, was present in higher plants. A protein with a molecular weight > 200 kDa was detected in Western blots of tobacco (Nicotiana tabacum L.) nuclear extracts with affinity-purified antibodies, raised against four different beta-galactosidase-PRP8 fusion proteins. The < 200 kDa protein was also immunoprecipitated by antibodies against the snRNA-specific trimethylguanosine cap structure and was, therefore, snRNP-associated. The presence of this protein in plants, in addition to yeast, Drosophila and humans, and the conservation of large size and epitopes highlights the importance of PRP8 in pre-mRNA splicing.


Assuntos
Proteínas Fúngicas/análise , Nicotiana/química , Proteínas de Plantas/análise , Plantas Tóxicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Spliceossomos/química , Western Blotting , Reações Cruzadas , Proteínas Fúngicas/química , Proteínas de Plantas/química , Testes de Precipitina , RNA Nuclear Pequeno/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5
11.
Toxicology ; 82(1-3): 3-20, 1993 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-8236279

RESUMO

In this report we describe the heterologous expression of glutathione S-transferase (GST) and cytochrome P450 reductase (Red) in E. coli and Salmonella typhimurium. The same expression vectors could be applied to both systems and high levels of catalytically active GST and Red were obtained. Interestingly the level of expression was invariably higher in S. typhimurium. The level of the alpha class GST being up to 20% of the total bacterial protein. A further advantage of the salmonella system is that strains were used which can be applied to mutagenicity tests. This system was validated by demonstrating increasing mutation frequency of halogenated hydrocarbons in strains expressing the GST and increased cytotoxicity of mitomycin C in cells expressing P450 reductase.


Assuntos
Glutationa Transferase/biossíntese , NADPH-Ferri-Hemoproteína Redutase/biossíntese , Preparações Farmacêuticas/metabolismo , Animais , Animais Geneticamente Modificados , Clonagem Molecular , Escherichia coli , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Testes de Mutagenicidade , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae , Salmonella typhimurium
12.
Biochem Soc Trans ; 33(Pt 3): 433-8, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15916535

RESUMO

Sm and Lsm proteins are ubiquitous in eukaryotes and form complexes that interact with RNAs involved in almost every cellular process. My laboratory has studied the Lsm proteins in the yeast Saccharomyces cerevisiae, identifying in the nucleus and cytoplasm distinct complexes that affect pre-mRNA splicing and degradation, small nucleolar RNA, tRNA processing, rRNA processing and mRNA degradation. These activities suggest RNA chaperone-like roles for Lsm proteins, affecting RNA-RNA and/or RNA-protein interactions. This article reviews the properties of the Sm and Lsm proteins and structurally and functionally related proteins in archaea and eubacteria.


Assuntos
Processamento Pós-Transcricional do RNA , RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Autoantígenos , Humanos , RNA/genética , Ribonucleoproteínas Nucleares Pequenas/química , Proteínas Centrais de snRNP
13.
Nature ; 275(5676): 104-9, 1978 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-357984

RESUMO

Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.


Assuntos
DNA Recombinante , Escherichia coli/genética , Plasmídeos , Saccharomyces cerevisiae/genética , Transformação Genética , Hibridização Genética , Métodos
14.
Nucleic Acids Res ; 19(20): 5483-9, 1991 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-1945827

RESUMO

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.


Assuntos
Proteínas Fúngicas/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , DNA Fúngico , Proteínas Fúngicas/efeitos da radiação , Dados de Sequência Molecular , Testes de Precipitina , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/efeitos da radiação , Raios Ultravioleta
15.
Mol Microbiol ; 5(4): 805-12, 1991 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1857205

RESUMO

The spp81/ded1 mutations were isolated as suppressors of a Saccharomyces cerevisiae pre-mRNA splicing mutation, prp8-1. The SPP81/DED1 gene encodes a putative ATP-dependent RNA helicase. While attempting to clone the wild-type SPP81/DED1 gene we isolated plasmids which were able to suppress the cold-sensitive growth defect of spp81 mutants. These plasmids encoded a gene (named DBP1) which mapped to chromosome XVI and not to the SPP81/DED1 locus on chromosome XV. The cloned gene suppressed the defect of spp81/ded1 mutants when present on both high and low copy-number plasmids but complemented spp81/ded1 null mutants only when present on high copy-number plasmids. In contrast to the SPP81/DED1 gene the DBP1 gene was not essential for cell viability. The nucleotide sequence of the DBP1 gene revealed that it also encoded a putative ATP-dependent RNA helicase which showed considerable similarity at the amino acid level to the SPP81/DED1 protein.


Assuntos
Genes Supressores , RNA Nucleotidiltransferases/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Sobrevivência Celular/genética , Clonagem Molecular , RNA Helicases DEAD-box , Genes Fúngicos , Dados de Sequência Molecular , Mutação , RNA Helicases , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia
16.
EMBO J ; 11(10): 3721-9, 1992 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1396567

RESUMO

Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro. In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes. In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snRNP complexes. Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form. As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes. As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically. We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events.


Assuntos
Proteínas Fúngicas/metabolismo , Precursores de RNA/genética , Splicing de RNA , RNA Fúngico/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Proteínas Fúngicas/genética , Cinética , Dados de Sequência Molecular , RNA Helicases , RNA Nucleotidiltransferases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Fatores de Tempo , Transcrição Gênica , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
17.
Nucleic Acids Res ; 18(22): 6559-64, 1990 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-2251118

RESUMO

PRP2 protein of Saccharomyces cerevisiae is required for the pre-mRNA splicing reaction but not for the early stages of spliceosome assembly. Using anti-PRP2 antibodies we demonstrate that PRP2 protein is associated with spliceosomes prior to, and throughout step 1 of the splicing reaction. Heat-inactivated prp2 protein, by contrast, does not seem to associate with spliceosomes. By elution of electrophoretically distinct spliceosomal complexes from non-denaturing gels we identify the specific complex with which PRP2 initially interacts in the pathway of spliceosome assembly.


Assuntos
Proteínas Fúngicas/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Saccharomyces cerevisiae/genética , Anticorpos/imunologia , Sequência de Bases , Proteínas Fúngicas/imunologia , Temperatura Alta , Cinética , Dados de Sequência Molecular , Testes de Precipitina
18.
J Gen Microbiol ; 103(1): 127-40, 1977 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-201725

RESUMO

Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a maximum at a specific growth rate of 0.2 h-1, but fell off at lower and higher growth rates. The specific activity in nitrogen-limited cultures was always lower and was inversely proportional to growth rate. There was severe repression of benzyl alcohol dehydrogenase during metabolism of L(+)-mandelate or phenylglyoxylate in batch cultures. Synthesis of benzyl alcohol dehydrogenase was followed in experiments where various compounds, including a gratuitous inducer and an anti-inducer of the mandelate enzymes, were added to uninduced or pre-induced cultures and to constitutive and blocked mutants. The results led to the conclusion that there were at least two types of repression. One was caused by phenylglyoxylate carbon-lyase (or a compound synthesized co-ordinately with it), but not by the other mandelate enzymes or by L(+)-mandelate, phenylglyoxylate, benzaldehyde or benzoate. A second type of repression was observed during rapid growth or after the addition of compound such as succinate which are rapidly and completely metabolized.


Assuntos
Acinetobacter/enzimologia , Oxirredutases do Álcool/biossíntese , Oxirredutases do Álcool/antagonistas & inibidores , Benzaldeídos/farmacologia , Álcoois Benzílicos/metabolismo , Cloranfenicol/farmacologia , AMP Cíclico/farmacologia , Indução Enzimática , Repressão Enzimática , Glutamatos/metabolismo , Glioxilatos/farmacologia , Ácidos Mandélicos/farmacologia , Rifampina/farmacologia
19.
Nucleic Acids Res ; 23(3): 320-6, 1995 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-7885825

RESUMO

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.


Assuntos
Proteínas Fúngicas/metabolismo , Splicing de RNA/genética , RNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Éxons/genética , Íntrons/genética , Dados de Sequência Molecular , Precursores de RNA/metabolismo , RNA Fúngico/genética , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Spliceossomos/metabolismo , Raios Ultravioleta
20.
EMBO J ; 13(4): 879-87, 1994 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-8112301

RESUMO

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.


Assuntos
Proteínas Fúngicas/metabolismo , Genes Dominantes , Mutação , RNA Nucleotidiltransferases/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae , Spliceossomos/metabolismo , Sequência de Bases , Cromatografia de Afinidade , Sequência Conservada , RNA Helicases DEAD-box , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Leucina/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , RNA Helicases , Saccharomyces cerevisiae , Serina/genética
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa