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1.
PLoS Genet ; 20(2): e1011114, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38346076

RESUMO

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species.


Assuntos
Podospora , Podospora/genética , Alelos , Lectinas/genética , Lectinas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Polimorfismo Genético
3.
Mol Cell Biol ; 31(5): 1088-97, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21189291

RESUMO

Increasing evidence implicates cohesin in the control of gene expression. Here we report the first analysis of cohesin-dependent gene regulation in fission yeast. Global expression profiling of the mis4-367 cohesin loader mutant identified a small number of upregulated and downregulated genes within subtelomeric domains (SD). These 20- to 40-kb regions between chromosome arm euchromatin and telomere-proximal heterochromatin are characterized by a combination of euchromatin (methylated lysine 4 on histone H3/methylated Tysine 9 on histone H3 [H3K4me]) and heterochromatin (H3K9me) marks. We focused our analysis on the chromosome 1 right SD, which contains several upregulated genes and is bordered on the telomere-distal side by a pair of downregulated genes. We find that the expression changes in the SD also occur in a mutant of the cohesin core component Rad21. Remarkably, mutation of Rad21 results in the depletion of Swi6 binding in the SD. In fact, the Rad21 mutation phenocopied Swi6 loss of function: both mutations led to reduced cohesin binding, reduced H3K9me, and similar gene expression changes in the SD. In particular, expression of the gene pair bordering the SD was dependent both on cohesin and on Swi6. Our data indicate that cohesin participates in the setup of a subtelomeric heterochromatin domain and controls the expression of the genes residing in that domain.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/genética , Regulação para Baixo , Eucromatina/genética , Eucromatina/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Heterocromatina/genética , Lisina/metabolismo , Metilação , Mutação , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/análise , Telômero/genética , Regulação para Cima , Coesinas
4.
Mol Cell Biol ; 30(5): 1145-57, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20028739

RESUMO

Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways.


Assuntos
Inativação Gênica , Splicing de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Sequência de Bases , Centrômero/genética , Centrômero/metabolismo , Primers do DNA/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , Exossomos/genética , Exossomos/metabolismo , Genes Fúngicos , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Especificidade da Espécie
5.
EMBO J ; 27(1): 111-21, 2008 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-18079700

RESUMO

Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Fase G1/genética , Fase G2/genética , Proteínas Nucleares/genética , Ligação Proteica , Fase S/genética , Schizosaccharomyces/citologia , Troca de Cromátide Irmã/genética , Coesinas
6.
Curr Biol ; 16(9): 875-81, 2006 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-16682348

RESUMO

Sister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae (Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them . Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequently established during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired . The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required . Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human . By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 as a Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Coesinas
7.
J Biol Chem ; 280(26): 24532-8, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15894541

RESUMO

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.


Assuntos
Bioquímica/métodos , Poli A/química , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/fisiologia , RNA Fúngico , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Proteínas Fúngicas/química , Immunoblotting , Técnicas In Vitro , Carioferinas/química , Microscopia de Fluorescência , Poliadenilação , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , beta Carioferinas
8.
EMBO J ; 22(11): 2831-40, 2003 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-12773397

RESUMO

Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non-protein-coding RNAs such as snRNAs and snoRNAs. These RNAs have to be processed at their 3' ends to be functional. mRNAs are matured by cleavage and polyadenylation that require a well-characterized protein complex. Small RNAs are also subject to 3' end cleavage but are not polyadenylated. Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre-mRNA 3' end processing complex, are essential for yeast snoRNA 3' end maturation. We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p-dependent pathway for 3' end formation of non-polyadenylated transcripts.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Mutação , Proteínas Serina-Treonina Quinases/genética , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Nucleolar Pequeno/genética , Proteínas de Ligação a RNA , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Supressão Genética , Temperatura , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
9.
EMBO J ; 21(7): 1800-10, 2002 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-11927564

RESUMO

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3'-end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)-binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)(+) RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail-binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Delta hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export.


Assuntos
Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Transporte Nucleocitoplasmático , RNA Fúngico/fisiologia , RNA Nuclear Heterogêneo/fisiologia , RNA Mensageiro/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Alelos , Proteínas Fúngicas/genética , Ribonucleoproteínas Nucleares Heterogêneas , Mutagênese , Proteínas de Ligação a Poli(A) , RNA Fúngico/metabolismo , RNA Nuclear Heterogêneo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
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