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1.
Curr Biol ; 22(20): 1932-7, 2012 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-22959349

RESUMO

Efficient chromosomal movements are important for the fidelity of chromosome segregation during mitosis; however, movements are constrained during interphase by tethering of multiple domains to the nuclear envelope (NE). Higher eukaryotes undergo open mitosis accompanied by NE breakdown, enabling chromosomes to be released from the NE, whereas lower eukaryotes undergo closed mitosis, in which NE breakdown does not occur. Although the chromosomal movements in closed mitosis are thought to be restricted compared to open mitosis, the cells overcome this problem by an unknown mechanism that enables accurate chromosome segregation. Here, we report the spatiotemporal regulation of telomeres in Schizosaccharomyces pombe closed mitosis. We found that the telomeres, tethered to the NE during interphase, are transiently dissociated from the NE during mitosis. This dissociation from the NE is essential for accurate chromosome segregation because forced telomere tethering to the NE causes frequent chromosome loss. The phosphorylation of the telomere protein Rap1 during mitosis, primarily by Cdc2, impedes the interaction between Rap1 and Bqt4, a nuclear membrane protein, thereby inducing telomere dissociation from the NE. We propose that the telomere dissociation from the NE promoted by Rap1 phosphorylation is critical for the fidelity of chromosome segregation in closed mitosis.


Assuntos
Segregação de Cromossomos/fisiologia , Membrana Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Mitose , Membrana Nuclear/genética , Fosforilação , Estrutura Terciária de Proteína , Schizosaccharomyces/metabolismo , Complexo Shelterina , Fuso Acromático/genética , Fuso Acromático/metabolismo
2.
Nat Struct Mol Biol ; 18(2): 213-21, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21217703

RESUMO

Repressor activator protein 1 (RAP1) is the most highly conserved telomere protein. It is involved in protecting chromosome ends in fission yeast and promoting gene silencing in Saccharomyces cerevisiae, whereas it represses homology-directed recombination at telomeres in mammals. To understand how RAP1 has such diverse functions at telomeres, we solved the crystal or solution structures of the RAP1 C-terminal (RCT) domains of RAP1 from multiple organisms in complex with their respective protein-binding partners. Our analysis establishes RAP1(RCT) as an evolutionarily conserved protein-protein interaction module. In mammalian and fission yeast cells, this module interacts with TRF2 and Taz1, respectively, targeting RAP1 to chromosome ends for telomere protection. In contrast, S. cerevisiae RAP1 uses its RCT domain to recruit Sir3 to telomeres to mediate gene silencing. Together, our results show that, depending on the organism, the evolutionarily conserved RAP1 RCT motif has diverse functional roles at telomeres.


Assuntos
Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células Cultivadas , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomycetales/química , Saccharomycetales/genética , Saccharomycetales/metabolismo , Schizosaccharomyces/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
3.
J Biochem ; 148(2): 157-70, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20421362

RESUMO

We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding approximately 200,000 shRNA sequences that targeted 47,400 human transcripts were inserted into a lentivirus vector pFIV-H1-puro, and a pool of pseudovirus particles with a complexity of approximately 200,000 were used to infect target cells. From the cells surviving apoptogenic Fas stimulation, four candidate shRNA sequences were obtained that provided resistance to Fas-induced cell death, including two shRNAs for caspase-8, an shRNA for Bid, and an shRNA for Fas. The reconstructed shRNAs with these sequences were shown to reduce expression of the respective gene products and increase survival after Fas stimulation. When similar selection was performed for tunicamycin-induced apoptosis, no shRNA strongly inhibiting tunicamycin-induced cell death was isolated, although a few reconstructed shRNAs led to a slight increase of survival. Thus, this genome-wide shRNA library proved useful for selection of genes that are involved in cell death, but some limitation was also revealed.


Assuntos
Morte Celular/genética , Estudo de Associação Genômica Ampla/métodos , Biblioteca Genômica , Interferência de RNA , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Caspase 8/genética , Vetores Genéticos , Humanos , Lentivirus/genética , RNA Interferente Pequeno/genética , Receptor fas/genética , Receptor fas/fisiologia
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