RESUMO
BACKGROUND: Schizosaccharomyces pombe cells lacking the catalytic subunit of telomerase (encoded by trt1+) lose telomeric DNA and enter crisis, but rare survivors arise with either circular or linear chromosomes. Survivors with linear chromosomes have normal growth rates and morphology, but those with circular chromosomes have growth defects and are enlarged. We report the global gene-expression response of S. pombe to loss of trt1+. RESULTS: Survivors with linear chromosomes had expression profiles similar to cells with native telomeres, whereas survivors with circular chromosomes showed continued upregulation of core environmental stress response (CESR) genes. In addition, survivors with circular chromosomes had altered expression of 51 genes compared to survivors with linear chromosomes, providing an expression signature. S. pombe progressing through crisis displayed two waves of altered gene expression. One coincided with crisis and consisted of around 110 genes, 44% of which overlapped with the CESR. The second was synchronized with the emergence of survivors and consisted of a single class of open reading frames (ORFs) with homology both to RecQ helicases and to dh repeats at centromeres targeted for heterochromatin formation via an RNA interference (RNAi) mechanism. Accumulation of transcript from the ORF was found not only in trt1- cells, but also in dcr1- and ago1- RNAi mutants, suggesting that RNAi may control its expression. CONCLUSIONS: These results demonstrate a correlation between a state of cellular stress, short telomeres and growth defects in cells with circular chromosomes. A putative new RecQ helicase was expressed as survivors emerged and appears to be transcriptionally regulated by RNAi, suggesting that this mechanism operates at telomeres.
Assuntos
DNA Fúngico/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Schizosaccharomyces/genética , Telômero/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Cromossomos Fúngicos/química , Cromossomos Fúngicos/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Genes Fúngicos/genética , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Interferência de RNA , RecQ Helicases , Recombinação Genética/genética , Schizosaccharomyces/classificação , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe , Telomerase/genéticaRESUMO
RecQ helicases play roles in telomere maintenance in cancerous human cells using the alternative lengthening of telomeres mechanism and in budding yeast lacking telomerase. Fission yeast lacking the catalytic subunit of telomerase (trt1(+)) up-regulate the expression of a previously uncharacterized sub-telomeric open reading frame as survivors emerge from crisis. Here we show that this open reading frame encodes a protein with homology to RecQ helicases such as the human Bloom's and Werner's syndrome proteins and that copies of the helicase gene are present on multiple chromosome ends. Characterization of the helicase transcript revealed a 7.6-kilobase RNA that was associated with polyribosomes, suggesting it is translated. A 3.6-kilobase domain of the helicase gene predicted to encode the region with catalytic activity was cloned, and both native and mutant forms of this domain were overexpressed in trt1(-) cells as they progressed through crisis. Overexpression of the native form caused cells to recover from crisis earlier than cells with a vector-only control, whereas overexpression of the mutant form caused delayed recovery from crisis. Taken together, the sequence homology, functional analysis, and site-directed mutagenesis indicate that the protein is likely a second fission yeast RecQ helicase (in addition to Rqh1) that participates in telomere metabolism during crisis. These results strengthen the notion that in multiple organisms RecQ helicases contribute to survival after telomere damage.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Homologia de Sequência de Aminoácidos , Telomerase/deficiência , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Cromossomos Fúngicos/enzimologia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Clonagem Molecular , DNA Helicases/genética , Regulação Fúngica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Polirribossomos/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RecQ Helicases , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Telomerase/genética , Telômero/enzimologia , Telômero/genética , Telômero/metabolismoRESUMO
An important goal after structural genomics is to build up the structures of higher-order protein-protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and RIalpha regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen/deuterium (H/2H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A- and B-helices of RIalpha is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.