RESUMO
Yeast sporulation is a highly regulated developmental program by which diploid cells generate haploid gametes, termed spores. To better define the genetic pathways regulating sporulation, a systematic screen of the set of ~3300 nonessential Schizosaccharomyces pombe gene deletion mutants was performed to identify genes required for spore formation. A high-throughput genetic method was used to introduce each mutant into an h(90) background, and iodine staining was used to identify sporulation-defective mutants. The screen identified 34 genes whose deletion reduces sporulation, including 15 that are defective in forespore membrane morphogenesis. In S. pombe, the total number of sporulation-defective mutants is a significantly smaller fraction of coding genes than in S. cerevisiae, which reflects the different evolutionary histories and biology of the two yeasts.
Assuntos
Estudo de Associação Genômica Ampla , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Esporos Fúngicos/genética , Regulação Fúngica da Expressão Gênica , Haploidia , Meiose/genética , Fenótipo , Deleção de SequênciaRESUMO
In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.
Assuntos
Genes Fúngicos , Meiose/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Bases , Análise por Conglomerados , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/efeitos dos fármacos , Genes Fúngicos/genética , Meiose/efeitos dos fármacos , Análise em Microsséries , Dados de Sequência Molecular , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Cyclin-dependent kinases (CDKs) are subunits of transcription factor (TF) IIH and positive transcription elongation factor b (P-TEFb). To define their functions, we mutated the TFIIH-associated kinase Mcs6 and P-TEFb homologs Cdk9 and Lsk1 of fission yeast, making them sensitive to inhibition by bulky purine analogs. Selective inhibition of Mcs6 or Cdk9 blocks cell division, alters RNA polymerase (Pol) II carboxyl-terminal domain (CTD) phosphorylation, and represses specific, overlapping subsets of transcripts. At a common target gene, both CDKs must be active for normal Pol II occupancy, and Spt5-a CDK substrate and regulator of elongation-accumulates disproportionately to Pol II when either kinase is inhibited. In contrast, Mcs6 activity is sufficient-and necessary-to recruit the Cdk9/Pcm1 (mRNA cap methyltransferase) complex. In vitro, phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. We propose that TFIIH primes the CTD and promotes recruitment of P-TEFb/Pcm1, serving to couple elongation and capping of select pre-mRNAs.
Assuntos
Fator B de Elongação Transcricional Positiva/genética , Capuzes de RNA/genética , Schizosaccharomyces/metabolismo , Fator de Transcrição TFIIH/genética , Transcrição Gênica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaAssuntos
Processamento Alternativo/genética , Ciclinas/genética , Regiões Promotoras Genéticas/genética , Schizosaccharomyces/genética , Animais , Regulação Fúngica da Expressão Gênica , Íntrons/genética , Meiose/genética , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
In a recent issue of Nature, Skotheim et al. (2008) show that a transcriptional positive feedback loop plays a key role in the commitment to enter the yeast cell cycle.