RESUMO
RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.
Assuntos
Regulação Fúngica da Expressão Gênica , Interferência de RNA , Schizosaccharomyces/genética , Transcriptoma/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Northern Blotting , Western Blotting , Endorribonucleases/genética , Endorribonucleases/metabolismo , Microscopia de Fluorescência , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , RNA Antissenso/genética , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Pequeno RNA não Traduzido/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.
Assuntos
Autoantígenos/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexo Mediador/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteína Centromérica A , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Cinetocoros/metabolismo , Complexo Mediador/genética , RNA Fúngico/genética , RNA não Traduzido/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transcrição GênicaRESUMO
The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5'-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1Delta cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity.
Assuntos
Centrômero/ultraestrutura , Regulação Fúngica da Expressão Gênica , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/fisiologia , Sequência de Bases , Centrômero/metabolismo , Heterocromatina/química , Dados de Sequência Molecular , Família Multigênica , Mutação , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , Schizosaccharomyces/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
The term epigenetics refers to heritable changes not encoded by DNA. The organization of DNA into chromatin fibers affects gene expression in a heritable manner and is therefore one mechanism of epigenetic inheritance. Large parts of eukaryotic genomes consist of constitutively highly condensed heterochromatin, important for maintaining genome integrity but also for silencing of genes within. Small RNA, together with factors typically associated with RNA interference (RNAi) targets homologous DNA sequences and recruits factors that modify the chromatin, commonly resulting in formation of heterochromatin and silencing of target genes. The scope of this review is to provide an overview of the roles of small RNA and the RNAi components, Dicer, Argonaute and RNA dependent polymerases in epigenetic inheritance via heterochromatin formation, exemplified with pathways from unicellular eukaryotes, plants and animals.
Assuntos
Epigênese Genética , Heterocromatina/metabolismo , Interferência de RNA , Animais , Plantas/genética , RNA Interferente Pequeno/metabolismo , Transcrição GênicaRESUMO
Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.
Assuntos
Cromatina/metabolismo , Interferência de RNA/fisiologia , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Centrômero/genética , Centrômero/metabolismo , Cromatina/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/fisiologia , RNA Polimerase II/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transcrição Gênica/fisiologiaRESUMO
RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1+) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximately 23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1+ was required for silencing of two genes containing a conserved motif.