RESUMO
Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/biossíntese , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Biologia Computacional , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Exorribonucleases/metabolismo , Genes Reporter , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos , Polímeros/metabolismo , Ligação Proteica , Estabilidade de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Terminação da Transcrição GenéticaRESUMO
Silencing of genomic repeats, including transposable elements, in Drosophila melanogaster is mediated by repeat-associated short interfering RNAs (rasiRNAs) interacting with proteins of the Piwi subfamily. rasiRNA-based silencing is thought to be mechanistically distinct from both the RNA interference and microRNA pathways. We show that the amount of rasiRNAs of a wide range of retroelements is drastically reduced in ovaries and testes of flies carrying a mutation in the spn-E gene. To address the mechanism of rasiRNA-dependent silencing of retrotransposons, we monitored their chromatin state in ovaries and somatic tissues. This revealed that the spn-E mutation causes chromatin opening of retroelements in ovaries, resulting in an increase in histone H3 K4 dimethylation and a decrease in histone H3 K9 di/trimethylation. The strongest chromatin changes have been detected for telomeric HeT-A elements that correlates with the most dramatic increase of their transcript level, compared to other mobile elements. The spn-E mutation also causes depletion of HP1 content in the chromatin of transposable elements, especially along HeT-A arrays. We also show that mutations in the genes controlling the rasiRNA pathway cause no derepression of the same retrotransposons in somatic tissues. Our results provide evidence that germinal Piwi-associated short RNAs induce chromatin modifications of their targets.