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Cutoff suppresses RNA polymerase II termination to ensure expression of piRNA precursors

ABSTRACT: Small non-coding RNAs called piRNAs serve as the sequence-specific guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. The adaptation of the piRNA pathway to novel transposons is believed to occur when active transposons integrate into piRNA clusters, special genomic regions, which encode piRNA precursors. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) binds chromatin of dual-strand piRNA clusters and is required for transcription of piRNA precursors, though the mechanism by which RDC license transcription remained unknown. Here we show that Cuff prevents premature termination of RNA polymerase II by two distinct mechanisms. First, Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Second, if processing does occur, Cuff protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually novel 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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE81090 | GEO | 2016/05/05

SECONDARY ACCESSION(S): PRJNA320547

REPOSITORIES: GEO

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The Rhino-Deadlock-Cutoff complex licenses non-canonical transcription of dual-strand piRNA clusters in Drosophila

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2014-06-08 | E-GEOD-55824 | biostudies-arrayexpress

The Rhino-Deadlock-Cutoff complex licenses non-canonical transcription of dual-strand piRNA clusters in Drosophila

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2014-07-26 | E-GEOD-59608 | biostudies-arrayexpress

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [cuff RNA-seq]

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. total RNA sequencing of RNA from ovaries of cuff+/- or cuff-/- flies

2014-07-26 | E-GEOD-59609 | biostudies-arrayexpress

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2012-11-26 | E-GEOD-41729 | biostudies-arrayexpress

A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway [smallRNA-Seq]

Project description:The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two inter-related branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborated pathway centered on the three gonad-specific Argonaute proteins Piwi, Aubergine, and Argonaute3. While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals new key factors of piRNA-mediated transposon silencing, including the novel piRNA biogenesis factors, CG2183 (GASZ) and Deadlock. Last, our data uncovers a previously unanticipated set of factors preferentially required for repression of different transposons types. Examination of small RNA levels from nos-GAL4 or tj-GAL4 driven UAS-dsRNA knockdowns of control genes and piRNA pathway components in ovaries of Drosophila melanogaster by deep sequencing (using Illumina HiSeq2000).

2013-05-10 | E-GEOD-46099 | biostudies-arrayexpress

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Project description:The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two inter-related branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborated pathway centered on the three gonad-specific Argonaute proteins Piwi, Aubergine, and Argonaute3. While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals new key factors of piRNA-mediated transposon silencing, including the novel piRNA biogenesis factors, CG2183 (GASZ) and Deadlock. Last, our data uncovers a previously unanticipated set of factors preferentially required for repression of different transposons types. Examination of total RNA levels from nos-GAL4 or tj-GAL4 driven UAS-dsRNA knockdowns of control genes and piRNA pathway components in ovaries of Drosophila melanogaster by deep sequencing (using Illumina HiSeq2000).

2013-05-10 | E-GEOD-46100 | biostudies-arrayexpress

The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline

Project description:In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters. The molecular mechanisms and the factors that govern the expression of these loci are largely unknown. We have preciously shown the Cutoff (Cuff), a protein with similarity to yeast Rai1, is a component of the piRNA pathway. In order to understand the function of the Cuff protein in piRNA production, we produced small RNA libraries from cn, bw (wt) and cuffwm25 mutant ovaties. The analysis of these libraries revealed that approximately 80% of the total piRNA population is depleted in the absence of a functional Cuff protein. We also determined that Cuff is mostly a nuclear protein and is enriched at the level of certain piRNA clusters. Our results point to a role for Cuff in the transcriptional regulation of piRNA generating loci and in the production of the proper piRNA complement during Drosophila oogenesis. piRNA profiling in ovaries from cn, bw and cuffwm25 mutant ovaries

2013-06-07 | E-GEOD-47738 | biostudies-arrayexpress

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [ChIP-seq]

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. ChIPseq of Rhino and Cutoff in Drosophila melanogaster ovaries The Rhino-BioTAP flies were made by fusing the BioTAP tag (Alekseyenko et al. 2014 )to the C-terminal region of the Rhino gene under the UASp promoter. The Cutoff-EGFP fly line (Nanos-GAL4/UASp-Cutoff-GFP), Cuff^wm25 and Cuff^qq37 were a generous gift from T. Schupbach.

2014-07-26 | E-GEOD-59604 | biostudies-arrayexpress

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