Drosophila melanogaster
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA344466 | ENA |
REPOSITORIES: ENA
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Project description:Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of Transposable Elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36%of protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.
2017-04-19 | GSE87346 | GEO
Project description:The PIWI interacting RNA pathway is a small RNA silencing system that keeps selfish genetic elements such as transposons under control in animal gonads. Several lines of evidence indicate that nuclear PIWI family proteins guide transcriptional silencing of their targets, yet the composition of the underlying silencing complex is unknown. Here we demonstrate that the double CHHC zinc finger protein Gtsf1 is an essential factor for Piwi mediated transcriptional repression in Drosophila. Cells lacking Gtsf1 contain nuclear Piwi loaded with piRNAs, yet Piwi's silencing capacity is ablated. Gtsf1 interacts stably with a sub-population of nuclear Piwi and loss of Gtsf1 phenocopies loss of Piwi in terms of deregulation of transposons, loss of H3K9me3 marks at euchromatic transposon insertions and deregulation of genes in proximity to repressed transposons. We propose that only a small fraction of nuclear Piwi interacts productively with a target RNA, resulting in assembly of a silencing complex with Gtsf1 as one core component. impact of loss of DmGtsf1 on transcription and H3K9m3 in ovarian somatic cells (OSC)
2013-09-06 | E-GEOD-48317 | biostudies-arrayexpress
Project description:PIWI-interacting RNAs (piRNAs) are animal gonad-specific small RNAs that control the activity of transposable elements. Long single stranded RNAs from a variety of sources are substrates for the nebulous primary processing pathway that converts these into thousands of 24-30 nucleotide (nt) piRNAs. How these transcripts are selected as precursors is not known. Here we show that targeting a transcript with PIWI slicer activity of cysosolic Ago3 is sufficient to trigger ~30-nt waves of non-overlapping primary piRNAs in the fly ovarian germline. The generated primary piRNAs are almost exclusively loaded into the nuclear PIWI protein, Piwi. In the fly ovarian somatic environment we find that an RNA fragment from the 5? end of a piRNA cluster is able to direct a heterologous sequence into primary processing. This piRNA trigger sequence (PTS) element drives generation of overlapping piRNAs from the transcript. Both mechanisms proceed with general 5?-3? directionality. We propose that the former pathway serves to link cytoplasmic silencing of a target to nuclear transcriptional repression, while the latter extracts silencing information from a wide variety of genomic sources including piRNA clusters, select protein coding and transposon transcripts. Total or immunoprecipitated small RNAs were purified from transfected BmN4 cells, Drosophila ovarian somatic cells (OSC) and from fly ovaries and high-throughput sequencing libraries were prepared. The mouse testicular RNAs were purified after ribozero treatment.
2015-07-09 | E-GEOD-69102 | biostudies-arrayexpress
Project description:piRNAs direct Piwi to repress transposons to maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) concentrate into perinuclear foci adjacent to Yb bodies, termed Flam bodies. Although flam expression is not required for Yb body formation, Yb, the core component of Yb bodies, is required for Flam body formation. Abolishment of the RNA-binding activity of Yb disrupts both Yb bodies and Flam bodies. Loss of Zucchini, an endoribonuclease necessary for piRNA maturation, enlarges Flam bodies, which now superimpose with Yb bodies. Yb directly binds flam, but not neighboring protein-coding gene, transcripts. Thus, Yb integrates piRNA processing factors and piRNA intermediates into Yb bodies and Flam bodies, respectively, through direct binding to enhance piRNA biogenesis and formation of piRNA-inducing silencing complexes. HITS-CLIP was performed using OSC (Ovarian Somatic Cells). The antibody for Drosophila Yb, which was generated in this study, was used. Obtained CLIP tags were analyzed using illumina HiSeq200.
2014-06-24 | E-GEOD-54875 | biostudies-arrayexpress
Project description:Piwi regulates niche and intrinsic mechanisms to maintain germline stem cells in Drosophila, yet how this regulation occurs remains elusive. Here, we show that Piwi interacts with the Polycomb Group Complexes PRC1 and PRC2 to maintain ovarian germline stem cells and oogenesis, as well as to repress retrotransposons. Piwi binds to PRC2 subunits Su(z)12 and Esc in vitro and forms a complex with PRC2 in vivo. Whole-genome analyses of PRC2-mediated histone 3 lysine 27 trimethylation (H3K27m3) in wild type and piwi mutant ovarian nuclei indicate that Piwi inhibits H3K27m3 at PRC2 target genes that are mostly regulators of development and transcription.
2017-01-03 | GSE40150 | GEO
Project description:piRNAs function in silencing retrotransposons by associating with the PIWI proteins, AGO3, Aub, and Piwi, in Drosophila germlines. Bioinformatics analyses of piRNAs in Drosophila ovaries suggested that piRNAs are produced by two systems, the primary processing pathway and the amplification loop, from repetitive genes and piRNA clusters in the genome. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, the primary processing pathway remains largely conceptual. Here we show that in ovarian somatic cells, which lack Aub and AGO3 but express Piwi, the primary processing pathway for piRNAs indeed exists. Keywords: Small RNA profiling by high throughput sequencing Piwi-associated small RNAs were extracted from Drosophila ovarian somatic cells and their deep sequencing was carried out.
2009-10-01 | E-GEOD-15137 | biostudies-arrayexpress
Project description:FLX sequencing of Piwi-associated small RNAs extracted from Drosophila ovarian somatic cells (OSC)
| PRJNA114895 | ENA
Project description:Drosophila Piwi-family proteins have been implicated in transposon control. Here, we examine piwi-interacting RNAs (piRNAs) associated with each Drosophila Piwi protein and find that Piwi and Aubergine bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs. As in mammals, the majority of Drosophila piRNAs are derived from discrete genomic loci. These loci comprise mainly defective transposon sequences, and some have previously been identified as master regulators of transposon activity. Our data suggest that heterochromatic piRNA loci interact with potentially active, euchromatic transposons to form an adaptive system for transposon control. Complementary relationships between sense and antisense piRNA populations suggest an amplification loop wherein each piRNA-directed cleavage event generates the 5’ end of a new piRNA. Thus, sense piRNAs, formed following cleavage of transposon mRNAs, may enhance production of antisense piRNAs, complementary to active elements, by directing cleavage of transcripts from master control loci. Keywords: small RNA libraries from Drosophila ovaries small RNAs (23-29nt) were isolated from total ovarian RNA or from immunopreciptated Piwi/Aubergine/Ago3 complexes. cDNA libraries were constructed after Pfeffer et al. 2005 (Nat. Methods) and sequenced at 454 Life Sciences. The used strain is OregonR. Only sequences matching the Release5 genome assembly (www.fruitfly.org) are considered.
2007-03-08 | E-GEOD-6734 | biostudies-arrayexpress
Project description:The PiwiM-bM-^@M-^SpiRNA complex (PiwiM-bM-^@M-^SpiRISC) in Drosophila ovarian somatic cells represses transposons transcriptionally to maintain genome integrity; however, the underlying mechanisms remain obscure. We performed mRNA-seq analysis from OSCs transfected with siRNAs against CG3893, the known piRNA pathway genes, Piwi, Maelstrom, HP1a and Armitage, and the control (EGFP), and PolII ChIP-seqanalysis from OSCs transfected with siRNAs against CG3893, Piwi, Mael and the control (EGFP). This result indicates that CG3893 is a novel factor for primary piRNA pathway in OSCs. RNA levels in wild-type (EGFP control knock-down) ovarian somatic cells (OSC) and RNAi knock-downs of Piwi, Armi, Mael, CG3893, and HP1a. RNA Polymerase II occupancy in wild-type (EGFP control knock-down) ovarian somatic cells (OSC) and RNAi knock-downs of Piwi, Mael, and CG3893.
2013-08-01 | E-GEOD-47006 | biostudies-arrayexpress
Project description:Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. Here we show that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3’s compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. We propose that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs. In order to investigate function of Krimp in piRNA pathway, sequencing of Piwi subfamily protein associated small RNAs was performed using adult Drosophila ovaries and Ovarian Somatic Cells (OSCs) depleted for Krimp or Aub.
2015-05-28 | E-GEOD-69293 | biostudies-arrayexpress