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FLX sequencing of Piwi-associated small RNAs extracted from Drosophila ovarian somatic cells (OSC)

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Toutai MITUYAMA

PROVIDER: E-GEOD-15137 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila.

Saito Kuniaki K Inagaki Sachi S Mituyama Toutai T Kawamura Yoshinori Y Ono Yukiteru Y Sakota Eri E Kotani Hazuki H Asai Kiyoshi K Siomi Haruhiko H Siomi Mikiko C MC

Nature 20091007 7268

PIWI-interacting RNAs (piRNAs) silence retrotransposons in Drosophila germ lines by associating with the PIWI proteins Argonaute 3 (AGO3), Aubergine (Aub) and Piwi. piRNAs in Drosophila are produced from intergenic repetitive genes and piRNA clusters by two systems: the primary processing pathway and the amplification loop. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, primary piRNA processing remains elusive. Here we analysed piRNA processing in a ...[more]

PMID: 19812547

Similar Datasets

Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline

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

FLX sequencing of Piwi-associated small RNAs extracted from Drosophila ovarian somatic cells (OSC)

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

2009-10-02 | GSE15137 | GEO

Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila.

Project description:Here, we analyzed small RNA libraries derived from ovarian tissues heterozygous or mutant for the Tudor gene, Vreteno. In the absence of vret, Piwi-bound piRNAs are lost without changes in piRNA precursor transcript production, supporting a role for Vret in primary piRNA biogenesis. In the germline, piRNAs can engage in an Aub/Argonaute 3 (AGO3)-dependent amplification in the absence of Vret, suggesting that Vret function can distinguish between primary piRNAs loaded into Piwi/Aub complexes and piRNAs engaged in the amplification cycle. We propose that Vret acts at an early step in primary piRNA processing where it plays an essential role in transposon regulation. Keyword : Epigenetics 2 libraries were analyzed, with 1 being a control (heterozygote).

2011-08-15 | E-GEOD-30088 | biostudies-arrayexpress

the analysis of piRNA shows the function of Slicer activity of AGO3 in piRNA biogenesis.

Project description:Piwi-interacting RNAs (piRNAs) silence transposons in animal germ cells. In Drosophila, the reciprocal “Ping-Pong” cycle of piRNA-directed RNA cleavage, catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (AGO3) through their Slicer activity, is believed to expand the population of antisense piRNAs in response to transposon expression. Whether and how the Slicer activity of AGO3/Aub promotes the process of the secondary piRNA amplification remain unclear. Here we generated transgenic flies that could express AGO3 Slicer mutant forms to ellucidate the Slicer activity of AGO3.

2014-06-30 | GSE49436 | GEO

High throughput sequencing of Piwi bound piRNAs from Drosophila ovaries in which key factors for primary piRNA biogenesis in somatic support cells were knocked down using RNAi

Project description:In Drosophila, PIWI proteins and bound PIWI interacting RNAs (piRNAs) form the core of a small RNA mediated defense system against selfish genetic elements. Within germline cells piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi nuclear accumulation. We show that Armitage and Yb interact physically and co-localize in cytoplasmic Yb-bodies, which flank P-bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb-bodies indicating that Yb-bodies are sites of primary piRNA biogenesis. small RNA libraries were prepared from Piwi immuno-precipitates of five different genotypes

2010-10-03 | E-GEOD-23560 | biostudies-arrayexpress

piRNA libraries from Drosophila melanogaster ovaries

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

the analysis of piRNA shows the function of Slicer activity of AGO3 in piRNA biogenesis.

2014-06-30 | E-GEOD-49436 | biostudies-arrayexpress

Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline

2015-05-28 | GSE69293 | GEO

Somatic Primary piRNA Biogenesis Driven by Cis-Acting RNA Elements and Trans- Acting Yb

Project description:Primary piRNAs in Drosophila ovarian somatic cells arise from piRNA cluster transcripts and the 3′ UTRs of a subset of mRNAs, including Traffic jam (Tj) mRNA. However, it is unclear how these RNAs are determined as primary piRNA sources. Here, we identify a cis-acting 100-nt fragment in the Tj 3′ UTR that is sufficient for producing artificial piRNAs from unintegrated DNA. These artificial piRNAs were effective in endogenous gene transcriptional silencing. Yb, a core component of primary piRNA biogenesis center Yb bodies, directly bound the Tj-cis-element. Disruption of this interaction markedly reduced piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis-element. Yb-CLIP revealed that Yb-binding correlated with somatic piRNA production but Tj-cis-element downstream sequences produced few artificial piRNAs. Thus, Yb determines primary piRNA sources by two modes of action; primary binding to cis-elements to specify substrates, and secondary binding to downstream regions to increase diversity in piRNA populations. HITS-CLIP of Yb in OSCs (Ovarian Somatic Cells) depleted for tj cis-element, and small RNA sequencing of Piwi-piRNAs in OSCs depleted for tj cis-element.

2015-06-05 | E-GEOD-69625 | biostudies-arrayexpress

RNA elements in precursors instruct directional and processive primary piRNA biogenesis

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

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