The analysis of piRNA shows the function of Slicer activity of AGO3 in piRNA biogenesis.
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE49436 | GEO | 2014/06/30
SECONDARY ACCESSION(S): PRJNA213903
REPOSITORIES: GEO
Similar Datasets
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. small-RNA libraries from 3 samples of D. mel. Ovaries.
2014-06-30 | E-GEOD-49436 | 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
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.
2015-05-28 | GSE69293 | GEO
Project description:In Drosophila, Piwi proteins associate with Piwi-interacting RNAs (piRNAs) and protect the germline genome by silencing mobile genetic elements. This defense system acts in germline and gonadal somatic tissue to preserve germline development. Genetic control for these silencing pathways varies greatly between tissues of the gonad. Here, we identified Vreteno (Vret), a novel gonad-specific protein essential for germline development. Vret is required for piRNA-based transposon regulation in both germline and somatic gonadal tissues. We show that Vret, which contains Tudor domains, associates physically with Piwi and Aubergine (Aub), stabilizing these proteins via a gonad-specific mechanism, absent in other fly tissues. 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. These studies show that vreteno (vret) has a role in germline development and primary piRNA regulation in Drosophila. Transposable element expression profiles from Drosophila ovaries mutant for vreteno, piwi and aubergine were compared using genome-wide mRNA expression profiling by Affymetrix GeneChip arrays (Drosophila 2.0). Key targets were validated by qPCR experiments.
2011-09-28 | E-GEOD-30360 | biostudies-arrayexpress
Project description:Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI- interacting RNAs (piRNAs)—the 22–30-nt-long guides for PIWI- clade Ago proteins that silence transposons in animal gonads— are generated independently of Dicer from single-stranded precursors. piRNA 5′ ends are defined either by Zucchini, the Drosophila homologue of mitoPLD—a mitochondria-anchored endonuclease, or by piRNA-guided target cleavage. Formation of piRNA 3′ ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3′ ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini- mediated cleavages directly define mature piRNA 3′ ends, Aub/ Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3′-to-5′ exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5′ and 3′ ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3′-end formation.
2016-11-16 | GSE83698 | GEO
Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. To understand the hierarchical order of primary versus secondary piRNA biogenesis in Drosophila ovaries, we sequenced piRNAs bound to total-Piwi, germline-Piwi, Aubergine and Argonaute3 from ovaries of germline specific knockdowns of control, piwi, aub, ago3 single knockdowns and aub/ago3 double knockdowns. To determine changes in Transposable Element (TE) transcription or TE RNA steady state in perturbed piRNA pathway conditions, we performed Pol2-ChIP-sequencing and polyA bound RNA-sequencing from ovaries of multiple germline knockdown genotypes. We also sequenced genomic DNA from ovaries of control knockdowns to experimentally estimate the TE copy number in our genetic background. Finally, we used CAP-seq from germline specific Piwi depletions to identify the Transcriptional Start Sites (TSS) in TEs in a deregulated background. Replicates are labeled with R1, R2, R3, R4 where indicated.
2015-08-31 | E-GEOD-71775 | biostudies-arrayexpress
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
Project description:In Drosophila, Piwi proteins associate with Piwi-interacting RNAs (piRNAs) and protect the germline genome by silencing mobile genetic elements. This defense system acts in germline and gonadal somatic tissue to preserve germline development. Genetic control for these silencing pathways varies greatly between tissues of the gonad. Here, we identified Vreteno (Vret), a novel gonad-specific protein essential for germline development. Vret is required for piRNA-based transposon regulation in both germline and somatic gonadal tissues. We show that Vret, which contains Tudor domains, associates physically with Piwi and Aubergine (Aub), stabilizing these proteins via a gonad-specific mechanism, absent in other fly tissues. 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. These studies show that vreteno (vret) has a role in germline development and primary piRNA regulation in Drosophila.
2011-09-29 | GSE30360 | GEO
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: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