Drosophila melanogaster
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA219479 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR989482.fastq.gz | Fastqsanger.gz | |||
| SRR989483.fastq.gz | Fastqsanger.gz |
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Project description:Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.
2016-06-08 | GSE50984 | 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: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:In Drosophila, Piwi proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two Piwi proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimp uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal essential functions of post-translational modifications in unstructured regions of Piwi proteins and of Tudor domains capable of discriminating between modification states in piRNA biogenesis and silencing.
2020-06-25 | GSE153156 | GEO
Project description:Silencing of transposons in the Drosophila ovary relies on three Piwi-family proteins, Piwi, Aubergine (Aub), and Ago3, acting in concert with their small RNA guides, the piRNAs. Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell-type specific knockdowns with measurements of steady state transposon mRNA levels, nascent RNA synthesis, and small RNA abundance. In somatic cells, Piwi loss lead to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on transcription but also participating in germ cell piRNA biogenesis.
2013-02-09 | GSE43360 | 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:Silencing of transposons in the Drosophila ovary relies on three Piwi-family proteins, Piwi, Aubergine (Aub), and Ago3, acting in concert with their small RNA guides, the piRNAs. Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell-type specific knockdowns with measurements of steady state transposon mRNA levels, nascent RNA synthesis, and small RNA abundance. In somatic cells, Piwi loss lead to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on transcription but also participating in germ cell piRNA biogenesis. Piwi function in transcriptional and post-transcriptional transposon silencing was probed using deep-sequencing of small RNAs, steady-state and nascent transcripts, and DNA associated with H3K9me3 chromatin mark. In all cases comparison of two samples was performed: Tj- or nos-driven knock down of piwi to respective knock down of white gene (control sample). RNA-seq dataset has two replicates.
2013-02-09 | E-GEOD-43360 | 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: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