Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA204938 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR866323.fastq.gz | Fastqsanger.gz | |||
| SRR866324.fastq.gz | Fastqsanger.gz |
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Project description:PIWI-clade Argonaute proteins repress transposable elements in animal gonads. Their sequence specificity is conferred via bound ~23-30nt long piRNAs, which are processed from single stranded precursor RNAs. How transcripts are specified as precursors and processed into stereotypical piRNA populations are central unresolved questions. Here we show that piRNA-guided RNA cleavage in Drosophila results not only in generation of a ping-pong partner piRNA but further triggers efficient 3′ directed and phased primary piRNA biogenesis. Phasing is a feature of primary piRNAs in somatic and germline cells and a consequence of consecutive endo-nucleolytic cleavage events catalyzed by Zucchini. Formation of 3′ and 5′ ends of flanking piRNAs is therefore tightly coupled. Zucchini also participates in 3′ end formation of secondary piRNAs but its function can be bypassed by additional downstream piRNA-guided cleavages and subsequent precursor trimming. Hallmarks of Zucchini-dependent phased piRNA biogenesis are also evident in mouse testes, pointing to an evolutionarily conserved mechanism of piRNA biogenesis. This study aims at understanding how piRNA biogenesis is intiated in the Drosophila germline and understanding the role of the nuclease Zucchini/MitoPLD in piRNA biogenesis in Drosophila/Mouse by analysing small RNA sequencing data of various genotypes and sensor constructs.
2015-06-07 | E-GEOD-64802 | biostudies-arrayexpress
Project description:PIWI-clade Argonaute proteins repress transposable elements in animal gonads. Their sequence specificity is conferred via bound ~23-30nt long piRNAs, which are processed from single stranded precursor RNAs. How transcripts are specified as precursors and processed into stereotypical piRNA populations are central unresolved questions. Here we show that piRNA-guided RNA cleavage in Drosophila results not only in generation of a ping-pong partner piRNA but further triggers efficient 3′ directed and phased primary piRNA biogenesis. Phasing is a feature of primary piRNAs in somatic and germline cells and a consequence of consecutive endo-nucleolytic cleavage events catalyzed by Zucchini. Formation of 3′ and 5′ ends of flanking piRNAs is therefore tightly coupled. Zucchini also participates in 3′ end formation of secondary piRNAs but its function can be bypassed by additional downstream piRNA-guided cleavages and subsequent precursor trimming. Hallmarks of Zucchini-dependent phased piRNA biogenesis are also evident in mouse testes, pointing to an evolutionarily conserved mechanism of piRNA biogenesis.
2015-06-07 | GSE64802 | 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:PIWI proteins and their bound piRNAs form the core of a gonad specific small RNA silencing pathway in animals that protects the genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology, where TUDOR domains of various proteins recognize and bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analyzed the Drosophila TUDOR protein family and identified three previously not characterized TUDOR domain-containing genes (CG4771, CG14303 and CG11133) as essential piRNA pathway members. We characterized CG4771 (Avocado) in detail and demonstrate a critical role for this protein during primary piRNA biogenesis in somatic and germline cells of the ovary. Avocado physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Avocado also interacts with the Tdrd12 orthologs CG11133 and CG31755, which are essential for primary piRNA biogenesis in the germline and probably functionally replace the related and soma specific factor Yb.
2011-08-16 | GSE30955 | GEO
Project description:PIWI proteins and their bound piRNAs form the core of a gonad specific small RNA silencing pathway in animals that protects the genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology, where TUDOR domains of various proteins recognize and bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analyzed the Drosophila TUDOR protein family and identified three previously not characterized TUDOR domain-containing genes (CG4771, CG14303 and CG11133) as essential piRNA pathway members. We characterized CG4771 (Avocado) in detail and demonstrate a critical role for this protein during primary piRNA biogenesis in somatic and germline cells of the ovary. Avocado physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Avocado also interacts with the Tdrd12 orthologs CG11133 and CG31755, which are essential for primary piRNA biogenesis in the germline and probably functionally replace the related and soma specific factor Yb. small RNA libraries were prepared from total RNA isolation of 8 different genotypes
2011-08-16 | E-GEOD-30955 | biostudies-arrayexpress
Project description:The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. Here we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.
2020-06-17 | E-MTAB-9090 | 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. 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: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.
2015-08-31 | GSE71775 | GEO
Project description:Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5ʹ→3ʹ directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5ʹ ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5ʹ→3ʹ helicase activity, and mutations that abolish it reduce piRNA processing. Another somatic piRNA pathway factor Yb, and an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.
2017-08-16 | GSE102013 | GEO