Drosophila melanogaster
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA271898 | ENA |
REPOSITORIES: ENA
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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 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: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:Purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that piRNA target sites with cleavage-site mismatches are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are identified by perfect complementarity base-pairing with the piRNA. Additionally, we find that such pachytene piRNA-guided MIWI/MILI slicing in vivo failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as a pre-piRNA and 17-19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis.
2023-10-23 | GSE219200 | GEO
Project description:piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire
| PRJNA292069 | ENA
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
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:Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to initiate biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing TEs. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that neither mechanism is required to efficiently produce TE antisense piRNAs in Drosophila. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested.
2022-09-19 | GSE213379 | GEO
Project description:Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to initiate biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing TEs. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that neither mechanism is required to efficiently produce TE antisense piRNAs in Drosophila. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested.
2022-09-19 | GSE213375 | GEO