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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

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE23560 | GEO | 2010/10/04

SECONDARY ACCESSION(S): PRJNA131811

REPOSITORIES: GEO

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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

HITS-CLIP analysis of Yb binding sites in Drosophila ovary cell line

Project description:piRNAs direct Piwi to repress transposons to maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) concentrate into perinuclear foci adjacent to Yb bodies, termed Flam bodies. Although flam expression is not required for Yb body formation, Yb, the core component of Yb bodies, is required for Flam body formation. Abolishment of the RNA-binding activity of Yb disrupts both Yb bodies and Flam bodies. Loss of Zucchini, an endoribonuclease necessary for piRNA maturation, enlarges Flam bodies, which now superimpose with Yb bodies. Yb directly binds flam, but not neighboring protein-coding gene, transcripts. Thus, Yb integrates piRNA processing factors and piRNA intermediates into Yb bodies and Flam bodies, respectively, through direct binding to enhance piRNA biogenesis and formation of piRNA-inducing silencing complexes. HITS-CLIP was performed using OSC (Ovarian Somatic Cells). The antibody for Drosophila Yb, which was generated in this study, was used. Obtained CLIP tags were analyzed using illumina HiSeq200.

2014-06-24 | E-GEOD-54875 | biostudies-arrayexpress

HITS-CLIP analysis of Yb binding sites in Drosophila ovary cell line

2014-06-24 | GSE54875 | GEO

Distinct and collaborative functions of Yb and Armitage in transposon-targeting piRNA biogenesis

Project description:PIWI-interacting RNAs (piRNAs) repress transposons to maintain germline genome integrity. Previous studies showed that artificial tethering of Armitage (Armi) to reporter RNAs induced piRNA biogenesis. However, lack of female sterile (1) Yb (Yb) in Drosophila ovarian somatic cells (OSCs) impaired production of transposon-targeting piRNAs even in the presence of Armi. Here, we show that specific interaction of Armi with RNA transcripts of flamenco piRNA cluster, the primary source of transposon-targeting piRNAs in OSCs, is strictly regulated by Yb. Lack of Yb allowed Armi to bind RNAs promiscuously, leading to the production of piRNAs unrelated to transposon silencing. The ATP hydrolysis-defective mutants of Armi failed to unwind RNAs and were retained on them, abolishing piRNA production. These findings shed light on distinct and collaborative requirements of Yb and Armi in transposon-targeting piRNA biogenesis. We also provide evidence supporting the direct involvement of Armi but not Yb in Zucchini-dependent piRNA phasing.

2019-03-28 | GSE128924 | GEO

Requirements for multivalent Yb body assembly in transposon silencing in Drosophila

Project description:Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci known as the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells. Yb consists of Helicase C-terminal (Hel-C), RNA helicase and extended Tudor (eTud) domains. We previously showed that the RNA helicase domain is necessary for Yb−RNA interaction, Yb body formation and piRNA biogenesis. Here, we investigated the functions of the two other domains and found that Hel-C and eTud are necessary for Yb to self-associate and to interact with RNAs and other Yb body components, respectively. Both domains were essential for Yb body formation and transposon silencing. Without eTud, piRNA production was completely impaired. Loss of Hel-C severely reduced the levels of transposon-targeting piRNAs, although genic piRNAs unrelated to transposon silencing were still produced. Similar phenotypes were observed when mutations in flamenco, the primary source of transposon-targeting piRNAs, led to the expression of attenuated RNAs. Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb self-association and widespread Yb−flamenco RNA binding.

2019-04-29 | GSE121605 | GEO

Novel piRNA Pathway Components Identified Among the Class of TUDOR Domain Containing Proteins

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

Novel piRNA Pathway Components Identified Among the Class of TUDOR Domain Containing Proteins

2011-08-16 | GSE30955 | GEO

The Yb Body, a Major Site for Piwi-associated RNA Biogenesis and a Gateway for Piwi Expression and Transport to the Nucleus in Somatic Cells

Project description:Despite exciting progress in understanding the Piwi-associ-ated RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mu- tants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways. Examination of the effect of Yb on piRNA pathway

2011-02-10 | E-GEOD-26407 | biostudies-arrayexpress

Daedalus and Gasz recruit Armitage to mitochondria, bringing piRNA precursors to the biogenesis machinery

Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-bodies and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor, Armitage (Armi). We find that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb-bodies in a manner that depends upon Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.

2019-04-18 | PXD013403 | Pride

Cloning of 50-75 nt small RNAs associated with Armitage in OSCs

Project description:Armitage (Armi) is a component of Yb bodies and functions in the primary piRNA processing pathway. Armi interaction with Piwi does not require Yb expression; however, without Yb, the Armi-Piwi complex does not localize at Yb bodies and contains few piR-ILs (small RNAs associated with Armitage complex). These results suggest that only upon its localization at Yb bodies does the Armi-Piwi-Yb complex gain an opportunity to interact with piR-ILs. Currently, however, it remains unclear how piR-ILs are loaded onto the Piwi complex at Yb bodies and which protein(s) within the complex is directly bound to them.

2010-10-25 | GSE23931 | GEO

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