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Characterization of active piRNAs and an piRNA machinery in WRR-1 cells deficient for the Hippo pathway (deficient for the warts tumor suppressor) and expressing activated RasV12 (small RNA)

ABSTRACT: We have found that oncogenic Ras combined with loss of the Hippo tumour-suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells, coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also to altered expression of some protein coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that at least in this context, the piRNA pathway may play a functional role in cancer.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE83871 | GEO | 2016/08/01

SECONDARY ACCESSION(S): PRJNA327243

REPOSITORIES: GEO

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Characterization of active piRNAs and piRNA machinery in WRR-1 cells deficient for the Hippo pathway (deficient for the warts tumor suppressor) and expressing activated RasV12 (RNA-seq)

2016-08-01 | GSE83843 | GEO

Characterization of active piRNAs and piRNA machinery in WRR-1 cells deficient for the Hippo pathway (deficient for the warts tumor suppressor) and expressing activated RasV12 (RNA-seq)

Project description:We have found that oncogenic Ras combined with loss of the Hippo tumour-suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells, coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also to altered expression of some protein coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that at least in this context, the piRNA pathway may play a functional role in cancer. Small RNAs cloned from whole cells, Piwi-bound small RNAs and long RNA-Seq were performed in wts-RNAi;RasV12 cells to identify piRNAs and gene expression changes compared to RasV12 cells, somatic OSS cells, and germline UAS-wts-RNAi;UAS-RasV12 control ovaries. After knock-down of components of the piRNA machinery in WRR-1 cells ([piwi, zuchini, armitage, aubergine, argonaute3) and GFP and/or dsRED control knock-downs, small RNAs were cloned and gene expression (RNA-Seq) profiles were established.

2016-08-01 | E-GEOD-83843 | biostudies-arrayexpress

Characterization of active piRNAs and an piRNA machinery in WRR-1 cells deficient for the Hippo pathway (deficient for the warts tumor suppressor) and expressing activated RasV12 (small RNA)

Project description:We have found that oncogenic Ras combined with loss of the Hippo tumour-suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells, coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also to altered expression of some protein coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that at least in this context, the piRNA pathway may play a functional role in cancer. Small RNAs cloned from whole cells, piwi-bound small RNAs and long RNA-Seq were performed in wts-RNAi;RasV12 cells to identify piRNAs and gene expression and compared to RasV12 cells, somatic OSS cells, and germline UAS-wts-RNAi;UAS-RasV12 control ovaries. After knock-down of several components of the piRNA machinery in WRR-1 cells (piwi, zuchini, armitage, aubergine, argonaute3) and GFP and/or dsRED control knock-downs, small RNAs were cloned and gene expression profiles (RNA-Seq) were established.

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Characterization of active piRNAs and piRNA machinery in WRR-1 cells deficient for the Hippo pathway (deficient for the warts tumor suppressor) and expressing activated RasV12

Project description:Characterization of active piRNAs and piRNA machinery in WRR-1 cells deficient for the Hippo pathway (deficient for the warts tumor suppressor) and expressing activated RasV12

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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

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Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. In Drosophila ovaries, piRNAs are produced from discrete genomic loci, called piRNA clusters, which are composed of inactive transposon copies and fragments and thus constitute a genetically encoded memory of past transposon challenges. Two types of piRNA clusters exist in flies: dual-strand clusters, expressed only in the germline via a highly specialised machinery, and uni-strand cluster, which are predominantly expressed in the somatic follicle cells. Flamenco (flam) is the major uni-strand piRNA cluster in Drosophila, giving rise to the majority of somatic piRNAs. Flam resembles a canonical RNA polymerase II transcriptional unit, nonetheless it can be specifically recognised by the piRNA pathway and directed to the biogenesis machinery. Recent work has implicated the RNA helicase Yb in the licensing of somatic piRNA production, however a detailed understanding of the molecular mechanisms underlying flam export and specification is still lacking. Here, we show that flam export triggers the assembly of peri-nuclear condensates of Yb and provide evidence that piRNA production from flam specifically requires subunits of the Nuclear Pore Complex (NPC). In the absence of some NPC subunits, transposons become de-silenced and piRNA biogenesis is compromised exclusively from flam. We also show that Yb transiently associates with the NPC to promote flam export. Taken together, our data shed light on how the export of uni-strand cluster transcripts is achieved and suggest the evolution of a specialised machinery that couples transcription, nuclear export and piRNA production.

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Requirement of Nuclear Pore Complex subunits for transposon silencing in Drosophila

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