MicroRNA-dependent transposable element transcriptional silencing in Drosophila somatic ovarian cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: SEVERINE CHAMBEYRON
PROVIDER: E-GEOD-60974 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:RNAi-related silencing mechanisms concern as diverse biological processes as gene regulation, mostly via the miRNA pathway, and defense against molecular parasites, mainly controlled by the siRNA and the piRNA pathways. In Drosophila somatic ovarian cells, transposable elements (TEs) are repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs). We show here that a functional miRNA pathway is required for this piRNA-mediated TE transcriptional silencing to operate in this tissue. A general miRNA depletion, caused by tissue- and stage-specific knock-down of either drosha (involved in miRNA biogenesis) or AG01 and gawky (both responsible for miRNA activity) resulted in chromatin-mediated TE transcriptional desilencing and piRNA loss. For unknown reasons, the amount of piRNA produced by the traffic jam 3' UTR was apparently unaffected in miRNA-defective somatic ovarian cells. Although weaker, similar phenotypes could also be observed upon individual titration (by expression of the complementary miR-sponge) of at least three miRNAs, miR-14, miR-34 and miR-989. This work adds the maintenance of genome stability, via the piRNA-mediated TE repression, to the list of the already reported miRNA-controlled biological functions. Study of small regulatory RNA populations present in ovaries expressing either wild-type Drosha protein or the trans-dominant negative Drosha protein coupled or not with the Tub-GAL80[ts]GAL4 inhibitor.
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Project description:The discovery of the small regulatory RNA populations has changed our vision of cellular regulations. Indeed, loaded on Argonaute proteins they formed ribonucleoprotein complexes that target complementary sequences and achieved widespread silencing mechanisms conserved in most eukaryotes. The recent development of deep sequencing approaches highly contributed to their detection. Small RNA isolation form cells and/or tissues remains a crucial stage to generate robust and relevant sequencing data. In 2006, a novel strategy based on anion-exchange chromatography has been purposed as an alternative to the standard size-isolation purification procedure. However, the eventual biases of such a method have been poorly investigated. Moreover, this strategy not only relies on advanced technical skills and expensive material but is time consuming and requires an elevated starting biological material amount. Using bioinformatic comparative analysis of six independent small RNA-sequencing libraries of Drosophila ovaries, we here demonstrate that anion-exchange chromatography purification prior to small RNA extraction unbiasedly enriches datasets in bona fide reads (small regulatory RNA reads) and depletes endogenous contaminants (ribosomal RNAs and degradation products). The resulting increase of sequencing depth provides a major benefit to study rare populations. We then developed a fast and basic manual procedure to purify loaded small non coding RNAs using anion-exchange chromatography at the bench. We validated the efficiency of this new method and used this strategy to purify small RNAs from various tissues and organisms. We moreover determined that our manual purification increases the output of the previously described anion-exchange chromatography procedure. Comparison of small regulatory RNA populations obtained after three different small RNA purification procedures
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Project description:Transposable elements (TEs), whose propagation can result in severe damage to the host genome, are silenced in the animal gonad by Piwi-interacting RNAs (piRNAs). piRNAs produced in the ovaries are deposited in the embryonic germline and initiate TE repression in the germline progeny. Whether the maternally transmitted piRNAs play a role in the silencing of somatic TEs is, however, unknown. Here we show that maternally transmitted piRNAs from the tirant retrotransposon in Drosophila are required for the somatic silencing of the TE and correlate with an increase in histone H3K9 trimethylation an active tirant copy. Comparison of tirant piRNAs in two Drosophila simulans natural populations.
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Project description:RNAi-related silencing mechanisms concern as diverse biological processes as gene regulation, mostly via the miRNA pathway, and defense against molecular parasites, mainly controlled by the siRNA and the piRNA pathways. In Drosophila somatic ovarian cells, transposable elements (TEs) are repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs). We show here that a functional miRNA pathway is required for this piRNA-mediated TE transcriptional silencing to operate in this tissue. A general miRNA depletion, caused by tissue- and stage-specific knock-down of either drosha (involved in miRNA biogenesis) or AG01 and gawky (both responsible for miRNA activity) resulted in chromatin-mediated TE transcriptional desilencing and piRNA loss. For unknown reasons, the amount of piRNA produced by the traffic jam 3' UTR was apparently unaffected in miRNA-defective somatic ovarian cells. Although weaker, similar phenotypes could also be observed upon individual titration (by expression of the complementary miR-sponge) of at least three miRNAs, miR-14, miR-34 and miR-989. This work adds the maintenance of genome stability, via the piRNA-mediated TE repression, to the list of the already reported miRNA-controlled biological functions. Analyses of RNA present in 3 different genetic backgounds with or without Drosha-IP: a control, a second one expressing the wild-type Drosha protein and a third one expressing the trans-dominant negative Drosha protein in the somatic cells.
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