Transcriptome and transposable element dynamics during PIWI regulation in Drosophila ovarian cell cultures
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE45112 | GEO | 2014/09/15
SECONDARY ACCESSION(S): PRJNA192902
REPOSITORIES: GEO
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Project description:This study examines the regulatory capacity of the Piwi protein in the Drosophila OSS cell. Piwi CLIP-Seq, mRNA-Seq and nascent RNAseq datasets were integrated to determine how Piwi proteins where using piRNAs and binding interactions to regulate the expression of transcripts. We also sequenced the genomes of various OSS cell lines. We first performed several replicates of a Piw CLIP-Seq experiment to isolate RNA fragments as CLIP-tags to discover which transcripts are preferentially bound by the Piwi protein. Then we performed several types of mRNA expression profiling experiments using several forms of mRNA-Seq library construction formats. Finally, we sequenced the genomes from various OSS cell lines. The genomic sequencing component of the study is represented by BioProject PRJNA240323. The genomic sequencing raw data have been deposited at SRA (SRP039565).
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Project description:This study examines the population of transcripts associated with the Xenopus Piwi proteins, Xiwi and Xili, from X.laevis and X.tropicalis. RIP-seq, CLIP-seq, piRNA-seq, and mRNA-seq datasets were integrated to determine how the Xenopus Piwi proteins where using piRNAs and binding interactions to associate with transcripts in gonadal cells. We performed several replicates of a Piw CLIP-Seq experiment to isolate RNA fragments as CLIP-tags to discover which transcripts are preferentially bound by the Piwi protein. Then we performed several types of mRNA expression profiling experiments using several forms of mRNA-Seq library construction formats. Finally, we sequenced the piRNAs from the OSS cells
2015-11-10 | E-GEOD-63228 | biostudies-arrayexpress
Project description:This study examines the population of transcripts associated with the Xenopus Piwi proteins, Xiwi and Xili, from X.laevis and X.tropicalis. RIP-seq, CLIP-seq, piRNA-seq, and mRNA-seq datasets were integrated to determine how the Xenopus Piwi proteins where using piRNAs and binding interactions to associate with transcripts in gonadal cells.
2015-11-10 | GSE63228 | GEO
Project description:Silencing of transposons in the Drosophila ovary relies on three Piwi-family proteins, Piwi, Aubergine (Aub), and Ago3, acting in concert with their small RNA guides, the piRNAs. Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell-type specific knockdowns with measurements of steady state transposon mRNA levels, nascent RNA synthesis, and small RNA abundance. In somatic cells, Piwi loss lead to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on transcription but also participating in germ cell piRNA biogenesis. Piwi function in transcriptional and post-transcriptional transposon silencing was probed using deep-sequencing of small RNAs, steady-state and nascent transcripts, and DNA associated with H3K9me3 chromatin mark. In all cases comparison of two samples was performed: Tj- or nos-driven knock down of piwi to respective knock down of white gene (control sample). RNA-seq dataset has two replicates.
2013-02-09 | E-GEOD-43360 | biostudies-arrayexpress
Project description:Silencing of transposons in the Drosophila ovary relies on three Piwi-family proteins, Piwi, Aubergine (Aub), and Ago3, acting in concert with their small RNA guides, the piRNAs. Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell-type specific knockdowns with measurements of steady state transposon mRNA levels, nascent RNA synthesis, and small RNA abundance. In somatic cells, Piwi loss lead to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on transcription but also participating in germ cell piRNA biogenesis.
2013-02-09 | GSE43360 | GEO
Project description:Transposable elements (TEs) are self-mobilizing elements that make up a large fraction of mammalian genomes. PIWI proteins and PIWI interacting RNAs (piRNAs) are a part of a system aimed at controlling and preventing TE proliferation. We examined the TE landscape and piRNA repertoires from three non-model Laurasiatherian mammals (dog, horse and big brown bat) with differing TE complements and proliferation patterns to address questions about the evolutionary relationships between piRNAs and TEs. We found that the genomic abundance of new TEs may not match TE transcription. We speculate that a higher rate of SINE targeting by piRNAs in the horse genome contributed to the reduced SINE insertion rate.
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Project description:To test whether the RNA Polymerase II factor PAF1 affects PIWI silencing, we examined the transcriptome expression levels in Drosophila OSS cells following siRNA knockdown of these factors.
2017-08-31 | GSE84766 | GEO
Project description:Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here we examined the contribution of structural domains in Piwi proteins towards defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5′ uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5’ end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis
2014-07-01 | GSE55451 | GEO
Project description:Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of Transposable Elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36%of protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.
2017-04-19 | GSE87346 | GEO
Project description:Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here we examined the contribution of structural domains in Piwi proteins towards defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5M-bM-^@M-2 uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5M-bM-^@M-^Y end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis Immunoprecipitated small RNA were purified from Bmn4 cells for preparation of high-throughput sequencing libraries.
2014-07-01 | E-GEOD-55451 | biostudies-arrayexpress