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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Shutdown is a component of the Drosophila piRNA biogenesis machinery (smallRNA)

ABSTRACT: In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping- pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown, a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germ line and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins, with domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones. Though the relevance of these domains to piRNA biogenesis is unknown, evolutionary comparisons raise questions about the integrity of these functions in the shutdown protein. Examination of small RNA levels from nos-GAL4 or tj-GAL4 driven UAS-dsRNA knockdowns of white, shu and piwi in ovaries of Drosophila melanogaster by deep sequencing (using Illumina GAII).

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Benjamin Czech

PROVIDER: E-GEOD-38089 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Shutdown is a component of the Drosophila piRNA biogenesis machinery (RNA-seq)

Project description:In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown, a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown. Analysis of mRNA expression in Drosophila OSS cells transfected with GFP dsRNA. One sample and replicate, used to establish the OSS baseline transcriptome in the presence of exogenous RNAi activity.

2012-06-30 | E-GEOD-38090 | biostudies-arrayexpress

A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway [smallRNA-Seq]

Project description:The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two inter-related branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborated pathway centered on the three gonad-specific Argonaute proteins Piwi, Aubergine, and Argonaute3. While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals new key factors of piRNA-mediated transposon silencing, including the novel piRNA biogenesis factors, CG2183 (GASZ) and Deadlock. Last, our data uncovers a previously unanticipated set of factors preferentially required for repression of different transposons types. Examination of small RNA levels from nos-GAL4 or tj-GAL4 driven UAS-dsRNA knockdowns of control genes and piRNA pathway components in ovaries of Drosophila melanogaster by deep sequencing (using Illumina HiSeq2000).

2013-05-10 | E-GEOD-46099 | biostudies-arrayexpress

Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline

Project description:Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. Here we show that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3’s compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. We propose that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs. In order to investigate function of Krimp in piRNA pathway, sequencing of Piwi subfamily protein associated small RNAs was performed using adult Drosophila ovaries and Ovarian Somatic Cells (OSCs) depleted for Krimp or Aub.

2015-05-28 | E-GEOD-69293 | biostudies-arrayexpress

Shutdown is a component of the Drosophila piRNA biogenesis machinery (RNA-seq)

2012-07-01 | GSE38090 | GEO

Shutdown is a component of the Drosophila piRNA biogenesis machinery (smallRNA)

Project description:In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping- pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown, a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germ line and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins, with domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones. Though the relevance of these domains to piRNA biogenesis is unknown, evolutionary comparisons raise questions about the integrity of these functions in the shutdown protein.

2012-07-01 | GSE38089 | GEO

piRNA-guided slicing specifies transcripts for Zucchini dependent, phased piRNA biogenesis

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

Major concepts of piRNA biogenesis revealed by the analysis of Shutdown, a co-chaperone with essential roles in the biogenesis of all Drosophila piRNA populations

Project description:In animal gonads, 23-30nt long PIWI interacting RNAs (piRNAs) guarantee genome integrity by guiding the sequence specific silencing of selfish genetic elements such as transposons. Two major branches of piRNA biogenesis, namely primary processing and ping-pong amplification, feed into the PIWI clade of Argonaute proteins. Despite our conceptual understanding of piRNA biogenesis, major gaps exist in the mechanistic understanding of the underlying molecular processes as well as in the knowledge of the involved players. Here, we demonstrate an essential role for the female sterility gene shutdown in the piRNA pathway. Shutdown, an evolutionarily conserved co-chaperone of the immunophilin class is the first piRNA biogenesis factor that is essential for all primary and secondary piRNA populations in Drosophila. Based on these findings, we define distinct groups of piRNA biogenesis factors and reveal the core concept of how PIWI family proteins are hard-wired into piRNA biogenesis processes. small-RNA libraries from 2 control samples and 7 knock-down samples of D. mel. ovaries and 2 small-RNA profiles from Piwi IP and Aub IP from OSCs.

2012-06-29 | E-GEOD-38728 | biostudies-arrayexpress

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [Rhino RNA-seq]

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. total RNA sequencing of RNA from ovaries of shWhite and Rhino knockdown flies

2014-07-26 | E-GEOD-59607 | biostudies-arrayexpress

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [run-on]

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. nuclear run-on in wildtype and Rhino knockdown to investigate the transcriptional activity in Rhino knockdown compared to wildtype

2014-07-26 | E-GEOD-59605 | biostudies-arrayexpress

Major concepts of piRNA biogenesis revealed by the analysis of Shutdown, a co-chaperone with essential roles in the biogenesis of all Drosophila piRNA populations

2012-06-30 | GSE38728 | GEO

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