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

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE30955 | GEO | 2011/08/16

SECONDARY ACCESSION(S): PRJNA146245

REPOSITORIES: GEO

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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

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

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

2010-10-04 | GSE23560 | 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

Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries

Project description:Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5ʹ→3ʹ directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5ʹ ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5ʹ→3ʹ helicase activity, and mutations that abolish it reduce piRNA processing. Another somatic piRNA pathway factor Yb, and an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.

2017-08-16 | GSE102013 | 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

2011-02-10 | GSE26407 | 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

Mechanistic divergence of piRNA biogenesis in Drosophila [ChIP-seq]

Project description:Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to initiate biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing TEs. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that neither mechanism is required to efficiently produce TE antisense piRNAs in Drosophila. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested.

2022-09-19 | GSE213379 | GEO

Mechanistic divergence of piRNA biogenesis in Drosophila [RNA-seq]

2022-09-19 | GSE213375 | GEO

Mechanistic divergence of piRNA biogenesis in Drosophila [ncRNA-seq]

2022-09-19 | GSE213369 | GEO