Bombyx mori strain:BmN4
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ABSTRACT:
PROVIDER: PRJDB17075 | ENA |
REPOSITORIES: ENA
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Project description:In germ cells, piRNAs are amplified through the Ping-Pong cycle that depends on reciprocal Slicer-mediated target RNA cleavage by two PIWI members. A germ-specific DEAD-box protein Vasa is required for the process. However, Vasa’s function is poorly understood. Here, we show that target RNAs cleaved by a Bombyx mori (silkworm) PIWI, Siwi, remain to be bound with the protein upon cleavage, but are released in the presence of Vasa in B. mori (BmVasa) and ATP. Under normal conditions, BmVasa co-purifies with Siwi, but not with second B. mori PIWI BmAgo3. However, when BmVasa loses the ATP-binding and RNA-unwinding activities, BmVasa avidly associates with Siwi and BmAgo3, which contains transposon transcripts predominantly in sense orientation, the sources of BmAgo3-piRNAs. Without BmVasa, BmAgo3 is devoid of piRNAs. Thus, BmVasa actively releases target RNAs from Siwi, upon its cleavage, to urge BmAgo3-piRNA complex formation in the Ping-Pong cycle, enabling continuous supply of piRNAs in germ cells.
2014-12-31 | GSE58221 | GEO
Project description:Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.
2016-06-08 | GSE50984 | GEO
Project description:Two Gtsf paralogs orthogonally activate their partner PIWI proteins for target cleavage in silkworms
| PRJDB13728 | ENA
Project description:piRNA profile of Zuc knockdown in BmN4 cells
| PRJDB17073 | ENA
Project description:Proof-of-concept for the ping-pong amplification cycle of PIWI-interacting RNAs
| PRJDB4324 | ENA
Project description:PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction.
2014-12-17 | GSE53919 | GEO
Project description:PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction.
2014-12-17 | GSE53915 | GEO
Project description:RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Previously, the BmN4-SID1 cell expressing Caenorhabditis ele gans SID-1 was established, in which soaking RNAi could induce effective gene silencing. To establish its utility, 6 cell cycle progression related cDNAs, CDK1, MYC, MYB, RNRS, CDT1, and GEMININ, were isolated from the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), and their expressions were further silenced by soaking RNAi in the BmN4-SID1 cells. The cell cycle progression analysis using flow cytometer demonstrated that the small amount of double stranded RNA was enough to arrest cell cycle progression at the specific cell phases. These data suggest that RNAi in the BmN4-SID1 cells can be used as a powerful tool for loss-of-function analysis of B. mori genes.
| S-EPMC4015410 | biostudies-literature
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.
2012-07-01 | GSE38090 | GEO
Project description:Germline-specific Piwi-interacting RNAs (piRNAs) protect the genome against selfish genetic elements and are essential for fertility in animals. piRNAs targeting active transposons are amplified by a feed-forward loop known as the Ping-pong cycle, which links endonucleolytic slicing of target RNAs by Piwi proteins to biogenesis of new piRNAs. However, the biochemical framework for this pathway remains elusive. Here, we describe the identification of a transient Amplifier complex mediating the biogenesis of secondary piRNAs in insect cells. This complex is nucleated by the RNA helicase Vasa and contains the two Piwi proteins participating in the Ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodelling by Vasa facilitates the transfer of 5'-sliced piRNA precursors between the Ping-pong partners, and failure to achieve this results in Drosophila female sterility.
2014-06-04 | GSE57420 | GEO