Production of Virus-Derived Ping-Pong-Dependent Piwi-like RNAs in the Mosquito Soma
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ABSTRACT:
PROVIDER: PRJNA147359 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR346384.fastq.gz | Fastqsanger.gz | |||
| SRR346385.fastq.gz | Fastqsanger.gz | |||
| SRR346386.fastq.gz | Fastqsanger.gz | |||
| SRR346387.fastq.gz | Fastqsanger.gz | |||
| SRR346388.fastq.gz | Fastqsanger.gz |
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Project description:The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma and a role for dsRNA in biogenesis. We also present evidence that suggests virus-derived piwi-like RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication. Comparison of virus derived small RNAs in several mosquito cell types
2012-01-06 | E-GEOD-32247 | biostudies-arrayexpress
Project description:The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma and a role for dsRNA in biogenesis. We also present evidence that suggests virus-derived piwi-like RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.
2012-01-06 | GSE32247 | 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 | 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: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
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: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. Immunoprecipitated small RNA were purified from untransfected or transfected Bmn4 cells for preparation of high-throughput sequencing libraries.
2014-06-04 | E-GEOD-57420 | 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. 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:PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Planarian PIWI proteins thus act as both nucleases and RNA-binding proteins, enabling planarians to repurpose piRNAs for critical roles in neoblast mRNA turnover.
2019-09-04 | GSE122199 | GEO
Project description:Belle has been known to be co-localized with piRNA-related proteins at the nuage of germline cells during Drosophila oogenesis. However, its role in piRNA biogenesis remains unclear. To reveal whether Belle is involved in regulating piRNA expression, we performed next-generation sequencing analysis of small non-coding RNAs on ovaries harvested from the wild type (W1118) and trans-heterozygous bel[74407/neo30] mutant. Small RNA-seq experiments were performed on three individual ovary samples with the same genotype. For piRNA expression analysis, we performed mapping of three sets of small RNA sequencing reads for each genotype to previously identified eight distinct piRNA clusters located in four different Drosophila chromosomes (from X to 4). Analysis of the piRNA expression profiling from these piRNA cluster loci indicates that some specific piRNA populations were either upregulated or downregulated in bel mutant ovaries compared with wild-type ovaries. Furthermore, we performed systematic analysis by mapping piRNA sequencing reads to sequences of all identified Drosophila transposable elements (TEs) to classify and measure piRNA reads based on their TE targets. Among 124 TE-classified piRNA populations, 9 and 20 of them were upregulated and downregulated (≥2 folds), respectively, in bel74407/neo30 mutant ovaries compared with those from wild-type ovaries. To examine the effect of the bel[74407/neo30] mutation on the ping-pong cycle for secondary piRNA biogenesis, analysis of the ping-pong signature of piRNAs specifically mapped to the retro-element Burdock was performed. The ping-pong signature for the generation of secondary piRNAs was not significantly altered in bel mutants compared with the wild type. These results, taken together, indicate that Bel is not required for primary and secondary piRNA biogenesis, but it is involved in regulating expression of specific subsets of piRNA populations.
2017-09-01 | GSE65565 | GEO