SiRNA generation in reponsse to exogenous dsRNA application in Paramecium
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB86902 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR14718309.fastq.gz | Fastqsanger.gz | |||
| ERR14718310.fastq.gz | Fastqsanger.gz | |||
| ERR14718311.fastq.gz | Fastqsanger.gz | |||
| ERR14718312.fastq.gz | Fastqsanger.gz | |||
| ERR14718313.fastq.gz | Fastqsanger.gz |
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Project description:We applied nanoparticle packed heteroduplex RNA to paramecia in order to dissect siRNA populations being produced from exogenous dsRNA or from newly synthesized strands by different RNA dependent RNA polymerases.
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Project description:Supply of exogenous dsRNA (exo-dsRNA), either by injection or by feeding, is a fast and powerful alternative to classical knockout studies. The biotechnical potential of feeding techniques is evident from the numerous studies focusing on oral administration of dsRNA to control pests and viral infection in crops/animal farming. We aimed to dissect the direct and indirect effects of exo-dsRNA feeding on the endogenous short interfering RNA (endo-siRNA) populations of the free-living ciliate Paramecium. We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. This interference of exo-dsRNA with endo-siRNAs warrants further investigations into secondary effects in target species/consumers, risk assessment of dsRNA feeding applications, and environmental pollution with dsRNA.
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Project description:A green fluorescence protein (GFP)-derived dsRNA (dsRNA-GFP) has been used as an exogenous control for Apis mellifera RNAi assays by multiple research groups. Its sequence does not share any significant homology with any known honey bee genes. Although dsRNA-GFP is not expected to trigger an RNAi response in treated bees, undesirable effects on gene expression, pupal pigmentation or developmental timing have been routinely observed. To better understand the multiple molecular and phenotypic effects of dsRNA-GFP in honey bees and to evaluate its use as a control for RNAi studies, we examined the impact of dsRNA-GFP on global gene expression patterns in developing workers. We found that dsRNA-GFP causes large-scale changes in gene expression associated with multiple biological processes. Furthermore, dsRNA-GFP exposure tended to preferentially decrease, rather than increase, expression of genes compared to controls.
2013-01-10 | GSE41004 | GEO
Project description:A green fluorescence protein (GFP)-derived dsRNA (dsRNA-GFP) has been used as an exogenous control for Apis mellifera RNAi assays by multiple research groups. Its sequence does not share any significant homology with any known honey bee genes. Although dsRNA-GFP is not expected to trigger an RNAi response in treated bees, undesirable effects on gene expression, pupal pigmentation or developmental timing have been routinely observed. To better understand the multiple molecular and phenotypic effects of dsRNA-GFP in honey bees and to evaluate its use as a control for RNAi studies, we examined the impact of dsRNA-GFP on global gene expression patterns in developing workers. We found that dsRNA-GFP causes large-scale changes in gene expression associated with multiple biological processes. Furthermore, dsRNA-GFP exposure tended to preferentially decrease, rather than increase, expression of genes compared to controls. Gene expression differences were analyzed using a dye-swap design. Second instar worker larvae received 0.5 M-NM-<g of dsRNA-GFP. Non-treated and dsRNA-GFP treated 7 day-old workers (W7d) were sampled.
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Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics. virus-derived siRNA (vsiRNA) expression comparison between control and 4 different virus-infected cells in control as well as 5 different RNAi pathway protein knock-downs in Drosophila dl1 cells
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Project description:Paramecium cells in stationary phase were treated for deciliation and total mRNA extracted at two time points (45 and 130 minutes) after deciliation. Keywords: Time course analysis of expression during reciliation
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Project description:In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4, and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.
2016-12-14 | GSE92309 | GEO
Project description:Extensive research has characterized distinct exogenous RNAi pathways interfering in gene expression during vegetative growth of the unicellular model ciliate Paramecium. However, role of RNAi in endogenous transcriptome regulation, and environmental adaptation is unknown. Here, we describe the first genome-wide profiling of endogenous sRNAs in context of different transcriptomic states (serotypes). We developed a pipeline to identify, and characterize 2602 siRNA producing clusters (SRCs). Our data show no evidence that SRCs produce miRNAs, and in contrast to other species, no preference for strand specificity of siRNAs. Interestingly, most SRCs overlap coding genes and a separate group show siRNA phasing along the entire open reading frame, suggesting that the mRNA transcript serves as a source for siRNAs. Integrative analysis of siRNA abundance and gene expression levels revealed surprisingly that mRNA and siRNA show negative as well as positive associations. Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Importantly, most SRCs depend on both RDRs, reminiscent to primary siRNAs in the RNAi against exogenous RNA, indicating mechanistic overlaps between exogenous and endogenous RNAi contributing to flexible transcriptome adaptation.
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Project description:5-methyl-cytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisfulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methyl-cytosine DNA methylation as an integral part of its epigenetic arsenal.
2018-04-01 | GSE111621 | GEO