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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNAi knock down of GW182 in Drososphila cells compared to cells with knock down of AGO1, a key effector of miRNA-mediated gene silencing

ABSTRACT: RNA silencing pathways are conserved gene regulation mechanisms that lead to degradation, translational repression or transcriptional silencing of target-transcripts selected based on complementarity with small RNA molecules. Using firefly luciferase reporters, we previously were able show that the RNA binding protein GW182 plays a role in microRNA-mediated gene silencing (Rehwinkel, J. et al., RNA J. 2005). In this study, we provide further evidence for the role GW182 in the miRNA pathway by determining targets regulated by this protein at the genomic level. We examined expression profiles in Drosophila cells depleted of GW182 and compared these profiles to data obtained from cells depleted of AGO1, a key effector of miRNA-mediated gene silencing.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Isabelle Behm-Ansmant

PROVIDER: E-MEXP-487 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes.

Behm-Ansmant Isabelle I Rehwinkel Jan J Doerks Tobias T Stark Alexander A Bork Peer P Izaurralde Elisa E

Genes & development 20060630 14

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. We show that depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector ...[more]

PMID: 16815998

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RNAi knock down of four Argonaute paralogs (AGO1, AGO2, PIWI and Aubergine) and the microRNA-processing enzyme Drosha in Drosophila

Project description:RNA silencing pathways are conserved gene regulation mechanisms that lead to degradation, translational repression or transcriptional silencing of target-transcripts selected based on complementarity with small RNA molecules. The fraction of the genome regulated by these pathways has not been established. Argonaute proteins play fundamental roles in RNA silencing. To identify their physiological targets at the genomic level, we examined expression profiles in Drosophila cells depleted of 4 Argonaute paralogs (i.e. AGO1, AGO2, PIWI or Aubergine). We also profiled cells depleted of the microRNA (miRNA)-processing enzyme Drosha.

2006-11-20 | E-MEXP-295 | biostudies-arrayexpress

RNAi knock down of mRNA decapping co-activators in Drosophila S2 cells

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2007-09-03 | E-MEXP-1012 | biostudies-arrayexpress

RNAi knock-down of CAF1-CCR4-NOT1 deadenylase complex or AGO1 in D. melanogaster S2 cells

Project description:miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. AGO1 is a key protein required for miRNA-mediated gene silencing. In animal cells, mRNA degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1 or NOT1. We show that approximately 45% of AGO1-targets are regulated by both CAF1 and NOT1, indicating deadenylation is a widespread effect of miRNA regulation.<br><br>We employed RNA interference using long double-stranded RNAs to deplete cultured S2 cells of AGO1 (CG6671, 2 independent samples), CAF1 (CG5684, 2 independent samples), or NOT1 (CG1884, 3 independent samples). Further, we used Affymetrix oligonucleotide microarrays to analyze expression profiles in these samples. We included the following controls: mock RNAi treatment and GFP dsRNA treatment (3 and 2 independent samples, respectively).<br>

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Expression data from Tnrc6a (GW182) mutant E18.5 lungs

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2017-05-01 | GSE89327 | GEO

Expression data from Tnrc6a (GW182) mutant yolk sac

2011-09-01 | GSE30244 | GEO

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila

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2013-12-04 | E-GEOD-51585 | biostudies-arrayexpress

Interactome of Nematostella GW182 in HEK293 cells

Project description:Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria. Despite this remarkable mechanistic conservation Argonaute and GW182 differentially co-evolved in bilaterians and cnidarians seemingly leading to distinct interaction surfaces.

2016-09-27 | PXD004668 | Pride

Expression data from Tnrc6a (GW182) mutant yolk sac

Project description:GW182 (Tnrc6a) is a key component of RISC (miRNA-Induced Silencing Complex) that plays a critical role in miRNA-mediated gene silencing. Here, we show that GW182 is expressed in the yolk sac endoderm, and that gene-trap disruption of GW182 leads to growth arrest of yolk sac endoderm, impaired hematopoiesis and embryonic lethality. To investigate roles of GW182 in the yolk sac endoderm, we assessed changes in mRNA expression in the yolk sac of E9.5 GW182gt/gt embryos using microarrays (Affymetrix). Yolk sac of wild type littermates and GW182gt/gt embryos at E9.5 was collected for total RNA isolation using Trizol (Invitrogen). RNAs were purified according to the manufacturer’s protocol before subjected to Mouse Gene 1.0 ST Whole Genome Array (Affymetrix) for mRNA expression profiling. Experiments were performed in triplicate. Differentially expressed mRNAs were identified using a two-sample t-test (P<0.05 considered significant).

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