Differential Argonaute-2 RNA immunoprecipitation of control cells versus cells overexpressing miR-146a followed by deep sequencing (Ago2 RIP-seq)
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PROVIDER: PRJNA430256 | ENA |
REPOSITORIES: ENA
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Project description:We undertook an unbiased approach based on differential Argonaute-2 (Ago2) RNA immunoprecipitation followed by deep sequencing (Ago2 RIP-seq) to unravel the molecular mechanism(s) underlying miR-146a function, namely the relevant mRNA target(s). Ago2 is a crucial protein that engages with both miRNA and its mRNA target(s) in the RISC complex. To overcome the problem of lack of commercially available high affinity antibodies against Ago2, we took advantage of a genetically modified mouse in which endogenous Ago2 is replaced with a N-terminal tagged version of the protein (3xFLAG- 6xHIS), thus allowing stringent purification (i.e., with low unspecific background) of the RNA molecules bound to Ago2 (upon UV-crosslinking), followed by RNA-sequencing. In order to enrich for miR-146a targets, we transduced purified CD3+ T cells (we could not use γδ T cells due to insufficient cell numbers for this approach) with a miR-146a-expressing retrovirus (with~12-fold overexpression of miR-146a). We thereby identified 225 putative mRNAs after differential Ago2-RIP-seq, from which 96 (42,7%) had a predicted miR-146a binding site in their 3’ UTR region.
2018-02-12 | GSE109263 | GEO
Project description:microRNA profiling in Argonaute 2 deficient mice bone marrow erythroblasts (ago2 fl/fl & ago2 -/-)
2010-05-26 | E-TABM-996 | biostudies-arrayexpress
Project description:The reduced sperm count observed in Ago2 cKO mice implies that AGO2 has non-redundant functions in the male germ line. Because AGO2 is a key protein in the RNA interference (RNAi) pathway, we first postulated that AGO2 loss disrupts normal transcriptional and translational dynamics of target mRNAs relevant to sperm maturation. To address this hypothesis, we took advantage of the apparently normal spermatogenesis in Ago2 cKO mice, which allowed us to purify matched meiotic and post-meiotic germ cells from Ago2 cKO and wild type controls. We examined changes in the transcriptome and proteome of these two spermatogenic stages in Ago2 cKO relative to control mice using RNA-seq and quantitative mass spectrometry (MS). To further examine if the changes in mRNA and protein levels detected in Ago2 cKO germ cells was due to a loss of regulation by the canonical AGO2-miRNA pathway, we characterized AGO2 protein interactors by AGO2 immunoprecipitation-mass spectrometry (IP-MS) in cytoplasmic and nuclear fractions of post-meiotic cells
2022-08-01 | PXD027935 | Pride
Project description:The goal of this study was to identify small RNA-ARGONAUTE interactions in Arabidopsis. Co-immunoprecipitation assays followed by high-throughput sequencing were done to identify small RNA associated with HA-AGO2 and HA-AGO7. Keywords: small RNA - ARGONAUTE interactions
2008-08-16 | GSE12037 | GEO
Project description:To determine the spectrum of miRNA targets regulated following Dicer deletion, we performed argonaute 2 (AGO2)-RNA Immunoprecipitation (RIP)-microarray in bone marrow-derived macrophages (BMDMs) from LysM-Cre/Dicerflox/flox/Apoe–/– and LysM-Cre/Dicerwt/wt/Apoe–/– mice. This analysis combined with miRNA profiling in Dicer wild type (WT) and knockout (KO) BMDMs may help to identify the miRNA targets regulated by Dicer deletion.
2018-05-23 | GSE87717 | GEO
Project description:Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and while its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo, using developing spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males exhibit abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.
2022-07-19 | GSE181264 | GEO
Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). To identify new regulators of the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity. Iterative rounds of screening revealed a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrated that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 revealed a dramatic expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
2017-01-17 | GSE89944 | GEO
Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). To identify new regulators of the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity. Iterative rounds of screening revealed a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrated that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 revealed a dramatic expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
2017-01-17 | GSE89943 | GEO
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
Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). To identify new regulators of the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity. Iterative rounds of screening revealed a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrated that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 revealed a dramatic expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
2017-01-17 | GSE89940 | GEO