Identification of high-affinity microRNA targets
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Mus musculus
SUBMITTER: Stanislas Werfel
PROVIDER: E-MTAB-5386 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We established a neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high throughput sequencing (AGO2-RIP-seq) to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this technique, we identified more than two thousand miRNA target genes in hippocampal neurons, regulating essential neuronal features such as axon guidance and transcription. Furthermore, we found that stable inhibition of the highly expressed miR-124 in hippocampal neurons led to significant changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. Our data suggest that target redundancies are common among microRNA families. Together, these findings greatly enhance our understanding of the mechanisms and dynamics through which miRNAs regulate their target genes in neurons. Analysis of the miRNA targetome in hippocampal neurons after inhibition of 2 different miRNAs. AAV5 injections into the hippocampus of adult C57BL/6 mice producing either of the following under a synapsin promoter: GFP only (Samples beginning with 'GFP124…' or 'GFP125…'), GFP-miR124sp (Samples beginning with 'miR124…'), GFP-miR125sp (Samples beginning with 'miR125…'), GFP-AGO2-miR292sponge (samples ending with '…292'), GFP-AGO2-miR124sponge (samples ending with '…124'), GFP-AGO2-miR125sponge (samples ending with '…125'). All other samples were sham-injected.
2015-07-31 | E-GEOD-65543 | biostudies-arrayexpress
Project description:MiR-21 is an important suppressor of T-cell apoptosis that is also widely overexpressed in many types of cancers. The exact mechanisms related to the anti-apoptotic effect of miR-21 is not well understood. In this study, we applied AGO2 RNA Immunoprecipitation followed by gene expression profiling (RIP-Chip) in Jurkat cells to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 resulted in reduced cell growth and induced apoptosis. Upon AGO2-RIP-Chip, we observed an overall increased enrichment of miR-21 target genes in the IP fraction of miR-21-overexpressing Jurkat cells as compared to the IP fraction of empty vector control cells. We noted a systematic decrease in transcript levels of predicted miR-21 target genes compared to EV control. We identified 72 genes that were 2-fold enriched in the AGO2-IP fraction of miR-21-overexpressing cells that contained a predicted miR-21 binding site. Of these, 71 were enriched 2-fold more in the miR-21-overexpressing cells as compared to EV Jurkat cells. The target gene for which the enrichment was most prominently increased upon miR-21 overexpression was the pro-apoptotic protein LATS1. Luciferase reporter assays confirmed direct targeting of the LATS1 3'UTR by miR-21. In line with the luciferase results, Western blot analysis revealed a decrease in LATS1 upon miR-21 inhibition. LATS1 qRT-PCR analysis in primary T-cells showed that LATS1 levels decrease upon T-cell stimulation while the miR-21 levels increase. Collectively, these data identify the miR-21 target LATS1 as a likely candidate whose inhibition contributes to the anti-apoptotic function of miR-21 in T-cells and perhaps also many types of cancers. Gene expression array on Jurkat cells overexpressing miR-21 and empty vector (EV).
2016-08-03 | E-GEOD-85116 | biostudies-arrayexpress
Project description:RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and as RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs. Flp-In T-REx HEK293 cells expressing FLAG/HA-tagged MOV10 WT, MOV10 K530A, MOV10 D645N and UPF1 were used to determine the protein-RNA interaction sites of RNA helicases MOV10 and UPF1 as well as MOV10 inactive variants using PAR-CLIP in combination with next generation sequencing. mRNA half-life changes of MOV10-targeted mRNA were determined by measuring mRNA half-lives by mRNA sequencing of mock and MOV10-depleted HEK293 cells.
2014-04-11 | E-GEOD-56748 | biostudies-arrayexpress
Project description:To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines. Further expression analyses using a large panel of HCC cell lines and primary tumors demonstrated four miRNAs, miR-101, -195, -378 and -497, as candidate TS-miRNAs frequently silenced in HCCs. Among them, two clustered miRNAs miR-195 and miR-497 showed significant growth-suppressive activity with induction of G1 arrest. Comprehensive exploration of their targets using Argonute2-immunoprecipitation-deep-sequencing (Ago2-IP-seq) and genome-wide expression profiling after their overexpression, successfully identified a set of cell-cycle regulators, including CCNE1, CDC25A, CCND3, CDK4, and BTRC. Our results suggest the molecular pathway regulating cell cycle progression to be integrally altered by downregulation of miR-195 and miR-497 expression, leading to aberrant cell proliferation in hepatocarcinogenesis. Identification of miR-195 and miR-497 target genes by sequencing Ago2-binding mRNAs and total mRNAs of miR-195 or miR-497 overexpressed, or non-treated Hep G2 cell. Deep sequencing of RNAs in Ago2-IP fraction and mRNAs extracted from miR-195 or miR-497 overexpressed, or non-treated Hep G2 cell.
2013-05-31 | E-GEOD-41074 | biostudies-arrayexpress
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. Experiment Overall Design: Sequencing-by-synthesis technology was used to sequence small RNA from input and co-immunoprecipitation fractions from cell lysates of inflorescence tissue from Arabidopsis plants transformed with HA epitope-tagged AGO2 or AGO7 constructs. Meaningful intepretation requires comparison between the input and co-immunoprecipitation datasets. Experiment Overall Design: Note: Raw data files requested, however, the submitter declined to provide them.
2009-08-30 | E-GEOD-12037 | biostudies-arrayexpress
Project description:Background: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequenced long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. Results: Analysis of these data sets revealed that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently catalogued. We further found that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs revealed that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. Conclusions: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon. PAR-CLIP profiling for snoRNP core proteins NOP56, NOP58, Fibrillarin, and Dyskerin in HEK293 cells. Small RNA profiling using RNA-seq in HEK293 and HeLa cells, small RNA profiling using IP-seq of Ago2 associated small RNAs.
2013-05-08 | E-GEOD-43666 | biostudies-arrayexpress
Project description:Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays, and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-Sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs, and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes, and upregulating pluripotency-associated genes such as Lin28. For RNA-IP experiments, cells were transfected with 300 pmol of miR-294 or cel-239b control miRNA (Dharmacon) and harvested 12-16 hr after transfection for immunoprecipitation. Following RNA-immunoprecipitation of Ago2-myc, RNA from the INPUT (total RNA) and IP were subjected to library preparation and sequenced by SOLiD. There are 10 samples from Dicer1-null mouse ES cells, which are essentially 5 sample pairs of INPUT (A) and its corresponding RNA-IP (B): Ago2-myc transfected with miR-294 (sampe 1), 2 replicates of Ago2-myc-MUT (catalytically inactive) transfected with miR-294 (sample 2 & 5), 2 replicates of Ago2-myc transfected with cel-239b (sample 3 & 4).
2010-02-27 | E-GEOD-20199 | biostudies-arrayexpress
Project description:This project aims to investigate the metabolic pathways expressed by the active microbial community occurring at the deep continental subsurface. Subsurface chemoLithoautotrophic Microbial Ecosystems (SLiMEs) under oligotrophic conditions are supported by H2; however, the overall ecological trophic structures of these communities are poorly understood. Some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa appear to support inverted trophic pyramids wherein methanogens contributing <5% of the total DNA apparently produce CH4 that supports the rest of the community. Here we show the active metabolic relationships of one such trophic structure by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Four autotrophic β-proteobacteria genera that are capable of oxidizing sulfur by denitrification dominate. They co-occur with sulfate reducers, anaerobic methane oxidizers and methanogens, which each comprises <5% of the total community. Defining trophic levels of microbial chemolithoautotrophs by the number of transfers from the initial abiotic H2-driven CO2 fixation, we propose a top-down cascade influence of the metabolic consumers that enhances the fitness of the metabolic producers to explain the inverted biomass pyramid of a multitrophic SLiME. Symbiotic partnerships are pivotal in the deep biosphere on and potentially beyond the Earth.
2018-10-27 | PXD004634 | Pride
Project description:miR-133a-3p is a highly abundant cardiomyocyte-enriched microRNA whose expression is persistently decreased in response to pressure overload (or transverse aortic constriction, TAC) in mice. Overexpression of miR-133a in cardiomyocytes of mouse hearts in vivo (under the control of the Myh6 promoter) decreases pressure overload-induced apoptosis and fibrosis. In previous studies using microarray platforms, we detected numerous mRNAs whose transcript levels were altered by either or both of miR-133a overexpression and pressure overload. The data set presented here builds upon our previous study in these mice by examining mRNA-RISC associations (using Ago2-immunoprecipitated RNA) and global mRNA abundances via RNA-sequencing procedures, and tests the hypothesis that mRNAs targeted by overexpressed miR-133a are dissimilar between sham and TAC contexts. Cardiac polyadenylated RNA (mRNA) profiles were generated from nontransgenic and transgenic mouse hearts of FVB/N background, on Illumina HiSeq 2000 instruments. Male mice 8-12 weeks of age were used in these studies, and subjected to sham surgery or 1 week of pressure-overload via transverse aortic constriction (TAC). 3 nontransgenic sham, 7 transgenic sham, 5 nontransgenic TAC, 4 transgenic TAC, each with mRNA-seq and RISC-seq (mRNA-seq of Ago2 immunoprecipitate) data.
2016-01-11 | E-GEOD-65141 | biostudies-arrayexpress
Project description:SILAC labeled human kidney cells (293 cells) or bat kidney cells (PakiT03cells)were infected with Hendra virus for 8 or 24 hours and compared to uninfected control cells. Protein identification and quantitation relied on a combination of Uniprot lists of proteins and Proteomics Informed by Transcriptomics (PIT) analysis whereby RNA extracted from the same samples was deep sequenced and the sequencing data was used to construct mRNA from which possible ORFS were inferred and used as a search space by MaxQuant.
2014-11-05 | PXD001165 | Pride