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MiRBShunter: a pipeline to identify miRNA binding sites from Ago2-CLIP data by de novo motif finding

ABSTRACT: The proposed method uses enriched de novo motifs on the peak sequences of Ago2-CLIP to determine miRNA-RNA heteroduplex for either canonical or non-canonical miRNA-binding sites. miRNA-RNA heteroduplex are ranked based on a duplex score. Since the quality of the peak detection is of pivotal importance for the final output of the proposed method, we also provide an illustrative comparative evaluation of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, for four publicly available Ago2-datasets and one previously unpublished in-house Ago2-dataset in stem cells. The experimental validation was carried out on the in-houseAgo2 dataset by gene reporter assay.

ORGANISM(S): Mus musculus

PROVIDER: GSE85219 | GEO | 2017/03/01

SECONDARY ACCESSION(S): PRJNA336567

REPOSITORIES: GEO

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Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates (part 3)

Project description:When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Ago2/microRNA machinery has been shown to participate in stress-induced translational upregulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by CLIP-seq. Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3` UTR and CDS sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2 binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA-seq. Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian microRNAs in mediating the translational component of the stress response. In this sub-series, CLIP and RNAseq data from the hippuristanol treatment experiment are presented. Experiments on 293S cells +/- hippuristanol treatment, in 2 biological replicates. Here, for each treatment/replicate sample, an aliquot was used for RNA-seq, and the rest was split into three aliquots to perform 3 parallel CLIP-seq protocols with different antibodies. So, each RNA-seq dataset here corresponds to 3 CLIP-seq datasets.

2013-07-15 | E-GEOD-44378 | biostudies-arrayexpress

Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates (part 2)

Project description:When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Ago2/microRNA machinery has been shown to participate in stress-induced translational upregulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by CLIP-seq. Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3` UTR and CDS sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2 binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA-seq. Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian microRNAs in mediating the translational component of the stress response. In this sub-series, CLIP and RNAseq data from the emetine treatment experiment are presented. Experiments on 293S cells +/- emetine treatment, in 1 biological replicate. Here, for each treatment/replicate sample, an aliquot was used for RNA-seq, and the rest was split into three aliquots to perform 3 parallel CLIP-seq protocols with different antibodies. So, each RNA-seq dataset here corresponds to 3 CLIP-seq datasets.

2013-07-15 | E-GEOD-44377 | biostudies-arrayexpress

ena-DATASET-DKFZ-IBIOS-17-08-2015-09:13:01:216-19 - samples

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| EGAD00001001468 | EGA

RNA-seq of miR-CLIP double purified, miR-124/miR-132 miR-CLIP probe treated HEK293T cells and corresponding mock treated and intermediate samples

Project description:Targetome of miR-124/miR-132 was enriched in HEK293T cells, a cell line which lacks the expression of these miRNA. The targetome was captured using miR-CLIP, a transfection-based double-purification crosslinking and immunoprecipitation (CLIP) method. Therefore HEK293T cells were transfected with miR-124/miR-132 miR-CLIP probes, which are trioxsalen, biotin bis-modified pre-miRNAs. Unspecific RNA-protein and trioxsalen mediated RNA-RNA crosslinking were induced by UV-irradiation before cell lysis. Total RNA (input samples) was enriched for AGO2-bound RNA using AGO2 immunoprecipitation, by depleting non-RISC associated RNAs. A second enrichment for specific targets of the transfected miRNA is performed using magnetic streptavidin-beads. This step relies on the interaction of the biotinylated probe, on which the RNA target is crosslinked, with the beads. The method was shown to deliver high-quality miRNA targetomes of both 5p and 3p miRNAs.

2020-11-23 | E-MTAB-8517 | biostudies-arrayexpress

Halo-enhanced Ago2 pulldown (HEAP) to identify miRNA targets in vivo

Project description:The identification of miRNA targets by Ago2-CLIP methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily translated to an in vivo setting. To overcome these limitations and to facilitate the investigation of miRNA functions in mice, we have developed a method (HEAP) to map miRNA-mRNA binding sites. This method uses a novel mouse strain harboring a conditional Halo-Ago2 allele expressed from the Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be efficiently purified from cells and tissues expressing Halo-Ago2. We demonstrate the reproducibility and sensitivity of this method in embryonic stem cells, in embryos, in adult tissues and in autochthonous mouse models of cancers. These tools and datasets are valuable resources to the scientific community and will facilitate the characterization of miRNA functions under physiological and pathological conditions.

2019-10-27 | GSE139344 | GEO

RNA-seq to measure gene expression changes caused by Dicer1 knockout in mouse embryonic stem cells (mESCs)

2019-10-27 | GSE139348 | GEO

RNA-seq to measure mRNA expression in normal lung tissues, KRasG12D; p53-null(KP)-driven and Eml4-Alk(EA)-driven lung adenocarcinomas

2019-10-27 | GSE139347 | GEO

Ribo-depleted RNA-seq in KRasG12D; p53-null (KP) lung adenocarcinoma cells to measure gene expression changes upon the expression of a T6B-YFP fusion

2019-10-27 | GSE139346 | GEO

Halo-enhanced Ago2 pulldown (HEAP) to identify miRNA targets in mouse embryonic stem cells (mESCs)

2019-10-27 | GSE139345 | GEO

Muscleblind-Like 2 mediated alternative splicing in the developing bain by mRNA sequencing

Project description:We have identified Mbnl2 mediated splicing events and mRNA expression regulation by comparing WT and Mbnl2 ΔE2/ΔE2 mouse hippocampii using Affymetrix Mouse Exon Junction Array and mRNA sequencing. The splicing microarray data has already been submitted under GSE37908 which also includes a re-analysis of RNA-seq data. The TableS1.xls contains Splicing microarray analysis data of Mbnl2+/+ vs. MBNL2 ΔE2/ΔE2 knockout hippocampus. The TableS2.xls files contain RNA-Seq, Gene Ontology, HITS-CLIP and CIMS summary of Mbnl2+/+ vs. MBNL2 ΔE2/ΔE2 knockout hippocampus. The file contents are descibed in the 'TableS1_S2_readme.pdf' and data processing details are included in the 'data_processing_readme.pdf' file. The Mbnl2_TableS2.xls contains data from exon junction microarrays for splicing analysis between WT and Mbnl2 delta2 mice. It also contains RNA-seq based splicing analysis between WT and Mbnl2 delta2 mice. Mbnl2_TableS2 further contains common targets between the two analyses, Gene Ontology and finally the HITS-CLIP data. CLIP tag summary describes read numbers for the three biological replicates. CLIP sig peaks contains significant HITS-CLIP unique reads/peaks after processing the data (BED files). Finally, CIMS analysis describes the Mbnl2 binding motif.

2012-08-16 | GSE38497 | GEO

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