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Elucidation of Transcriptome-Wide MicroRNA Binding Sites in Human Cardiac Tissues by Ago2 HITS-CLIP

ABSTRACT: We deciphered Ago2:RNA interactions in post-mortem human heart tissues using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83410 | GEO | 2016/08/09

SECONDARY ACCESSION(S): PRJNA325822

REPOSITORIES: GEO

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Elucidation of Transcriptome-Wide MicroRNA Binding Sites in Human Cardiac Tissues by Ago2 HITS-CLIP

Project description:We deciphered Ago2:RNA interactions in post-mortem human heart tissues using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Profiling AGO2-associated RNAs in cardiac tissues obtained from 6 donors with heart diseases of differing etiologies

2016-08-09 | E-GEOD-83410 | biostudies-arrayexpress

Transcriptome-wide discovery of microRNA binding sites in human brain by Ago2 HITS-CLIP [Ago2-miRNA complexes]

Project description:Here, seeking to gain insight into the array of transcripts engaged with miRNAs in human brain, we performed HITS-CLIP to profile transcriptome-wide Ago2:RNA interactions in a panel of eleven post-mortem adult human brain samples harvested from adult motor cortex and cingulate gyrus, regions associated with movement and psychiatric disorders . High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) Eleven post-mortem adult human brain tissues were subjected to ultraviolet radiation to crosslink proteins with nucleic acids, and HITS-CLIP was performed as previously described by Chi et al Nature. 2009 Jul 23;460(7254):479-86 using an anti-Ago2 polyclonal antibody and some additional modifications.

2014-01-03 | E-GEOD-52083 | biostudies-arrayexpress

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Transcriptome-wide discovery of microRNA binding sites in human brain by Ago2 HITS-CLIP [Ago2-miRNA-target mRNA complexes]

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Elucidation of transcriptome-wide microRNA binding sites in human cardiac tissues by Ago2 HITS-CLIP.

Project description:MicroRNAs (miRs) have emerged as key biological effectors in human health and disease. These small noncoding RNAs are incorporated into Argonaute (Ago) proteins, where they direct post-transcriptional gene silencing via base-pairing with target transcripts. Although miRs have become intriguing biological entities and attractive therapeutic targets, the translational impacts of miR research remain limited by a paucity of empirical miR targeting data, particularly in human primary tissues. Here, to improve our understanding of the diverse roles miRs play in cardiovascular function and disease, we applied high-throughput methods to globally profile miR:target interactions in human heart tissues. We deciphered Ago2:RNA interactions using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Our initial exploration of this interactome revealed an abundance of miR target sites in gene coding regions, including several sites pointing to new miR-29 functions in regulating cardiomyocyte calcium, growth and metabolism. Also, we uncovered several clinically-relevant interactions involving common genetic variants that alter miR targeting events in cardiomyopathy-associated genes. Overall, these data provide a critical resource for bolstering translational miR research in heart, and likely beyond.

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Human Ago2-HITS-CLIP

Project description:mRNA and microRNA profile of Endothelial cells (HUVEC and HUAEC) were generated by HITS-CLIP assay, in triplicate

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From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data.

Project description:Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification.

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mRNAs associated with Ago2: Ago2, Ago2+miR-1, Ago2+miR-124, mock transfected

Project description:As a core RISC component, Ago2 associates with miRNAs and target mRNAs. To identify these mRNAs, we ran lysate from HEK293T cells over a FLAG resin from 2 conditions: +FLAG-Ago2, +mock transfection. To identify mRNAs associated with specific miRNAs, we ran lysate from HEK293T cells over a FLAG resin from 2 conditions: +FLAG-Ago2 & miR-1, and +FLAG-Ago2 & miR-124. Set of arrays that are part of repeated experiments Compound Based Treatment: mock transfected Keywords: Biological Replicate

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A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

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

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