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Ago2-CLASH reveals a wide repertoire of small RNA mediated regulation [CLASH-seq]

ABSTRACT: Here we present the CLASH analysis of AGO2 in HEK293 cells to address the small RNA repertoire and uncover their physiological targets. We developed an optimized bioinformatics approach of chimeric read identification to detect chimeras of high confidence. We report thousands of Ago2 target sites driven by microRNAs, but also a substantial number of Ago2 ‘drivers’ derived from fragments of other small RNAs such as tRNAs, snoRNAs, rRNAs and others. Target validation of several miRNAs delivered by 3’ Quantseq RNA-Seq.

ORGANISM(S): Homo sapiens

PROVIDER: GSE221572 | GEO | 2023/12/24

REPOSITORIES: GEO

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Ago2-CLASH reveals a wide repertoire of small RNA mediated regulation [RNA-Seq]

Project description:Here we present the CLASH analysis of AGO2 in HEK293 cells to address the small RNA repertoire and uncover their physiological targets. We developed an optimized bioinformatics approach of chimeric read identification to detect chimeras of high confidence. We report thousands of Ago2 target sites driven by microRNAs, but also a substantial number of Ago2 ‘drivers’ derived from fragments of other small RNAs such as tRNAs, snoRNAs, rRNAs and others. Target validation of several miRNAs delivered by 3’ Quantseq RNA-Seq.

2023-12-24 | GSE221573 | GEO

Mapping targets for small nucleolar RNAs in human cells [CLASH/FLASH]

Project description:We report the identification of potential novel RNA targets for box C/D snoRNAs in human HEK293 cells, using the approaches of UV crosslinking and sequencing of hybrids (CLASH), and formaldehyde crosslinking and sequencing of hybrids (FLASH).

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Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [CLASH]

Project description:By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3’UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.

2020-05-01 | GSE123049 | GEO

PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) analysis to detect RNA-RNA interaction at poly(A) site region

Project description:In an attempt to discover potential RNA-RNA interactions in mRNA 3’ processing regulation, we developed PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) method, which combines reported CLASH and poly(A) site sequencing protocols. Briefly, HeLa cells were treated with AMT, or without AMT as control, and irradiated at 365 nm for crosslinking. Following nuclear RNA fragmentation and purification, the potential proximate RNAs/PASs were ligated on the oligo d(T)25 beads, hybrids were reverse crosslinked and amplified with the QuantSeq Rev 3' mRNA sequencing library prep kit (Lexogen, Cat. 016.24) and sequenced with Hiseq, producing 150 bp paired end reads.

2019-01-22 | GSE125398 | GEO

Ago2-CLASH reveals a wide repertoire of small RNA mediated regulation

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-12-24 | GSE221574 | GEO

miRNA* loading on Ago2

Project description:In flies, microRNAs (miRNAs) are synthesized as a short imperfect duplex, where the miRNA is paired to another small RNA, dubbed the "miRNA*". Usually, miRNA*s are degraded in the last steps of miRNA maturation, but we found evidence that some of them can be loaded on the Ago2 protein. Small RNAs (18-29 nt) from fly heads (WT, ago2 mutants, dcr-2 homozygous and heterozygous mutants) were sequenced using a Solexa Genome Analyzer instrument. The ago2 cDNA library was loaded twice on the instrument, yielding two technical replicates.

2010-08-02 | E-GEOD-12462 | biostudies-arrayexpress

Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [RNA-seq]

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CLASH for 15.5K and FBL proteins in HEK293T cells

Project description:We performed CLASH for 15.5K and FBL in HEK293T cells which overexpressed 15.5K and FBL, respectively.

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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

2022-08-01 | PXD027935 | Pride

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