PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) analysis to detect RNA-RNA interaction at poly(A) site region
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ABSTRACT:
PROVIDER: PRJNA516215 | ENA |
REPOSITORIES: ENA
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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
Project description:Purpose: Identification of miRNA-mRNA interactions in human cells. Method: We used the CLASH technique with PTH-tagged AGO1 protein as a bait. Results: In 6 experiments we identified more than 18,000 miRNA-mRNA interactions in human HEK293 cells, corresponding to targets of 399 miRNAs. Conclusions: The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, non-seed basepairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA basepairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding. 6 samples (E1-E6) were prepared with comparable but not identical protocols, described in more detail in Helwak et al. Cell 2013. Samples E7-E10 were used for the determination of the background of the method resulting from RNA-RNA interactions formed after cell lysis.
2013-08-30 | E-GEOD-50452 | biostudies-arrayexpress
Project description:High-throughput identification of miRNA targets using CLASH (crosslinking, ligation and sequencing of hybrids)
| PRJNA217521 | ENA
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).
2021-07-21 | GSE114825 | GEO
Project description:Purpose: Identification of miRNA-mRNA interactions in human cells. Method: We used the CLASH technique with PTH-tagged AGO1 protein as a bait. Results: In 6 experiments we identified more than 18,000 miRNA-mRNA interactions in human HEK293 cells, corresponding to targets of 399 miRNAs. Conclusions: The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, non-seed basepairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA basepairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding.
2013-08-30 | GSE50452 | GEO
Project description:To understand how CFIm25, an mRNA 3' processing regulator, affects global mRNA alternative polyadenylation (APA) profiling in H9 cells. We performed PAS-seq (polyadenylation site sequencing) in control (2 samples) and CFIm25 KD cells (3 samples). PAS-seq library was prepared using a lexogen kit (3'-seq, Catolog. 016.024). Sequencing was performed using Novaseq PE150.
2022-04-21 | GSE176343 | GEO
Project description: Not available
| S-EPMC3969109 | biostudies-literature
Project description:Alternative polyadenylation (APA) is a regulatory mechanism that controls gene expression level and function through the usage of different transcription termination sites, but how APA is regulated to influence epidermal tissue differentiation remains under-characterized. Leveraging 3’READS+, we identified prevalent APA in keratinocytes. We further identified a high-confidence list of genes that alters their usages of polyadenylation site (PAS) during keratinocyte differentiation. To begin to understand the mechanisms that modulates PAS selection within a given gene, we identified that the expression of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex is down regulated during keratinocyte differentiation. Suppression of CPSF using RNAi or CRISPRi strongly impaired epidermal regeneration with upregulation of terminal differentiation marker gene expression. Mechanistically, we identified that CPSF suppression decreased the usage of the GRHL3 proximal PAS and upregulated full-length GRHL3 mRNA production. Knockdown of GRHL3 in the context of CPSFi partially restored differentiation marker gene expression. We also found CPSF interacts with RNA-binding proteins to control PAS usage. Thus, our data suggest a model where CPSF binding preference and PAS choices regulate epidermal terminal differentiation.
2020-11-02 | GSE127223 | GEO
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
Project description:Mammalian chromosomes are folded into an intricate hierarchy of structural domains, within which topologically associating domains (TADs) and CTCF-associated loops partition the physical interactions between regulatory sequences. Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after crosslinking of chromatin. To measure chromosome structure in vivo, quantitatively and without relying on crosslinking and ligation, we have implemented a new method named damC. DamC combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and a biophysical model of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of TADs and CTCF loops, confirms 3C-based measurements of the scaling of contact probabilities within TADs, and provides evidence that mammalian chromatin in vivo is essentially rigid below 5 kilobases. Combining damC with transposon-mediated genomic engineering shows that new loops can be formed between ectopically introduced and endogenous CTCF sites, which alters the partitioning of physical interactions within TADs. This establishes damC as a crosslinking- and ligation-free framework to measure and modify chromosome interactions combined with a solid theoretical background for rigorous data interpretation. This orthogonal approach to 3C validates the existence of key structural features of mammalian chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.
2019-05-16 | GSE128014 | GEO