Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

RBProximity-CLIP enables subcellular mapping of RNA-binding protein interactions at nucleotide resolution [CLIP]

ABSTRACT: RNA-binding proteins (RBPs) orchestrate post-transcriptional gene regulatory (PTGR) processes through specific interactions with RNA molecules, which are tightly regulated in both space and time, including through the subcellular compartmentalization of RBPs and their target RNAs. To characterize these interactions in specific subcellular compartments, we developed RBProximity-CLIP, an approach that integrates targeted APEX2-based proximity labeling with 4-thiouridine–enhanced RNA–protein crosslinking to enable the isolation and profiling of target sites of individual RBPs in a compartment-specific manner . Using this approach, we profiled three RBPs—AGO2, YBX1, and ELAVL1—within the cytoplasmic, nuclear, and nucleolar compartments. RBProximity-CLIP enabled robust isolation of compartment specific RBP-RNA interactome and new insights of compartmental specific gene regulation of distinct RBPs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE311747 | GEO | 2026/01/12

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

RBProximity-CLIP enables subcellular mapping of RNA-binding protein interactions at nucleotide resolution [RNA-seq]

Project description:RNA-binding proteins (RBPs) orchestrate post-transcriptional gene regulatory (PTGR) processes through specific interactions with RNA molecules, which are tightly regulated in both space and time, including through the subcellular compartmentalization of RBPs and their target RNAs. To characterize these interactions in specific subcellular compartments, we developed RBProximity-CLIP, an approach that integrates targeted APEX2-based proximity labeling with 4-thiouridine–enhanced RNA–protein crosslinking to enable the isolation and profiling of target sites of individual RBPs in a compartment-specific manner . Using this approach, we profiled three RBPs—AGO2, YBX1, and ELAVL1—within the cytoplasmic, nuclear, and nucleolar compartments. RBProximity-CLIP enabled robust isolation of compartment specific RBP-RNA interactome and new insights of compartmental specific gene regulation of distinct RBPs.

2026-01-12 | GSE311748 | GEO

Proximity-CLIP provides a snapshot of occupied cis-acting elements on RNA in different subcellular compartments on a transcriptome-wide scale

Project description:Many cellular RNAs localize to specific subcellular compartments. Currently, methods to systematically study subcellular RNA localization are limited and lagging behind proteomic approaches. Here, we combined APEX2-mediated proximity biotinylation of proteins with PAR-CLIP to simultaneously profile the proteome and the transcriptome bound by RNA binding proteins in any given subcellular compartment. Our approach is fractionation-independent and does not rely on additional RNA manipulation and labeling steps, thus making it easy to apply. Furthermore, it enables to study the locali-zation of RNA processing intermediates, as well as the identification of regulatory RNA cis-acting elements occupied in different cellular compartments. In a proof-of-concept study we studied RNA and protein localization in the nucleus, cytoplasm and at cell-cell interfaces using Proximity-CLIP. These experiments revealed among other in-sights frequent transcriptional readthrough continuing for several kilobases down-stream of the canonical cleavage and polyadenylation site, a differential binding pat-tern of nuclear and cytoplasmic mRNAs, as well as the localization of mRNAs contain-ing 3’UTR CUG sequence elements at cell-cell interfaces, of which many encode regulatory proteins.

2018-11-26 | GSE110380 | GEO

Subcellular refinement of RNA binding protein target interactions with colocalization CLIP [CLIP-seq]

Project description:RNA binding proteins (RBPs) are essential for RNA metabolism and have profound impacts in both health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization, and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

2023-10-25 | GSE245209 | GEO

Subcellular refinement of RNA binding protein target interactions with colocalization CLIP [RNA-seq]

2023-10-25 | GSE245210 | GEO

A fast PAR-CLIP variant using phenolic extractions

Project description:We performed PAR-CLIP in three variants using ELAVL1 (HuR) as example.

2019-02-02 | GSE113628 | GEO

Global Target mRNA Specification and Regulation by RNA-Binding Protein ZFP36/Tristetraprolin/TTP [PAR-CLIP]

Project description:Tristetraprolin/ZFP36/TTP and ELAVL1/HuR are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embyonic kidney cells and examined combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. Among the targets ZFP36 binds and negatively regulates the mRNA of genes encoding proteins necessary for immune function and cancer, and other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance from ZFP36 overexpression independent of effects from confounding features, such as 3M-bM-^@M-^Y UTR length. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes. ZFP36 preferentially interacts with and regulates AU-rich sequences while ELAVL1 prefers predominantly U- and CU-rich sequences. RNA target specificity identified by global in vivo ZFP36-mRNA interactions were quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 both bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to untangle the in vivo combinatorial regulation by RNA-binding proteins. FLAG-HA ZFP36

2013-12-24 | E-GEOD-53184 | biostudies-arrayexpress

Unstressed HeLa cells and ELAVL1/HuR knock down conditions: polyA RNA-Seq, small RNA-Seq, and PAR-CLIP

Project description:Post-transcriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a method based on RNA-protein crosslinking, to identify transcriptome wide ~26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in splicing. Upon HuR knock down, mRNA levels and protein synthesis of thousands of target genes was down regulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knock down triggered strong and specific up regulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing. PolyA mRNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression and splicing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq. PARCLIP was performed as in Hafner et. Al 2010 but with an antibody against endogenous HuR (3A2, Santa Cruz, sc-5261) in unstressed HeLa cells. We used, independently, 4-thiouridine (4SU) and 6-thioguanosine (6SG) to assess a possible nucleotide bias. As our proteomics measurements required labeling of cells in a special medium we also performed PAR-CLIP on cells grown in SILAC medium. Altogether we performed three PAR-CLIP experiments: 4SU labeling in standard DMEM medium, 4SU labeling in SILAC medium (as a replicate) and 6SG labeling in SILAC medium. Small RNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression. Sequencing was performed on Illumina GAII using the standard sRNA 36cycle protocol.

2011-06-29 | E-GEOD-29943 | biostudies-arrayexpress

A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins (CLIP)

Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions. We performed duplicate experiments for each variant of the CLIP protocol (CLIP, PAR-CLIP), each protein (HuR, Ago2), and enzymatic digestion (complete T1 digestion, mild MNase digestion). In addition, we performed a single PAR-CLIP experiment with mild T1 digestion.

2011-05-15 | E-GEOD-28859 | biostudies-arrayexpress