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

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: GSE311748 | GEO | 2026/01/12

REPOSITORIES: GEO

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

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 | GSE311747 | 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 [RNA-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 | GSE245210 | GEO

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

2023-10-25 | GSE245209 | GEO

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Project description:Recent methodological advances allowed the identification of an increasing number of RNAbinding proteins (RBPs) and their RNA-binding sites. RNA interactome capture is, however, limited to proteins interacting with polyadenylated RNAs while RBPs associating with nonadenylate RNA classes cannot be purified. Moreover, the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria) are not amenable to RNA interactome capture studies. To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to poly-A RNA, but also proteins associating with non-adenylate RNA species (rRNA, tRNA) as short as 30nt. PTex can be used to simplify complex work ows such as PAR-CLIP and reliably recovers RBPs from tissues and bacteria thus significantly expanding the experimental toolbox to species that could previously not be assessed experimentally.

2019-03-12 | PXD009571 | Pride

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

In vivo CRISPR screening unveils RNA binding protein dependencies for leukemic stem cells and identifies ELAVL1 as a potential therapeutic target [eCLIPseq]

Project description:RNA binding protein (RBP)-directed post-transcriptional control of stem cell fate represents an underexplored mechanism driving hematopoietic stemness and transformation. We show that a unique set of RBPs are specifically enriched in leukemic stem cells (LSCs) of human primary acute myeloid leukemia (AML) but repressed in normal hematopoietic stem cells. Using an in vivo CRISPR-Cas9-mediated screening approach, we identify 33 key RBPs specifically essential for LSC-mediated MLL-AF9/NrasG12D AML. Knock-down/genetic ablation of the hit RBP Elavl1 in genetically distinct leukemias significantly reduced LSC numbers, and hampered leukemic engraftment. In human AML we show impairment of LSC-driven in vivo leukemic reconstitution and selective depletion of AML progenitors upon ELAVL1 targeting, highlighting the potential clinical importance of our findings. Profiling of the leukemic ELAVL1-mRNA interactome revealed hematopoietic differentiation, RNA splicing and mitochondrial metabolism as major pathways impacted by ELAVL1. Altogether, this work demonstrates that Elavl1 and other RBPs are critical regulators of LSC-survival and self-renewal.

2023-01-18 | GSE127944 | GEO

In vivo CRISPR screening unveils RNA binding protein dependencies for leukemic stem cells and identifies ELAVL1 as a potential therapeutic target [RNA-seq]

2023-01-18 | GSE127743 | GEO

Implementation of PAR-CLIP to characterize RNA-protein interactions in prokaryotes at nucleotide resolution

Project description:The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as Crosslinking and Immunoprecipitation (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP) in vivo. Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA and for the isolation of RNA crosslinked to RNA-BPs of interest. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.

2022-10-18 | MSV000090547 | MassIVE

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

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