Subcellular refinement of RNA binding protein target interactions with colocalization CLIP [CLIP-seq]
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ABSTRACT:
PROVIDER: PRJNA1027370 | ENA |
REPOSITORIES: ENA
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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
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
Project description:Subcellular refinement of RNA binding protein target interactions with colocalization CLIP [RNA-seq]
| PRJNA1027368 | ENA
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
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
Project description:Integrase CLIP-seq experiments were conducted on wild-type and eccentric HIV-1 virions generated in the presence of allosteric integrase inhibitors and IN K264/266A and R269/K273A mutations Integrase CLIP-seq experiments were conducted by immunoprecipitation of integrase-RNA complexes from fully formed mature and eccentric virus particles. Libraries of RNA molecules bound by integrase were generated and sequenced by Illumina Hi-Seq2000 and 2500 platforms.
2016-08-25 | E-GEOD-83588 | biostudies-arrayexpress
Project description:hnRNPA1 CLIP-seq experiments were conducted on HEK293T cells transiently expressing WT hnRNPA1 and hnRNPA1-DSB mutant
2023-12-31 | GSE238202 | GEO
Project description:Integrase CLIP-seq experiments were conducted on wild-type and eccentric HIV-1 virions generated in the presence of allosteric integrase inhibitors and IN K264/266A and R269/K273A mutations
2016-08-25 | GSE83588 | GEO
Project description:UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are the most frequently used methods to study protein-RNA interactions in the intact cells and tissues, but their relative advantages or inherent biases have not been evaluated. To benchmark CLIP and iCLIP method, we performed iCLIP with Nova protein, which is the most extensively studied protein by CLIP. Further, we assessed UV-C-induced cross-linking preferences, by exploiting the UV-independent formation of covalent RNA cross-links of the mutant RNA methylase NSUN2.
2012-07-20 | E-MTAB-1008 | biostudies-arrayexpress