Temporal iCLIP captures co-transcriptional RNA-protein interactions
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ABSTRACT:
PROVIDER: PRJNA837938 | ENA |
REPOSITORIES: ENA
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Project description:Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5’end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3’end processing performed by RBM7. Altogether, our data provide an unprecedented temporal view of RNA-protein interactions during the early phases of transcription.
2023-01-13 | GSE202980 | GEO
Project description:Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5'end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3'end processing performed by RBM7. Altogether, our data provide a temporal view of RNA-protein interactions during the early phases of transcription.
| S-EPMC9908952 | biostudies-literature
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.
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Project description:This experiment identifies hnRNP A1 binding sites transcriptome-wide in Hela cells. HeLa cells with inducible expression of T7-tagged hnRNP A1 were grown to approximately 90% confluence and then subject to iCLIP analysis (following the protocol from Huppertz et al. 2014 (iCLIP: protein-RNA interactions at nucleotide resolution)). The iCLIP library was sequenced using Illumina's HighSeq 1500
2016-06-23 | E-MTAB-3612 | biostudies-arrayexpress
Project description:This experiments was performed in HeLa cells according to the iCLIP protocol with the following modifications: no antiRNase was used and the concentration of RNase I was 0.5 U/ml. In iCLIP4, the dephosphorylation step was omitted from the standard protocol. The rest of the protocol was identical to the previously published iCLIP protocol )Huppertz I, Attig J, D'Ambrogio A, Easton LE, Sibley CR, Sugimoto Y, Tajnik M, Knig J, Ule J: iCLIP: protein-RNA interactions at nucleotide resolution. Methods 2014, 65:274-287).
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Project description:We used iCLIP to investigate the RNA interactions of the RNA binding protein SAFB1
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Project description:The studies of spliceosomal interactions are challenging due to their dynamic nature. Here we developed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs) to simultaneously map the spliceosomal binding to human snRNAs and pre-mRNAs. This identified 9 distinct regions on pre-mRNAs, which overlap with position-dependent binding patterns of 15 RBPs. Using spliceosome iCLIP, we additionally identified >50,000 branchpoints (BPs) that have canonical features, unlike those identified by RNA-seq. The iCLIP BPs generally overlap with the computationally predicted BPs, and alternative BPs are associated with extended regions of structurally accessible RNA. We find that the position and strength of BPs defines the binding patterns of SF3 and U2AF complexes, whereas the RNA structure around BPs affects the sensitivity of exons to perturbation of these complexes. Our findings introduce spliceosome iCLIP as a new method for transcriptomic studies of BPs and splicing mechanisms.
2019-08-30 | E-MTAB-6950 | biostudies-arrayexpress
Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei. 3 (2) biological replicates of U1C (U1-70K) -specific co-immunoprecipitated RNA after UV-crosslinking
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Project description:Nine patients with temporal lobe epilepsy (TLE) underwent hippocampal resection. Six had hippocampal sclerosis, three did not show signs of hippocampal sclerosis. Resected hippocampi were used for Ago iCLIP to detect miRNA targeting in the resected tissue.
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Project description:In this study, we employed a combination of RIP-seq and short- and long-wave iCLIP technologies to identify transcripts associated with cytoplasmic RNPs containing the RNA-binding protein Drosophila Imp. We also made a Imp knockdown vs luciferase control experiment. Two biological iCLIP-seq, as well as two mRNAseq made on cells harvested on the same day. Two biological PAR-iCLIP-seq. Two biological RIP-seq, as well as two mRNAseq made on cells harvested on the same day. Three biological mRNAseq of imp dsRNA treated cells as well as three control mRNAseq of cells treated with Luciferase dsRNA.
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