IrCLIP-RNP reveals patterns of dynamic protein associations on RNA [RNA-seq]
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ABSTRACT:
PROVIDER: PRJNA1130389 | ENA |
REPOSITORIES: ENA
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Project description:RNA binding proteins (RBPs) interact with RNA targets to control an array of processes, including RNA splicing, stability, transport, and translation1-3. Dysfunctional RNA-RBP interactions contribute to pathogenesis of a plethora of human diseases1,4,5, underscoring the need for a greater understanding of the nature and dynamics of RNA-protein assemblies. The capacity to study native RNA-dependent protein assemblies in living cells, however, has been limited. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation6 was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with specific RBPs of interest. irCLIP-RNP defined landscapes of complex and multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP associations on RNA. This included cell-type-selective patterned relationships between RDAPs and primary RBPs, such as cell context-dependent reciprocal impacts of HNRNPU and NONO on each other’s RDAP landscapes. irCLIP-RNP also defined dynamic RDAP remodeling patterns in response to epidermal growth factor (EGF) and uncovered EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of mRNAs that mediate cell proliferation. The development of sequential immunoprecipitation irCLIP (RE-irCLIP) supported the same-RNA-molecule co-localization of irCLIP-RNP-identified associations. Thus, irCLIP-RNP and RE-irCLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
2025-01-28 | PXD053494 | Pride
Project description:RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
2025-01-28 | GSE271168 | GEO
Project description:RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
2025-01-28 | GSE271167 | GEO
Project description:irCLIP-RNP reveals patterns of dynamic protein associations on RNA [CLIP-seq]
| PRJNA1130390 | ENA
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Project description:We performed irCLIP-seq to determine the mRNA targets of YTHDF2 in mouse hematopoietic progenitor cell line HPC-7
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Project description:we performed infrared crosslinking immunoprecipitation followed by sequencing (irCLIP-seq) (Zarnegar et al., 2016) for Rbm15 to directly map its binding sites on RNA. As a principle of concept, the cells were engineered to simultaneously express emGFP-Rbm15 and Xist RNA together, as Rbm15 strongly interacts with Xist A-repeat to deposit the m6A methylation downstream. Cross-linking induced truncation site (CITS or RT stops) is the main signature occurring at our irCLIP-seq datasets. RNA meta-profile plot against normalized transcripts shows that these CITSs reside across the transcript, with 2 main peaks in transcript starts and near the stop-codon regions, in agreement with the RBM15/15B binding profile in human cells (Patil et al., 2016).Motif analysis against CITSs revealed that Rbm15 binding sites prefer U-rich stretches, namely 3 or 4 consecutive Us. This is also true for crosslinking induced mutations (CIMS).
2020-09-15 | GSE154708 | GEO
Project description:To fine-map the position of lnc-CCTT that directly interact with CENP-C, we performed irCLIP-seq (infrared crosslingking and immunoprecipitation followed by high throughput RNA sequencing), which utilize ultraviolet (UV) light to induce zero-length covalent bonds between RNA and the directly attached protein and an infrared-dye-conjugated and biotinylated ligation adaptor to isolate RNA fragments. irCLIP-seq identified a possible CENP-C binding region of lnc-CCTT ranging from nucleotides 127-177nt.
2022-04-20 | GSE149530 | GEO