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).
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.
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.
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.
Project description:RBM15 interactome has been determined in mouse Embryonic Stem cells. The cells express inducible emGFP-PreScission-RBM15 alongside with inducible Xist RNA.
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.
Project description:Immune checkpoint blockade (ICB) offers therapeutic options for patients with advanced colorectal cancer; however, only a subset of patients with microsatellite instability-high (MSI-H) tumors respond to treatment. Therefore, strategies to enhance immunotherapy sensitivity are urgently needed. In this study, we demonstrate that RBM15 is highly expressed in colorectal cancer and is associated with poor prognosis. Loss of RBM15 increases the expression of fumarate hydratase (FH), leading to a reduction in its substrate fumarate, which acts as a suppressor of anti-tumor immunity. Notably, RBM15 depletion delays tumor growth by promoting CD8+ T cell infiltration. Our findings identify RBM15 as a key suppressor of anti-tumor immunity, suggesting that targeting RBM15 could be a promising therapeutic strategy in colorectal cancer.