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
Project description:A key function for RNA-binding proteins in orchestrating plant development and environmental responses is well established. However, the lack of a genome-wide view on their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) for genome-wide determining the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7. We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues.
Project description:iCLIP experiment to assess the binding of the highly abundant nuclear RNA-binding protein hnRNP C and core splicing factor U2AF65 on a genomic scale. To investigate how both proteins compete for binding at a subset of sites, U2AF65 iCLIP experiments were performed from both HNRNPC knockdown and control HeLa cells.
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.
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).
Project description:iCLIP of HeLa cells with anti-TFIP11 antibody to investigate RNA binding locations of TFIP11 as part of a larger study on the function of TFIP11.
Project description:Our ChIP resuls suggested that coilin association with U3, snRNA and histone genes might be dependent on coilin-RNA interaction. We used iCLIP of coilin-GFP expressed in HeLa and P19 cell lines at endogenous levels to identify coilin RNA targets and investigate RNA-binding specificity. P19 cells expressing GFP fused to a nuclear localization signal (GFP-NLS) was used as a negative control. iCLIP results revealed that coilin binds several classes of ncRNA including snRNAs, U3 snoRNA and scaRNAs. Interestlignly the majority of coilin targets were intronic snoRNAs, suggesting a novel role of CBs in snoRNA biogenesis.
Project description:TDP-43 is an important RNA binding protein. To better understand its binding targets in human neurons, we performed TDP-43 iCLIP on SHSY5Y cells.