Project description:We performed iCLIP of CPSF-160- GFP as CPSF160 was believed to be the main RNA-binding subunit of CPSF. To this end, we used a stable HeLa cell line that expresses GFP-CPSF160 in a doxycycline (Dox)-dependent manner. After GFP-CPSF160 was induced, we irradiated the cells with UV-C (wavelength=254nm) to induce protein-RNA crosslinks and immunoprecipitated the complex with a GFP antibody.
Project description:hiCLIP protocol was followed to isolate RNA bound to 3XFLAG-STAU1 protein and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction, and are specified below for each sequencing run. Random barcodes were also incoperated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina paired-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3
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.
Project description:Monosome and disome profiling was performed on Flag-STAU1 Flp-In 293 T-REx to study the causes of ribosomal collisions, and whether this may be modulated by the presence/absence of Staufen-1. Cells were treated with either an siRNA targeting STAU1 transcript (4x samples) or a control siRNA (2x samples). Two of the four samples treated with the STAU1 siRNA had siRNA-resistant STAU1 mRNA expression induced by doxycycline (rescue). Sequencing libraries from monosome and disome fractions were generated in parallel from the same samples. Note that unique molecular identifiers/random barcodes (UMIs/RBCs) were included in the sequencing experiment. Each UMI has been moved to the fastq read name of each read. For example \\"xxxxxxrbc:AGCCAAT\\" in the read name signifies that the given read had a UMI of \\"AGCCAAT\\". Using these UMIs, PCR duplicates can be removed with UMI-Tools following read alignment.
Project description:RNA abundance decreases linearly from the 5′ to 3′ end of long introns to create “saw-tooth” patterns, and these can be used to infer locations of major splicing events. FUS binds across entire pre-mRNAs with limited sequence specificity, permitting examination of these saw-tooth patterns. We used FUS iCLIP from the human brain to identify deviations from the expected linear decrease of reads across long introns that correspond to recursive splicing events. iCLIP protocol was followed to isolate RNA bound to FUS protein and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction, and are specified in the library construction protocol for each sequencing run. Random barcodes were also incorporated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina paired-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3
Project description:This experiment uses iCLIP to identify the binding pattern of the spliceosomal protein PRPF8 on RNA. The data shows that PRPF8 binds strongly and specifically in the region 12 to 14nt upstream of 5' splice sites (5ss). Due to PRPF8's role in the formation of the catalytically active spliceosome, this data can be used as a readout of 5ss selection. Here, we performed iCLIP on HeLa cells treated with control or EIF4A3 siRNA, with 4 replicate samples per condition and eIF4A3 protein levels reduced ~50% in knockdown. We investigated the role of the exon junction complex (EJC) in suppressing 5ss that are reconstituted at the junction of two canonical exons (RS-5ss) - selection of these splice sites would result in recursive splicing of canonical exons. We plotted the crosslink sites of reads that span an exon-exon junction, seperating reads that span RS-5ss from those that do not. We found that reads that span an RS-5ss are enriched at the 12-14nt window associated with 5ss selection, while reads that span other exon-exon junctions are not enriched. This effect is magnified greatly by knockdown of eIF4A3. The results indicate that RS-5ss can be used by the spliceosome, but that this process is usually repressed by the EJC. This data is evidence of recursive splicing of canonical exons and the role of the EJC in repressing recursive splicing.
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: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:We report the knockdown and rescue of RNPS1, a component of the ASAP and PSAP complexes and, hence, an auxiliary component of the exon junction complex. The studied conditions show an extensive change in alternative splicing and the cell's transcriptome. For the knockdown, HeLa cells were transfected with siRNAs targeting RNPS1 or the Luciferase control. Total RNA was extracted with peqGOLD TriFast, underwent ribosomal depletion and strand-specific library preparation (TruSeq R Stranded Total RNA LT). Sequencing occurred in an Illumina HiSeq4000 sequencer with 2×75bp, producing roughly 35 million read-pair per sample.