ICLIP and iCLAP for TIA1 and TIAL1 proteins
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Project description:Individual nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) and individual nucleotide resolution UV-crosslinking and affinity purification (iCLAP) were used to identify the global RNA binding sites for TIA1 and TIAL1 proteins. HeLa cells were UV crosslinked before lysing and digested with DNase and low concentration of RNase I. The protein-RNA complex were either immunoprecipitated with specific antibodies against either TIA1 or TIAL1 proteins and ligated to 3 prime adapter before separated by SDS-PAGE. For iCLAP, the His/Strep tagged proteins were overexpressed in HeLa cells, and the protein-RNA complexes were purified via Strep and His tag affinity purification. The proteins were digested by proteinase K and the RNA was reverse transcribed and self-circularised. The cDNA library was prepared by PCR with solexa primers compatible for high-throughput sequencing. This method allowed specific identify of crosslinked nucleotides, and genome-wide targets for TIA1 and TIAL1 not identified before. This method also allowed comparison between the 2 homologous proteins, whose binding sites and functions could be redundent. iCLAP provided an independent way to validate the binding sites identified by iCLIP, since no antibody was used in the iCLAP method.
2011-07-28 | E-MTAB-432 | biostudies-arrayexpress
Project description:HeLa cells were cultured in DMEM, supplemented with 10% (v/v) FCS and penicillin/streptomycin under 5% CO2 at 37C. For iCLIP, HeLa cells expressing GFP fusion proteins were induced with doxycycline to adjust the level of recombinant protein to the level of the endogenous counterpart and irradiated with 150 mJ/cm2 UV light (254 nm). The iCLIP cDNA libraries were sequenced with 50 bp on an Illumina HiSeq 2000 instrument. RNASeq was performed as a control with 50 bp paired-end on an Illumina HiSeq 2000 instrument.
2016-07-28 | E-MTAB-4215 | biostudies-arrayexpress
Project description:Germinal centres (GC) are essential for the establishment of long-lasting antibody responses. In there, GC B cells rely on post-transcriptional RNA mechanisms for translating activation-associated transcriptional programs into functional changes in the cell proteome. However, we still lack knowledge about which are the critical proteins driving these key mechanisms. Here we show that the RNA binding proteins TIA1 and TIAL1 are required for the generation of long-lasting GC responses. TIA1 and TIAL1- deficient GC B cells fail to undergo antigen-mediated positive selection, expansion and differentiation into B cell clones producing high-affinity antibodies. Mechanistically, TIA1 and TIAL1 control the transcriptional identity of dark and light zone GC B cells and enable timely expression of the pro-survival molecule MCL1. Altogether, we demonstrate here that TIA1 and TIAL1 are key players in the post-transcriptional program that selects high-affinity antigen-specific GC B cells.
2023-08-29 | GSE235655 | GEO
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.
2014-05-16 | GSE43848 | GEO
Project description:We used GFP-tagged SR proteins expressed at endogenous levels and iCLIP to compare the extent and pattern of SR protein binding to expression-matched lincRNAs and protein-coding RNAs
2018-10-31 | GSE113813 | GEO
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
2014-05-16 | E-GEOD-43848 | biostudies-arrayexpress
Project description:We used GFP-tagged SR proteins expressed at endogenous levels and iCLIP to identify and compare endogenous RNA targets of individual SR proteins, map the preferential sites of binding, compare binding pattern and binding motifs between family members and to NXF1 and quantify binding of SR proteins and NXF1 to spliced versus unspliced RNAs to study the role of SR proteins in mRNA export via NXF1.
2016-03-07 | GSE69689 | GEO