| 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. |