![]() ![]() Next, the cells are lysed and the RNAs are partially digested to obtain RNA fragments in an optimal size range ( Fig. The iCLIP protocol starts with UV irradiation, which forms covalent bonds at sites of protein–RNA interactions and thereby preserves the in vivo binding pattern ( Fig. In the last step, high-throughput sequencing generates reads in which the barcode sequences are immediately followed by the last nucleotide of the cDNA. Linearization generates suitable templates for PCR amplification. The free RT primers are removed by size selection and circularization of the cDNA is carried out. The RT primer introduces two cleavable adapter regions and barcode sequences. The protein is then digested by proteinase K, and reverse transcription (RT) is performed truncating at the remaining polypeptide. The complexes are separated by SDS–PAGE and isolated from a nitrocellulose membrane according to the expected size. For the library preparation and visualization, the RNA is dephosphorylated, a 3′ end adapter is ligated and the 5′ end is radioactively labeled. This is followed by partial RNase digestion and an immunoprecipitation with protein-specific antibodies. Cells are irradiated with UV-C light on ice, leading to formation of a covalent bond between protein and RNA. Schematic representation of the iCLIP procedure identifying RNA–protein interactions in intact cells. ![]() Importantly, in addition to identifying the RBP binding sites of an RBP, iCLIP can also quantitate genome-wide changes in protein–RNA interactions for instance, it was demonstrated that the splicing factor U2AF65 gains access to hundreds of Alu elements after knockdown of hnRNP C, which prevents their erroneous recognition under normal conditions. It determined high-resolution RNA splicing maps of different RBPs, which enabled to assess how the position of RBP binding around alternative exons determines their splicing function. iCLIP has been successfully used to study the function of RBPs in alternative splicing, alternative polyadenylation, RNA methylation and mRNA stability. We previously developed individual-nucleotide resolution CLIP (iCLIP), which enables PCR amplification of truncated cDNAs, and thereby identifies protein–RNA crosslink sites with nucleotide resolution ( Fig. However, in over 80% of cases, the reverse transcriptase stalls at the short polypeptide left at the UV-induced crosslink site, resulting in truncated cDNAs that lack the 5′ adapter, and are therefore not amplified in CLIP. In the original CLIP approach, reverse transcription needs to proceed from a universal 3′ ligated adapter to a universal 5′ ligated adapter, since both adapters are required for PCR amplification. Combined with high-throughput sequencing, CLIP became the standard tool for the genome-wide analysis of protein–RNA interactions. ![]() Development of in vivo UV-crosslinking and immunoprecipitation (CLIP) enabled the study of protein–RNA interactions with high positional resolution and specificity. However, these approaches were prone to identifying non-physiologic or indirect interactions and their low resolution made it difficult to narrow down actual binding sites. The first approaches to investigate protein–RNA complexes in vivo employed affinity purification or immunoprecipitation combined with microarray analysis (RIP-CHIP). In particular, RBPs cooperate and compete when binding to RNA therefore it is crucial to study protein–RNA interactions in the cellular environment. Although many RBDs recognize RNA in a sequence-specific manner, sequence information is not sufficient to reliably predict RBP binding sites throughout the transcriptome. Their RNA binding is mediated by modular RNA-binding domains (RBDs), such as the RNA recognition motif (RRM), hnRNP K-homology domain or zinc fingers (Znf). RNA-binding proteins (RBPs) are the primary regulatory factors of the various post-translational stages, including alternative splicing, polyadenylation, mRNA localization, translation and degradation. Post-transcriptional regulation critically contributes to the ability of cells to adjust gene expression in the face of a changing external or internal environment. ![]()
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