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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