The advantage of CLIP lies in allowing identification of interactions within cells (where the crosslinking occurs) versus interactions that might occur after cells are lysed. In summary, studying RNA-protein interactions is necessary to achieve a systematic understanding of transcription, translation and other biological processes.ĬLIP (cross-linking immunoprecipitation) is a molecular biology technology that employs ultraviolet (UV) cross-linking and immunoprecipitation in order to identify RBP–RNA interactions ( 17, 18). In addition, lack of Fragile X mental retardation protein (FMRP) results in a deficiency in human cognition and premature ovarian insufficiency ( 14) and the FUS, EWSR1, and TAF15 (FET) protein family is responsible for RNA editing and plays important roles in many diseases ( 15, 16). For instance, the RBP Musashi1 was found to be related to many cancer types, including those of the breast, colon, medulloblastoma and glioblastoma, as well as to neurogenesis and neurodegenerative diseases ( 13). RBPs have been implicated in various types of human diseases ( 1, 10–13). RBPs may influence the global coordination of gene expression by organizing nascent groups of RNAs into downstream chains of the post-transcriptional modification process, through what is known as the ‘RNA-operon’ theory ( 9). Binding of RBPs with RNA targets can also be regulated through competition with other RBPs and non-coding RNAs ( 7, 8). For example, the AUF1 protein recognizes RNAs through a signature motif composed of 29–39 nt with high A and U contents and a secondary structure specific to the RNAs ( 6). RBPs bind target RNAs by recognizing their sequences or/and RNA secondary structures through RNA-binding motifs. Cytoplasmic RBPs, on the other hand, include Unr, which has been shown to be required for internally initiating the translation of human rhinovirus RNA ( 5). One example of an intra-nuclear RBP is Yra1p, which has been found to be involved in mRNA export ( 4). For example, the TIAR protein has been shown to be transported from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death ( 3). Located in either the nucleus or cytoplasm, or both, they engage in every step of the post-transcriptional modification process, including alternative splicing, regulation of mRNA levels, transport between cellular compartments, alternative polyadenylation, transcript stability, etc. RNA-binding proteins (RBPs) are proteins that bind to double- or single-stranded RNAs in cells and form ribonucleoprotein complexes with the bound RNAs. The diversity of RNA in sequence and structure underpins much of cell heterogeneity and complexity. To assist readers in choosing optimal procedures for their analysis, we also review experimental design and procedures that affect bioinformatics analyses. Specifically, we compare the strengths and weaknesses, as well as the scopes, of various bioinformatics tools. In this review, we discuss the genome-wide CLIP technology and focus on bioinformatics analysis. Meanwhile, numerous bioinformatics pipelines for handling the genome-wide CLIP data have also been developed. This technology has evolved through the development of three distinct versions: HITS-CLIP, PAR-CLIP and iCLIP. The advent of cross-linking immunoprecipitation coupled with high-throughput sequencing (genome-wide CLIP) technology has recently enabled the investigation of genome-wide RBP–RNA binding at single base-pair resolution.

Systematic identification of RBP functional targets is among the key biomedical research questions and provides a new direction for drug discovery. Defects in the functions of these RBPs underlie a broad spectrum of human pathologies. RNAs are regulated by RNA-binding proteins (RBPs) at all post-transcriptional stages, including splicing, transportation, stabilization and translation. The past decades have witnessed a surge of discoveries revealing RNA regulation as a central player in cellular processes.

Iclip technique