Īlthough several tools have been recently developed, there is still a lack of a comprehensive publicly available pipeline for analyzing CLIP-seq data. Instead, cDNA is circularized and then linearized at specific restriction sites, so that the truncation positions are used to locate candidate RBP binding positions.
In addition, individual-nucleotide resolution CLIP (iCLIP) was developed to identify cross-linking sites independently of experimentally induced mutations. These cross-linking-induced mutations in HITS-CLIP and PAR-CLIP can be used as markers to identify the precise RBP binding sites. PAR-CLIP introduces a distinct spectrum of substitutions (T-to-C for 4SU and G-to-A for 6SG). For example, HITS-CLIP utilizes UV cross-linking of proteins with RNA, which introduces either insertions, deletions, or substitutions, depending on the RBPs. This cross-linking process usually introduces mutations in sequence tags at RBP binding sites. PAR-CLIP introduces photoactivatable ribonucleoside analogs, such as 4-thiouridine (4SU) and 6-thioguanosine (6SG), into the RNA of cultured cells to enhance cross-linking efficiency. To increase detection sensitivity, photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) was also developed. For example, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) was used to identify approximately 30 to 60 nucleotide regions around the peaks of CLIP read clusters that represent binding sites of RNA-binding proteins (RBPs). Recent technological developments, especially the technique of crosslinking immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), have provided powerful tools for studying the roles of RNA regulation in the control of gene expression and the generation of phenotypic complexity.
RNA’s diversity in sequence and structure endows it with crucial roles in cell biology.
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