Abstract
The pervasive transcription of the genome creates many types of non-coding RNAs (ncRNAs). However, we know very little regarding the functions and the regulatory mechanisms of these ncRNAs. Exploring the interactions of RNA and RNA binding proteins (RBPs) is vital because it can allow us to truly understand how these ncRNAs behave in vivo. High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP or CLIP-seq) and its variants have been successfully used as systemic techniques to study RBP binding sites. In this review, we will explain the major differences between the CLIP techniques, summarize successful applications of these techniques, discuss limitations of CLIP, present some suggested solutions and project their promising future roles in studying the RNA world.
Highlights
The pervasive transcription of the genome creates many types of non-coding RNAs
High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP or CLIP-seq) and its variants have been successfully used as systemic techniques to study RNA binding proteins (RBPs) binding sites
A method known as cross-linking immunoprecipitation (CLIP), and modified forms of this method, such as photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP), individual-nucleotide resolution cross-linking and immunoprecipitation, cross-linking and analysis of cDNAs (CRAC) and cross-linking, ligation, and sequencing of hybrids (CLASH), have been successfully used
Summary
Many biochemical technologies are invented to study the interactions between RNAs and RBPs. To overcome the intrinsic disadvantages of RIP, Darnell and co-workers [6,7] developed a complex protocol based on RIP, named UV cross-linking and immunoprecipitation (CLIP). They combined emerging high-throughput sequencing with CLIP and named the technique HITS-CLIP [8,9]. The fragmentation of RNAs makes it convenient to perform sequencing and motif analysis, in addition to avoiding pulling down undesirable protein-RNAprotein complexes Rigorous purification, such as multiple washings between each reaction, SDS-PAGE and transfer to nitrocellulose membrane were added.
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