HITS-CLIP

High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a variant of CLIP^[1] for genome-wide mapping protein–RNA binding sites or RNA modification sites in vivo.^[2][3][4] HITS-CLIP was originally used to generate genome-wide protein-RNA interaction maps for the neuron-specific RNA-binding protein and splicing factor NOVA1 and NOVA2;^[3] since then a number of other splicing factor maps have been generated, including those for PTB,^[5] RbFox2,^[6] SFRS1,^[7] hnRNP C,^[8] and even N6-Methyladenosine (m6A) mRNA modifications.^[4][9]

HITS-CLIP of the RNA-binding protein Argonaute has been performed for the identification of microRNA targets^[10] by decoding microRNA-mRNA and protein-RNA interaction maps in mouse brain,^[11][12] and subsequently in Caenorhabditis elegans,^[13] embryonic stem cells^[14] and tissue culture cells.^[15]

As a novel modification of HITS-CLIP, m6A-CLIP was developed to precisely map N6-Methyladenosine(m6A) locations in mRNA by UV-crosslinking m6A antibody to the target RNA.^[4][9] Recently, improved bioinformatics applied to Argonaute HITS-CLIP enables identification of binding sites with single nucleotide resolution.^[16]

Similar methods

• PAR-CLIP, for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) in tissue culture cells.
• iCLIP, for a thorough amplification of the cDNA library, including truncated cDNAs, thus also enabling an additional means to identify crosslink sites.