Argonaute HITS-CLIP Decodes Microrna–mrna Interaction Maps

MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; however, as miRNA activity requires base pairing with only 6-8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein–RNA interaction sites. Here we use HITS-CLIP to covalently crosslink native argonaute (Ago, also called Eif2c) protein–RNA complexes in mouse brain. This produced two simultaneous data sets—Ago–miRNA and Ago–mRNA binding sites—that were combined with bioinformatic analysis to identify interaction sites between miRNA and target mRNA. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. Ago HITS-CLIP provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA–mRNA interactions. The importance of gene silencing induced by inhibitory microRNAs (miRNAs) in the regulation of gene expression is now readily apparent. But since miRNA regulation of a messenger RNA requires only a short (eight-nucleotide or fewer) match in their sequences, it has proved almost impossible to definitively determine which among many predicted mRNA binding sites is the in vivo target for each miRNA. Now a refinement of the HITS-CLIP method that focuses on miRNA and mRNA interaction with Argonaute proteins, ubiquitous endonucleases that are part of the RNA-induced silencing complex, has been used to decode a precise map linking miRNA binding sites to brain mRNA transcripts. The method is generally applicable and should provide a new approach to understanding the role of miRNAs in biology. In addition the maps allow target site determination for RNA interference therapy on clinically relevant mRNAs. MicroRNAs (miRNAs) are short RNAs that are ubiquitous, potent regulators of gene expression; however, as miRNA activity requires base pairing with only 6–8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. By using a method known as HITS-CLIP, combined with bioinformatic analysis, it has now been possible to demonstrate how the in vivo interactions between miRNAs and the mRNA targets can be validated.