Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding

TLDR

MicroRNAs regulate gene expression, yet identifying their targets experimentally remains challenging, and noncanonical targeting of mRNAs is common and functional. The study aims to provide an unbiased view of human miRNA targets. The authors developed a technique for ligation and sequencing of miRNA‑target RNA duplexes associated with human AGO1. They identified over 18,000 high‑confidence miRNA‑mRNA interactions, finding that ~60 % of seed matches are noncanonical with bulges or mismatches, most interactions include additional nonseed pairing, 18 % involve the miRNA 3′ end, and distinct overrepresented motifs suggest miRNA‑specific targeting patterns that may influence RISC activity.

Abstract

Highlights•A experimental approach identifies in vivo targets for miRNAs•miRNAs show frequent noncanonical targeting of mRNAs•Nonseed interactions are common and functional•miRNA base pairing shows distinct patterns and overrepresented motifsSummaryMicroRNAs (miRNAs) play key roles in gene regulation, but reliable bioinformatic or experimental identification of their targets remains difficult. To provide an unbiased view of human miRNA targets, we developed a technique for ligation and sequencing of miRNA-target RNA duplexes associated with human AGO1. Here, we report data sets of more than 18,000 high-confidence miRNA-mRNA interactions. The binding of most miRNAs includes the 5′ seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, nonseed base pairing. 18% of miRNA-mRNA interactions involve the miRNA 3′ end, with little evidence for 5′ contacts, and some of these were functionally validated. Analyses of miRNA:mRNA base pairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding.Graphical abstract