Elucidating the Dark Side of the MicroRNA

In animals, microRNAs (miRNAs) direct Argonaute (AGO) proteins to target RNAs through imperfect base pairing. To identify endogenous miRNA target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed region (nucleotides 2-8) emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3ʹ sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families that share identical 5ʹ sequences. By editing an endogenous target site, we demonstrate that 3ʹ pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions in the endogenous context. Finally, we present a simplified method, Chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis of miRNA-target chimeras revealed that sequences in the 5ʹ as well as the 3ʹ regions of a miRNA provide the information necessary for stable and specific miRNA target interactions in vivo.