Pairing beyond the Seed Supports MicroRNA Targeting Specificity

• Published in: Molecular cell 2016-10, Vol.64 (2), p.320-333
• Main Authors: Broughton, James P., Lovci, Michael T., Huang, Jessica L., Yeo, Gene W., Pasquinelli, Amy E.
• Format: Article
• Language: English
• Subjects: ALG-1; Animals; Base Pairing; Base Sequence; Binding Sites; C. elegans; Caenorhabditis elegans - genetics; Caenorhabditis elegans - metabolism; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; chimeras; Exons; Gene Expression Regulation; iCLIP; Immunoprecipitation - methods; Introns; microRNA; MicroRNAs - classification; MicroRNAs - genetics; MicroRNAs - metabolism; miRNA family; Protein Binding; RNA, Helminth - genetics; RNA, Helminth - metabolism; RNA, Long Noncoding - genetics; RNA, Long Noncoding - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Nucleolar - genetics; RNA, Small Nucleolar - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2016.09.004

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 emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3′ sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3′ pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5′ as well as the 3′ regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo. [Display omitted] •AGO iCLIP miRNA-target chimeras reveal miRNA targeting landscape in C. elegans•miRNA families target non-overlapping sets of target sites•miRNA 3′ end interactions contribute to target site specificity in vivo•Chimera PCR (ChimP) identifies specific miRNA-target sites of interest Argonaute iCLIP produces miRNA-target chimeras that identify miRNA targets. Broughton et al. analyze these chimeras, reporting that 3′ regions of miRNAs confer specificity to miRNA-target interactions in vivo. They develop a simple, versatile, and economic method called chimera PCR (ChimP) for testing miRNA-target interactions.