title: Pairing beyond the Seed Supports MicroRNA Targeting Specificity.
abstract: 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.
authors.affiliation: Division of Biology, University of California, San Diego, La Jolla, CA 92093-0349, USA.
authors.forename: James P
authors.initials: JP
authors.lastname: Broughton
authors.affiliation: Department of Cellular and Molecular Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Lovci
authors.affiliation: Division of Biology, University of California, San Diego, La Jolla, CA 92093-0349, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Huang
authors.affiliation: Department of Cellular and Molecular Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.
authors.forename: Gene W
authors.initials: GW
authors.lastname: Yeo
authors.affiliation: Division of Biology, University of California, San Diego, La Jolla, CA 92093-0349, USA. Electronic address: apasquinelli@ucsd.edu.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Pasquinelli
mesh_terms: D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005786:Gene Expression Regulation; D047468:Immunoprecipitation / Q000379:methods; D007438:Introns; D035683:MicroRNAs / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D018510:RNA, Helminth / Q000235:genetics* / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C558782:ALG-1 protein, C elegans; D029742:Caenorhabditis elegans Proteins; D035683:MicroRNAs; D018510:RNA, Helminth; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D020537:RNA, Small Nucleolar; D016601:RNA-Binding Proteins
keywords: ALG-1; C. elegans; chimeras; iCLIP; miRNA family; microRNA