miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity

Nat Commun. 2015 Nov 25;6:8864. doi: 10.1038/ncomms9864.


microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA-target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3'-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3'-end pairing is a general determinant of AGO binding specificity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Argonaute Proteins / metabolism*
  • Base Pairing
  • Binding Sites
  • Blotting, Northern
  • Blotting, Western
  • Brain / metabolism
  • Cell Line, Tumor
  • Chimera / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Flow Cytometry
  • Humans
  • Immunoprecipitation
  • Mice
  • MicroRNAs / metabolism*
  • RNA Interference*
  • RNA, Messenger / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction


  • Argonaute Proteins
  • MicroRNAs
  • RNA, Messenger