MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome

Nucleic Acids Res. 2021 Jan 11;49(1):25-37. doi: 10.1093/nar/gkaa1117.


Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Amino Acid Motifs
  • Argonaute Proteins / chemistry
  • Argonaute Proteins / metabolism*
  • Base Sequence
  • Binding Sites
  • Biotin
  • Cross-Linking Reagents / pharmacology
  • DNA, Complementary / genetics
  • GTP-Binding Proteins / genetics
  • Gene Expression Regulation*
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • MicroRNAs / antagonists & inhibitors
  • MicroRNAs / genetics*
  • Models, Genetic*
  • Nuclear Proteins / genetics
  • Nucleic Acid Conformation
  • Photochemistry
  • Sequence Analysis, DNA
  • Streptavidin
  • Trioxsalen / radiation effects


  • 3' Untranslated Regions
  • AGO2 protein, human
  • Argonaute Proteins
  • Cross-Linking Reagents
  • DNA, Complementary
  • GNL3L protein, human
  • MIRN124 microRNA, human
  • MIRN1243 microRNA, human
  • MIRN132 microRNA, human
  • MicroRNAs
  • Nuclear Proteins
  • Biotin
  • Streptavidin
  • GTP-Binding Proteins
  • Trioxsalen