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Comprehensive Identification of miRNA Target Sites in Live Animals

  • Dimitrios G. ZisoulisEmail author
  • Gene W. Yeo
  • Amy E. Pasquinelli
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 732)

Abstract

MicroRNAs (miRNAs) are small RNA molecules that posttranscriptionally regulate the expression of protein-coding genes. The mature miRNAs are loaded into Argonaute-containing protein complexes (miRISC, miRNA Induced S  ilencing Complex), and guide these complexes to the 3′ UTR of targeted mRNA transcripts via base-pairing interactions. However, the imperfect complementarity that characterizes the interactions between animal miRNAs and target sites complicates the identification of direct target genes. We developed a biochemical method to identify on a large scale the target sequences recognized by miRISC in vivo. The mRNA sites bound by miRISC are stabilized by cross-linking and isolated by immunoprecipitation of Argonaute-containing complexes. The bound RNA molecules are trimmed to the regions protected by Argonaute, subjected to a series of isolation and linker ligation steps and identified by high-throughput sequencing methods.

Key words

MicroRNAs Argonaute Protein CLIP-seq Parallel sequencing miRNA target sequences Posttranscriptional gene regulation 

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Copyright information

© Humana Press 2011

Authors and Affiliations

  • Dimitrios G. Zisoulis
    • 1
    Email author
  • Gene W. Yeo
  • Amy E. Pasquinelli
  1. 1.Department of BiologyUniversity of CaliforniaSan DiegoUSA

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