Unambiguous Identification of miRNA: Target site interactions by different types of ligation reactions

Stefanie Grosswendt, Andrei Filipchyk, Mark Manzano, Filippos Klironomos, Marcel Schilling, Margareta Herzog, Eva Gottwein, Nikolaus Rajewsky*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

165 Scopus citations

Abstract

To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (CLIP) assays are state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C.elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in the absence of the exogenous ligase. Our invivo data set and reanalysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded ~17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, noncanonical, and nonconserved miRNA:targets. About 80% of miRNA interactions have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, invivo discovery of miRNA binding.

Original languageEnglish (US)
Pages (from-to)1042-1054
Number of pages13
JournalMolecular cell
Volume54
Issue number6
DOIs
StatePublished - Jun 19 2014

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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