Unambiguous Identification of miRNA:target site Interactions by Different Types of Ligation Reactions

  • Stefanie Grosswendt (Contributor)
  • Andrei Filipchyk (Contributor)
  • Mark Manzano (Contributor)
  • Filippos Klironomos (Contributor)
  • Marcel Schilling (Contributor)
  • Margareta Herzog (Contributor)
  • Eva Gottwein (Contributor)
  • Nikolaus Rajewsky (Contributor)

Dataset

Description

Accession Number: GSE56180

Platform:
GPL13657: Illumina HiSeq 2000 (Caenorhabditis elegans)

Organism: Caenorhabditis elegans

Published on 2014-05-23

Summary:
To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (“re 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 absence of the exogenous ligase. Our in vivo dataset and re-analysis 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, non-canonical, and non-conserved miRNA interactions. Our data suggest that ~80% of miRNA:targets have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, in vivo discovery of miRNA interactions.

Overall Design:
In vivo PAR-CLIP basically as described previously (Jungkamp et al. 2011) using GFP-tagged ALG-1 expressing worms in L3 stage. Worm lysate was treated with RNase T1. Following immunoprecipitation and a second RNase T1 digest, it was proceeded as described in Hafner et al. 2010. For the modified iPAR-CLIP ligation samples and its control samples immuno-purified complexes were treated with PNK phospathase minus, subjected to ligation with T4 RNA ligase/no ligase added and subsequently phosphorylated with PNK. Protein purification and RNA library preparation essentially as described in Hafner et al., but with the selection of longer RNA products.

Contact:
Name: Nikolaus Rajewsky
Organization: Max Delbrück Center for Molecular Medicine
Laboratory: Systems Biology of Gene Regulatory Elements
Deparment: Berlin Institute for Medical Systems Biology
Address: Robert-Rössle-Straße 10 Berlin Berlin 13125 Germany
Email: [email protected]
Phone: +49 30 9406-2998

Organization: GEO
Address: USA
Date made availableMar 25 2014
PublisherGene Expression Omnibus

Cite this