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Unambiguous identification of miRNA:target site interactions by different types of ligation reactions

Item Type:Article
Title:Unambiguous identification of miRNA:target site interactions by different types of ligation reactions
Creators Name:Grosswendt, S. and Filipchyk, A. and Manzano, M. and Klironomos, F. and Schilling, M. and Herzog, M. and Gottwein, E. and Rajewsky, N.
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 in vivo 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, in vivo discovery of miRNA binding.
Keywords:Argonaute Proteins, Binding Sites, Cell Line, Chimera, Embryonic Stem Cells, HEK293 Cells, MicroRNAs, Protein Interaction Mapping, RNA-Binding Proteins, Animals, Mice, Caenorhabditis elegans
Source:Molecular Cell
ISSN:1097-2765
Publisher:Cell Press / Elsevier (U.S.A.)
Volume:54
Number:6
Page Range:1042-1054
Date:19 June 2014
Official Publication:https://doi.org/10.1016/j.molcel.2014.03.049
PubMed:View item in PubMed

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