TY - JOUR
T1 - Unambiguous Identification of miRNA
T2 - Target site interactions by different types of ligation reactions
AU - Grosswendt, Stefanie
AU - Filipchyk, Andrei
AU - Manzano, Mark
AU - Klironomos, Filippos
AU - Schilling, Marcel
AU - Herzog, Margareta
AU - Gottwein, Eva
AU - Rajewsky, Nikolaus
N1 - Funding Information:
We thank F. Slack for the GFP::ALG-1 transgenic strain and M. Simard for the ALG-1 antibody. We thank all members of the Rajewsky lab for discussions and support. We acknowledge P. Shamulailatpam for technical assistance in validation experiments for viral miRNA interactions. C. Langnick and M. Feldkamp (W. Chen lab, MDC) performed sequencing runs. A.F. thanks the DFG Graduate School “Computational Systems Biology” CBS-GRK 1772 for a fellowship. F.K. acknowledges funding from DEEP (Deutsches Epigenom Programm), and M.S. acknowledges funding from the e:Bio program. Validation experiments for viral miRNA interactions in this publication were supported by the National Cancer Institute of the National Institutes of Health under Award Number U54CA143869. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
PY - 2014/6/19
Y1 - 2014/6/19
N2 - 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.
AB - 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.
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U2 - 10.1016/j.molcel.2014.03.049
DO - 10.1016/j.molcel.2014.03.049
M3 - Article
C2 - 24857550
AN - SCOPUS:84903616729
SN - 1097-2765
VL - 54
SP - 1042
EP - 1054
JO - Molecular cell
JF - Molecular cell
IS - 6
ER -