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Series GSE56180 Query DataSets for GSE56180
Status Public on May 23, 2014
Title Unambiguous Identification of miRNA:target site Interactions by Different Types of Ligation Reactions
Organism Caenorhabditis elegans
Experiment type Non-coding RNA profiling by high throughput sequencing
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.
Contributor(s) Grosswendt S, Filipchyk A, Manzano M, Klironomos F, Schilling M, Herzog M, Gottwein E, Rajewsky N
Citation(s) 24857550
Submission date Mar 25, 2014
Last update date May 15, 2019
Contact name Nikolaus Rajewsky
Phone +49 30 9406-2998
Organization name Max Delbrück Center for Molecular Medicine
Department Berlin Institute for Medical Systems Biology
Lab Systems Biology of Gene Regulatory Elements
Street address Robert-Rössle-Straße 10
City Berlin
State/province Berlin
ZIP/Postal code 13125
Country Germany
Platforms (1)
GPL13657 Illumina HiSeq 2000 (Caenorhabditis elegans)
Samples (7)
GSM1359318 standard iPAR-CLIP (replicate 1)
GSM1359319 modified iPAR-CLIP ligation (replicate 1a)
GSM1359320 modified iPAR-CLIP ligation (replicate 1b)
BioProject PRJNA242650
SRA SRP040587

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Supplementary file Size Download File type/resource
GSE56180_interactions_all-samples.gff.gz 100.0 Kb (ftp)(http) GFF
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Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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