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Cited by 282MicroRNA in disease regulationRNA Research and SplicingPlant Molecular Biology Research

Unambiguous Identification of miRNA:Target Site Interactions by Different Types of Ligation Reactions

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Grosswendt, Stefanie, Filipchyk, Andrei, Manzano, Mark, Klironomos, Filippos, Schilling, Marcel, Herzog, Margareta, Gottwein, Eva, Rajewsky, Nikolaus·2014·Molecular Cell
DOI 10.1016/j.molcel.2014.03.049PMC PMC4181535
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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.

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  • Chimeric CLIP-seq approaches incorporate a phosphorylation step and an additional ligation step to increase the frequency of chimeric fragments to as much as 5.3%, although it often remains less than 2%.

    “chimeric CLIP-seq approaches (including CLASH, CLEAR-CLIP, and modified iPAR-CLIP) have modified this approach by incorporating both a phosphorylation step (using 3' phosphatase minus T4 Polynucleotide Kinase) as well as an additional ligation step (without adapters) to encourage proximity-based ligation and increase the frequency of chimeric fragments to as much as 5.3%, although it often remains”

    From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Grosswendt et al., 2014

    The Experimental Procedures section (page unavailable) describes an added T4 RNA ligase reaction in modified iPAR-CLIP, supporting that part of the claim. Our copy does not document the specified phosphorylation step or a 5.3% chimeric-fragment frequency; the Discussion instead reports a ligation efficiency of 0.14% of reads. The Results’ “less than 2%” refers to chimeras containing bacterial sequences, not the overall frequency of chimeric fragments. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • A suite of chimeric CLIP methods including CLASH, modified iPAR-CLIP, and CLEAR-CLIP were developed to enable direct experimental mapping of microRNA:target interactions.

    “To enable this direct experimental mapping of microRNA:target interactions, a suite of chimeric CLIP methods including CrossLinking And Sequencing of Hybrids (CLASH), modified iPAR-CLIP, and CLEAR-CLIP (Broughton et al., 2016;Grosswendt et al., 2014;Helwak et al., 2013;Moore et al., 2015) were developed.”

    From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Grosswendt et al., 2014

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Authors
Grosswendt, S., Filipchyk, A., Manzano, M., Klironomos, F., Schilling, M., Herzog, M., Gottwein, E., & Rajewsky, N.
Affiliations
  • Max Delbrück Center (DE)
  • Northwestern University (US)
Corresponding author
Rajewsky, Nikolaus
ORCID
Venue
Molecular Cell
Publisher
Elsevier BV
Published
2014-06
Volume
54
Issue
6
Pages
1042-1054
ISSN
1097-2765 (ISSN-L)
DOI
10.1016/j.molcel.2014.03.049
PMC
PMC4181535
OpenAlex
W2045685910
Cited by
282
References
61
Citations by year
2026: 9 · 2025: 13 · 2024: 12 · 2023: 14 · 2022: 18 · 2021: 31 · 2020: 26 · 2019: 34 · 2018: 31 · 2017: 27 · 2016: 30 · 2015: 21 · 2014: 16
FWCI
9.37
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Cell (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (submittedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (CBS-GRK 1772)
  • National Institutes of Health (U54CA143869)
  • National Cancer Institute
  • Connecticut Department of Energy and Environmental Protection
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
Keywords
Argonaute proteins, viral miRNAs, RNA silencing, Caenorhabditis elegans
MeSH terms
Animals, Binding Sites, Cell Line, Chimera, Humans, RNA-Binding Proteins, Caenorhabditis elegans, Protein Interaction Mapping, MicroRNAs, Mice, Embryonic Stem Cells, HEK293 Cells, Argonaute Proteins

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