miRNA–target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity
Open accessAbstract
microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO–miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA–target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3′-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo , leading to the discovery that miRNA 3′-end pairing is a general determinant of AGO binding specificity.
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Targets identified using CLASH or similar chimeric ligation approaches showed enrichment for miRNA-dependent changes in RNA decay and specific functional categories, confirming that these methods yield high-quality sets of miRNA targets.
“targets identified using CLASH or similar chimeric ligation approaches showed enrichment for miRNA-dependent changes in RNA decay and specific functional categories, confirming that these methods yield high-quality sets of miRNA targets (Moore et al., 2015).”
From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Moore et al., 2015
The Results section, “CLEAR-CLIP-identified sites are functional” (page unavailable), reports that chimera-identified sites correlate with miRNA-dependent changes in transcript levels and polysome association. The Discussion also describes low false-target identification rates for CLEAR-CLIP. The copy does not establish enrichment for changes in RNA decay specifically or for particular functional categories. (LLM, conf=0.86)
Experimental — accuracy not yet measuredRequiring crosslinking of both miRNA and mRNA target would reduce post-lysis interactions, but low efficiency of UV crosslinking currently limits such efforts.
“Requiring crosslinking of both miRNA and mRNA target would reduce post-lysis interactions, but low efficiency of UV crosslinking currently limits such efforts (Moore et al., 2015).”
From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Moore et al., 2015
The Results section (page unavailable) says ligation to non-cross-linked targets could falsely identify interactions and tests for post-lysis reassociation, supporting the concern about post-lysis artifacts. It does not propose requiring UV cross-linking of both the miRNA and mRNA target. The Discussion (page unavailable) identifies miRNA–target ligation efficiency, not low UV-cross-linking efficiency, as the limiting factor. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe number of chimeras recovered per miRNA correlates well with miRNA abundance.
“However, the fact that the number of chimeras recovered per miRNA correlates well with miRNA abundance (Moore et al., 2015)”
From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Moore et al., 2015
We observed high correlation between miRNA-only and both miRNA:chimera reads and independent small RNA-seq.
“We observed high correlation between miRNA-only and both miRNA:chimera reads and independent small RNA-seq (Sup.Fig. 1C-E), consistent with previous chimeric CLIP methods (Moore et al., 2015).”
From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Moore et al., 2015
Prior chimeric CLIP experiments using spike-in of E. coli, Drosophila, or yeast lysate suggested a low (1-5%) rate of interactions formed in vitro after cell lysis.
“Prior chimeric CLIP experiments using spike-in of E. coli, Drosophila, or yeast lysate (Helwak et al., 2013;Moore et al., 2015) suggested a low (1-5%) rate of interactions formed in vitro after cell lysis”
From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Moore et al., 2015
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- Authors
- Moore, M. J., Scheel, T. K. H., Luna, J. M., Park, C. Y., Fak, J. J., Nishiuchi, E., Rice, C. M., & Darnell, R. B.
- Affiliations
- Howard Hughes Medical Institute (US)
- University of Copenhagen (DK)
- Rockefeller University (US)
- Corresponding author
- Moore, Michael J.
- Darnell, Robert B.
- ORCID
- Venue
- Nature Communications
- Published
- 2015-11-25
- Volume
- 6
- Issue
- 1
- Pages
- 8864-8864
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC4674787
- OpenAlex
- W2180388440
- Cited by
- 326
- References
- 69
- Citations by year
- 2026: 19 · 2025: 28 · 2024: 21 · 2023: 35 · 2022: 31 · 2021: 45 · 2020: 35 · 2019: 33 · 2018: 32 · 2017: 28 · 2016: 18 · 2013: 1
- FWCI
- 8.71
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- Simons Foundation (SFARI 240432)
- Jane Coffin Childs Memorial Fund for Medical Research
- Starr Foundation
- Simons Foundation Autism Research Initiative
- Danmarks Frie Forskningsfond
- National Institutes of Health (NS034389)
- National Cancer Institute (R01CA057973, CA057973)
- National Institute of Allergy and Infectious Diseases (AI091707, R01AI091707, R01AI090055, R01AI116943, AI090055)
- National Institute of Diabetes and Digestive and Kidney Diseases (DK085713, R01DK085713)
- National Institute of Neurological Disorders and Stroke (R01NS081706, NS081706, R01NS034389, R56NS034389, NS034389)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Keywords
- microRNA, Argonaute proteins, miRNA-target interactions, HITS-CLIP, post-transcriptional gene silencing, mouse brain, hepatoma cells
- MeSH terms
- Animals, Binding Sites, Brain, Chimera, Electrophoresis, Polyacrylamide Gel, Flow Cytometry, Humans, RNA, Messenger, Blotting, Northern, Blotting, Western, Base Pairing, Reverse Transcriptase Polymerase Chain Reaction, RNA Interference, MicroRNAs, Cell Line, Tumor, Immunoprecipitation, Mice, Argonaute Proteins
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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