Abstract
Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.
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Acknowledgements
This work was supported by the Swiss National Science Foundation (in part) with a joint Sinergia grant (CRSII3_127454) to A.P.G., M.Z. and J.H. as well as a grant to J.H. (CRS205321_124720). We thank B. Schoser (Ludwig-Maximilians-Universität, Munich) for myoblasts, M. Lucic and Y. Wang for help with assays and M. Zimmermann and U. Pradère for synthesis of RNAs. We thank H. Towbin and J.A. Zagalak for helpful discussions and Luca Gebert for graphical layout. We are grateful to Y. Huang (Yale Stem Cell Center) for sharing pH19 expression plasmids.
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All authors designed experiments and analyzed data. A. Brunschweiger designed, synthesized and characterized probes. J.I. performed cell assays with myoblasts. B.G. and P.T. performed reporter assays. A. Brunschweiger, J.I. and S.K. performed pull-down experiments. N.M. generated deep-sequencing libraries. A. Brümmer and M.Z. analyzed sequencing data. J.I., A. Brunschweiger, A. Brümmer, M.Z. and J.H. wrote the paper.
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Supplementary Text and Figures
Supplementary Results, Supplementary Tables 1–5, Supplementary Figures 1–18 and Supplementary Notes. (PDF 3144 kb)
Supplementary Data Set 1
Table of abundance levels of RNAs in HeLa cells (input), associated with Ago-2 (Mock), associated with Ago-2 after transfection of the miR-106a capture probe RNA-7 (Ago2 IP), and captured by the miR-106a capture probe RNA-7 (miRCLIP); list of 644 significantly enriched transcripts in miR-CLIP vs. Ago2-IP identified using DESeq. (XLS 2464 kb)
Supplementary Data Set 2
Table of abundance levels of RNAs in HeLa cells (input), associated with Ago-2 (Mock), associated with Ago-2 after transfection of let-7g capture probe RNA-11 (Ago2 IP), and captured by let-7g capture probe RNA-11 (miRCLIP). (XLSX 1566 kb)
Supplementary Data Set 3
Table of abundance levels of lncRNAs in HeLa cells (input), associated with Ago-2 (Mock), associated with Ago-2 after transfection of the miR-106a capture probe RNA-7 (Ago2 IP), and captured by miR-106a capture probe RNA-7 (miRCLIP). (XLS 653 kb)
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Imig, J., Brunschweiger, A., Brümmer, A. et al. miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19–miR-106a interaction. Nat Chem Biol 11, 107–114 (2015). https://doi.org/10.1038/nchembio.1713
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DOI: https://doi.org/10.1038/nchembio.1713
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