Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps

Abstract

MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; however, as miRNA activity requires base pairing with only 6-8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein–RNA interaction sites. Here we use HITS-CLIP to covalently crosslink native argonaute (Ago, also called Eif2c) protein–RNA complexes in mouse brain. This produced two simultaneous data sets—Ago–miRNA and Ago–mRNA binding sites—that were combined with bioinformatic analysis to identify interaction sites between miRNA and target mRNA. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. Ago HITS-CLIP provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA–mRNA interactions.

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Figure 1: Argonaute HITS-CLIP.
Figure 2: Distribution of mRNA tags correlates with seed sequences of miRNAs from Ago CLIP.
Figure 3: Ago–miRNA ternary clusters in validated miR-124 mRNA targets.
Figure 4: Meta-analysis of Ago–mRNA clusters in large-scale screens of miR-124-regulated targets.
Figure 5: Ago–miR-124 ternary maps in brain and transfected HeLa cells.
Figure 6: Ago–miRNA ternary maps.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The microarray data have been deposited in the GEO database under accession number GSE16338.

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Acknowledgements

Acknowledgments We thank members of the Darnell laboratory for discussion, J. Fak for help with exon arrays, C. Zhang for bioinformatic discussions, and G. Dunn, D. Licatalosi, C. Marney, M. Frias, T. Eom, J. Darnell, M. Yano and C. Zhang for critical review of the manuscript. We also thank Z. Mourelatos for supplying the 2A8 antibody and communicating unpublished results; G. Hannon for discussions; and S. Dewell for help with high-throughput sequencing. This work was supported in part by grants from the NIH (R.B.D.), the Cornell/Rockefeller/Sloan-Kettering Tri-Institutional Program in Computational Biology and Medicine (S.W.C.) and MD-PhD Program (J.B.Z.). R.B.D. is an Investigator of the Howard Hughes Medical Institute.

Author Contributions S.W.C. and R.B.D conceived, designed and supervised the experiments, analysed the data and wrote the paper. J.B.Z. did the initial experiments with the 7G1-1* antibody. A.M. helped with all HITS-CLIP experiments.

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Correspondence to Robert B. Darnell.

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Chi, S., Zang, J., Mele, A. et al. Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature 460, 479–486 (2009). https://doi.org/10.1038/nature08170

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