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An alternative mode of microRNA target recognition

Abstract

MicroRNAs (miRNAs) regulate mRNA targets through perfect pairing with their seed region (positions 2–7). Recently, a precise genome-wide map of miRNA interaction sites in mouse brain was generated by high-throughput sequencing and analysis of clusters of ~50-nucleotide mRNA tags cross-linked to Argonaute (Ago HITS-CLIP). By analyzing Ago HITS-CLIP 'orphan clusters'—Ago binding regions from HITS-CLIP that cannot be explained by canonical seed matches—we have now identified an alternative binding mode used by miRNAs. Specifically, G-bulge sites (positions 5–6) are often bound and regulated by miR-124 in brain. More generally, bulged sites comprise ≥15% of all Ago-miRNA interactions in mouse brain and are evolutionarily conserved. We call position 6 the 'pivot' nucleotide and suggest a model in which a transitional 'nucleation bulge' leads to functional bulge mRNA-miRNA interactions, expanding the number of potential miRNA regulatory sites.

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Figure 1: Identification of G-bulge sites pairing to miR-124 by Ago HITS-CLIP analysis.
Figure 2: Validation of G-bulge sites in Ago–miR-124 clusters.
Figure 3: Nucleation bulges are widely used and evolutionarily conserved as functional miRNA target sites.
Figure 4: Functional nucleation bulges in let-7 and miR-708, and gene ontology (GO) analysis.
Figure 5: Pivot pairing and transitional nucleation models.

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Acknowledgements

We thank the members of the Darnell and Hannon laboratories for helpful discussions. This work was supported in part by grants from the US National Institutes of Health (R.B.D. and G.J.H.) and a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare (A111989, to S.W.C.). R.B.D. and G.J.H. are investigators of the Howard Hughes Medical Institute.

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S.W.C. carried out the experiments and computational analyses. S.W.C. and R.B.D. analyzed the data. S.W.C., G.J.H. and R.B.D. designed the research and wrote the paper.

Corresponding authors

Correspondence to Sung Wook Chi or Robert B Darnell.

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The authors declare no competing financial interests.

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods (PDF 2261 kb)

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Chi, S., Hannon, G. & Darnell, R. An alternative mode of microRNA target recognition. Nat Struct Mol Biol 19, 321–327 (2012). https://doi.org/10.1038/nsmb.2230

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