Abstract
Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5′-AGGAGAU-3′. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5′-NGNNG-3′ consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.
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Acknowledgements
We are grateful to M. Blatter for assistance in the structure calculations, M. Zimmermann for synthesis of DNA templates, M. Roos for cell culture preparations and P. Wenter (Eurofins MWG Operon) for synthesis of biotinylated RNAs. This work was supported by an Eidgenössische Technische Hochschule (ETH) postdoctoral fellowship to F.E.L., Sinergia grant CRSII3_127454 from the Swiss National Science Foundation (SNF) to F.H.-T.A. and J.H., Krebsforschung Schweiz (KFS) grant 02648-08-2010 to F.H.-T.A. and J.H., and SNF grant 205321_124720 to J.H.
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F.H.-T.A., F.E.L. and J.H. designed the project; F.E.L. prepared protein and RNA samples for structural studies; F.E.L. and F.H.-T.A. collected and analyzed NMR data; F.E.L. carried out the structure calculations and the ITC measurements; H.T. and A.B. did the Lin28 binding assay with miRNAs and L.F.R.G. did the quantitative PCR in cell assays. All authors discussed the results, wrote and approved the manuscript.
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Loughlin, F., Gebert, L., Towbin, H. et al. Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28. Nat Struct Mol Biol 19, 84–89 (2012). https://doi.org/10.1038/nsmb.2202
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DOI: https://doi.org/10.1038/nsmb.2202
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