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Noncanonical G recognition mediates KSRP regulation of let-7 biogenesis

Nature Structural & Molecular Biology volume 19, pages 1282–1286 (2012)Cite this article

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Abstract

Let-7 is an important tumor-suppressive microRNA (miRNA) that acts as an on-off switch for cellular differentiation and regulates the expression of a set of human oncogenes. Binding of the human KSRP protein to let-7 miRNA precursors positively regulates their processing to mature let-7, thereby contributing to control of cell proliferation, apoptosis and differentiation. Here we analyze the molecular basis for KSRP–let-7 precursor selectivity and show how the third KH domain of the protein recognizes a G-rich sequence in the pre–let-7 terminal loop and dominates the interaction. The structure of the KH3–RNA complex explains the protein recognition of this noncanonical KH target sequence, and we demonstrate that the specificity of this binding is crucial for the functional interaction between the protein and the miRNA precursor.

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Figure 1: KSRP, pre–let-7a and their interaction.
Figure 2: Structure of the KH3–RNA complex.
Figure 3: Comparison of the KSRP KH3–AGGGU complex with three representative KH–nucleic acid complexes (that is, the Nova KH3, SF1 KH3 and hnRNPK KH3 complexes with nucleic acid).
Figure 4: KH3 interaction with an AGGGU RNA in the let-7-precursor hairpin domain is essential for KSRP function in let-7 biogenesis.

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NCBI Reference Sequence

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Acknowledgements

All the NMR experiments were recorded at the Medical Research Council Biomedical NMR Centre at the Medical Research Council National Institute for Medical Research, and we thank T. Frenkiel and A. Oregioni for assistance. We thank S. Howell for mass spectrometry analysis of protein samples. The work of A.R. has been funded by the Medical Research Council (MRC) grant U117574558 and the Wellcome Trust Grant WT082088MA. The work of S.R.M. and G.K. has been funded by MRC grants U117570592 and U117533887, respectively. The work of P.B. and R.G. has been supported by grants from the Association for International Cancer Research (10-0527) and Associazione Italiana per la Ricerca sul Cancro (I.G. 10090). We thank B. Faust for help in the expression and purification of the KH3 K368R mutant.

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Authors and Affiliations

  1. Molecular Structure Division, Medical Research Council National Institute for Medical Research, London, UK

    Giuseppe Nicastro, María Flor García-Mayoral, David Hollingworth & Andres Ramos

  2. Departamento de Química Física Biológica, Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain

    María Flor García-Mayoral

  3. Medical Research Council Biomedical NMR Centre, Medical Research Council National Institute for Medical Research, London, UK

    Geoff Kelly

  4. Physical Biochemistry Division, Medical Research Council National Institute for Medical Research, London, UK

    Stephen R Martin

  5. Gene Expression Regulation Laboratory IRCCS Azienda Ospedaliera Universitaria San Martino–IST, Genova, Italy

    Paola Briata & Roberto Gherzi

Authors
  1. Giuseppe Nicastro
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Contributions

Cloning and RNA synthesis were performed by D.H. Protein purification was performed by G.N. and D.H. Recording and analysis of the NMR spectra were performed by G.N., M.F.G.-M. and G.K. Structure calculations and recording of the isothermal titration calorimetry, CD and BLI data were performed by G.N. Analysis of the CD and isothermal titration calorimetry data was performed by S.R.M. and G.N. RIP and pri-miRNA processing assays were performed by P.B. and R.G. The manuscript was written by A.R. and G.N.

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Correspondence to Roberto Gherzi or Andres Ramos.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5 and Supplementary Table 1 (PDF 17418 kb)

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Nicastro, G., García-Mayoral, M., Hollingworth, D. et al. Noncanonical G recognition mediates KSRP regulation of let-7 biogenesis. Nat Struct Mol Biol 19, 1282–1286 (2012). https://doi.org/10.1038/nsmb.2427

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