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Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins

Nature Chemical Biology volume 12, pages 717–723 (2016)Cite this article

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Abstract

The RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with well-defined specificity would provide valuable tools and an exacting test of the current understanding of specificity. We have redesigned the specificity of an RRM using rational methods and demonstrated retargeting of its activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of a microRNA precursor (pre-miR-21) with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of the tumor suppressor PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of rationally engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications.

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Figure 1: Engineering the RNA-binding specificity of Rbfox-RRM by rational design.
Figure 2: Secondary structures of free pre-miR-21 and its complex with Fox-RRM*.
Figure 3: Engineered Fox-RRM* inhibits pri- and pre-miR-21 processing.
Figure 4: Engineering G Dicer to target pre-miR-21.
Figure 5: Engineered RRM*-Dicer reduces cellular mature miR-21 levels, increases expression of PDCD4 and reduces cancer cell viability.

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Acknowledgements

The authors are grateful to J. Mandic and other members of G.V.'s laboratory for technical assistance. We thank the Analytical Biopharmacy Core for access to ITC in the School of Pharmacy at the University of Washington. This work was supported by US National Institutes of Health Grant 1R01 GM103834 to G.V., the University of Trento (Progetto Biotecnologie, P.M.) and the Autonomous Province of Trento (Madelena Project, P.M.).

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  1. Yu Chen

    Present address: Present address: Seattle Children's Research Institute, Seattle, Washington, USA.,

Authors and Affiliations

  1. Department of Chemistry, University of Washington, Seattle, Washington, USA

    Yu Chen, Fan Yang, Tom Pavelitz, Wen Yang, Katherine Godin, Matthew Walker, Suxin Zheng & Gabriele Varani

  2. Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy

    Lorena Zubovic & Paolo Macchi

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Contributions

Y.C. conceived the project and designed the experiments, performed protein design, biochemical and cell-based assays, analyzed the data and wrote the paper; F.Y. performed ITC and NMR analysis of protein–RNA interactions; L.Z. and T.P. performed the cell biological assays; W.Y. cloned and expressed G Dicer proteins; K.G. performed SHAPE analysis; M.W. performed Dicer processing assays. S.Z. performed protein structural modeling; L.Z. and P.M. analyzed the data and wrote the paper; G.V. conceived the project, analyzed the data and wrote the paper.

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Correspondence to Yu Chen or Gabriele Varani.

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Chen, Y., Yang, F., Zubovic, L. et al. Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins. Nat Chem Biol 12, 717–723 (2016). https://doi.org/10.1038/nchembio.2128

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