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Structural determinants of RNA recognition and cleavage by Dicer

Nature Structural & Molecular Biology volume 14, pages 934–940 (2007)Cite this article

Abstract

A hallmark of RNA interference is the production of short double-stranded RNA (dsRNA) molecules 21–28 nucleotides in length by the specialized RNase III protein Dicer. Dicer enzymes uniquely generate RNA products of specific lengths by mechanisms that have not been fully elucidated. Here we show that the PAZ domain responsible for dsRNA end recognition confers this measuring ability through both its structural position and RNA-binding specificity. Point mutations define the dsRNA-binding surface and reveal a protein loop important for cleavage of substrates containing perfect or imperfect base pairing. On the basis of these results, we reengineered Dicer with a U1A RNA-binding domain in place of the PAZ domain to create an enzyme with altered end-recognition specificity and RNA product length. These results explain how Dicer functions as a molecular ruler and provide a structural basis for modifying its activity in cells.

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Figure 1: Role of the PAZ domain and helical end structure in cleavage site selection by Dicer.
Figure 2: Contributions of positively charged surface residues to dsRNA substrate recognition.
Figure 3: Kinetic measurements of Dicer surface mutants.
Figure 4: Role of the positioning loop in dsRNA recognition and cleavage.
Figure 5: Activity of an engineered Dicer with altered RNA recognition specificity.

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Acknowledgements

We thank the members of the Doudna lab for comments and suggestions. This work was supported by the Howard Hughes Medical Institute and US National Institutes of Health grant 5R01GM073794-02.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.,

    Ian J MacRae, Kaihong Zhou & Jennifer A Doudna

  2. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.,

    Ian J MacRae, Kaihong Zhou & Jennifer A Doudna

  3. Department of Chemistry, University of California, Berkeley, California 94720, USA.,

    Jennifer A Doudna

  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.,

    Jennifer A Doudna

Authors
  1. Ian J MacRae
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  2. Kaihong Zhou
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  3. Jennifer A Doudna
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Contributions

I.J.M. designed and performed experiments and wrote the manuscript, K.Z. performed experiments, and J.A.D. discussed results and wrote the manuscript.

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Correspondence to Jennifer A Doudna.

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MacRae, I., Zhou, K. & Doudna, J. Structural determinants of RNA recognition and cleavage by Dicer. Nat Struct Mol Biol 14, 934–940 (2007). https://doi.org/10.1038/nsmb1293

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