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An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3

Nature Structural & Molecular Biology volume 18, pages 680–687 (2011)Cite this article

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Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) chromosomal loci found in prokaryotes provide an adaptive immune system against bacteriophages and plasmids. CRISPR-specific endoRNases produce short RNA molecules (crRNAs) from CRISPR transcripts, which harbor sequences complementary to invasive nucleic acid elements and ensure their selective targeting by CRISPR-associated (Cas) proteins. The extreme sequence divergence of CRISPR-specific endoRNases and their RNA substrates has obscured homology-based comparison of RNA recognition and cleavage mechanisms. Here, we show that Cse3 type CRISPR-specific endoRNases bind a hairpin structure and residues downstream of the cleavage site within the repetitive segment of cognate CRISPR RNA. Cocrystal structures of Cse3–RNA complexes reveal an RNA-induced conformational change in the enzyme active site that aligns the RNA strand for site-specific cleavage. These studies provide insight into a catalytically essential RNA recognition mechanism by a large class of CRISPR-related endoRNases.

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Figure 1: Structure of Cse3 bound to SL+2 RNA at 1.8-Å resolution.
Figure 2: The active site of Cse3 with RNA bound.
Figure 3: Structures of Cse3 bound to SL+1 RNA.
Figure 4: Single-turnover cleavage analysis of crRNA or Cse3 mutants.
Figure 5: Binding of crRNA and Cse3 mutants.
Figure 6: Multiple-turnover cleavage analysis of crRNA and Cse3 mutants.
Figure 7: Comparison of Cse3 structure with Cas6 and Csy4.

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Acknowledgements

We thank E. Underbakke (UC Berkeley) for protein mass spectrometry; B. Wiedenheft, R. Haurwitz, S. Sternberg, K. Berry (UC Berkeley) and S. Coyle (UCSF) for helpful discussions; members of the Doudna laboratory for critical reading of the manuscript; and C. Ralston and J. Holton (Beamlines 8.2.1 and 8.3.1, Advanced Light Source, Lawrence Berkeley National Laboratory) for assistance with X-ray data collection. D.G.S. is supported by a Damon Runyon Cancer Research Foundation fellowship. M.J. is supported by a Human Frontier Science Program Long-Term Fellowship. This work was supported in part by grants from the National Science Foundation and the Bill and Melinda Gates Foundation to J.A.D. J.A.D. is a Howard Hughes Medical Institute investigator.

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Authors and Affiliations

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

    Dipali G Sashital, Martin Jinek & Jennifer A Doudna

  2. Howard Hughes Medical Institute, University of California, Berkeley, California, USA

    Jennifer A Doudna

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

    Jennifer A Doudna

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

    Jennifer A Doudna

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  1. Dipali G Sashital
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Contributions

D.G.S. and J.A.D. designed experiments. D.G.S. did all assays, crystallized the Cse3–RNA complexes and determined their structures. M.J. assisted with X-ray data collection and structure determination. D.G.S. and J.A.D. wrote the manuscript.

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Correspondence to Jennifer A Doudna.

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Sashital, D., Jinek, M. & Doudna, J. An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3. Nat Struct Mol Biol 18, 680–687 (2011). https://doi.org/10.1038/nsmb.2043

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