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Recognition and maturation of effector RNAs in a CRISPR interference pathway

Nature Structural & Molecular Biology volume 18, pages 688–692 (2011)Cite this article

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Abstract

In bacteria and archaea, small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by an RNA endonuclease generates the mature effector RNAs that interfere with phage gene expression. Here we describe structural and functional analyses of the Thermus thermophilus CRISPR Cse3 endonuclease. High-resolution X-ray structures of Cse3 bound to repeat RNAs model both the pre- and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene silencing.

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Figure 1: Processing of pre-crRNA by Cse3.
Figure 2: Structure of the Cse3 substrate–RNA complex.
Figure 3: Structural basis for cleavage of pre-crRNA by Cse3.
Figure 4: Modular organization of pre-crRNA recognition and processing revealed by comparison of Cse3 and Csy4 RNA complexes.

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Acknowledgements

This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to A.M.M. We are also grateful for the support of the Alberta Synchrotron Institute during the early stages of this work.

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  1. Emily M Gesner and Matthew J Schellenberg: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

    Emily M Gesner, Matthew J Schellenberg, Erin L Garside, Mark M George & Andrew M MacMillan

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  1. Emily M Gesner
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Contributions

E.M.G. cloned, expressed and biochemically characterized Cse3 and Cse3 mutants, purified the proteins and protein–RNA complexes and grew the crystals. M.J.S. solved the structures. E.L.G. and M.M.G. cloned, expressed and purified Cse3 mutants. E.M.G., M.J.S., E.L.G. and A.M.M. designed the experiments and contributed to writing the manuscript.

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Correspondence to Andrew M MacMillan.

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The authors declare no competing financial interests.

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Gesner, E., Schellenberg, M., Garside, E. et al. Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat Struct Mol Biol 18, 688–692 (2011). https://doi.org/10.1038/nsmb.2042

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