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Recognition and maturation of effector RNAs in a CRISPR interference pathway

Nature Structural & Molecular Biology volume 18, pages 688692 (2011) | Download Citation

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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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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Author notes

    • Emily M Gesner
    •  & Matthew J Schellenberg

    These authors contributed equally to this work.

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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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Andrew M MacMillan.

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DOI

https://doi.org/10.1038/nsmb.2042

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