Article | Published:

Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers

Nature volume 548, pages 543548 (31 August 2017) | Download Citation

Abstract

In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    et al. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 353, aad5147 (2016)

  2. 2.

    CRISPR–Cas immunity in prokaryotes. Nature 526, 55–61 (2015)

  3. 3.

    , & CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem. 82, 237–266 (2013)

  4. 4.

    , & Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017)

  5. 5.

    et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015)

  6. 6.

    et al. Co-transcriptional DNA and RNA cleavage during Type III CRISPR-Cas immunity. Cell 161, 1164–1174 (2015)

  7. 7.

    , , & Conditional tolerance of temperate phages via transcription-dependent CRISPR–Cas targeting. Nature 514, 633–637 (2014)

  8. 8.

    , , , & A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus. Mol. Microbiol. 87, 1088–1099 (2013)

  9. 9.

    et al. Bipartite recognition of target RNAs activates DNA cleavage by the type III-B CRISPR-Cas system. Genes Dev. 30, 447–459 (2016)

  10. 10.

    , & RNA-activated DNA cleavage by the type III-B CRISPR-Cas effector complex. Genes Dev. 30, 460–470 (2016)

  11. 11.

    , , , & Spatiotemporal control of type III-A CRISPR-Cas immunity: coupling DNA degradation with the target RNA recognition. Mol. Cell 62, 295–306 (2016)

  12. 12.

    , & Type III CRISPR-Cas immunity: major differences brushed aside. Trends Microbiol. 25, 49–61 (2017)

  13. 13.

    , , , & CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems. Front. Genet. 5, 102 (2014)

  14. 14.

    , , , & Comprehensive analysis of the HEPN superfamily: identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing. Biol. Direct 8, 15 (2013)

  15. 15.

    , , & Genetic characterization of antiplasmid immunity through a type III-A CRISPR-Cas system. J. Bacteriol. 196, 310–317 (2014)

  16. 16.

    , & Degradation of phage transcripts by CRISPR-associated RNases enables type III CRISPR-Cas immunity. Cell 164, 710–721 (2016)

  17. 17.

    & Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6. RNA 22, 318–329 (2016)

  18. 18.

    , , & The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease. RNA 22, 216–224 (2016)

  19. 19.

    et al. RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Mol. Cell 56, 518–530 (2014)

  20. 20.

    et al. Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. Mol. Cell 56, 506–517 (2014)

  21. 21.

    , , , & A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs. J. Biol. Chem. 288, 27888–27897 (2013)

  22. 22.

    , , , & Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling. Nucleic Acids Res. 43, 10633–10654 (2015)

  23. 23.

    , & Crystal structures of CRISPR-associated Csx3 reveal a manganese-dependent deadenylation exoribonuclease. RNA Biol. 12, 749–760 (2015)

  24. 24.

    & Recognition of a pseudo-symmetric RNA tetranucleotide by Csx3, a new member of the CRISPR associated Rossmann fold superfamily. RNA Biol. 13, 254–257 (2016)

  25. 25.

    et al. Activation of RNase L by 2′,5′-oligoadenylates. Kinetic characterization. J. Biol. Chem. 272, 19193–19198 (1997)

  26. 26.

    , , & Phosphoregulation of Ire1 RNase splicing activity. Nat. Commun. 5, 3554 (2014)

  27. 27.

    et al. Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus. Mol. Cell 52, 135–145 (2013)

  28. 28.

    , , , & Target RNA capture and cleavage by the Cmr type III-B CRISPR-Cas effector complex. Genes Dev. 28, 2432–2443 (2014)

  29. 29.

    et al. Evolution and classification of the CRISPR–Cas systems. Nat. Rev. Microbiol. 9, 467–477 (2011)

  30. 30.

    , & Crystal structure of the Cmr2-Cmr3 subcomplex in the CRISPR-Cas RNA silencing effector complex. J. Mol. Biol. 425, 3811–3823 (2013)

  31. 31.

    et al. Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, (p)ppGpp signaling in bacteria and implications in pathogenesis. Chem. Soc. Rev. 42, 305–341 (2013)

  32. 32.

    , , & OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids. Nat. Rev. Immunol. 14, 521–528 (2014)

  33. 33.

    et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273 (2016)

  34. 34.

    , , , & CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b. Nucleic Acids Res. 45, 1902–1913 (2017)

  35. 35.

    et al. The structure of the CRISPR-associated protein Csa3 provides insight into the regulation of the CRISPR/Cas system. J. Mol. Biol. 405, 939–955 (2011)

  36. 36.

    FX cloning: a simple and robust high-throughput cloning method for protein expression. Methods Mol. Biol. 1116, 153–164 (2014)

  37. 37.

    et al. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305, 709–712 (1983)

  38. 38.

    & Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268 (2004)

  39. 39.

    et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)

  40. 40.

    , , & ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)

  41. 41.

    , , , & The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protocols 10, 845–858 (2015)

Download references

Acknowledgements

We thank members of the Jinek and Marraffini laboratories for discussions and comments on the manuscript. We thank D. Swarts and P. Sledz for technical assistance and sharing reagents. We thank U. Manzau for technical assistance during A4>P synthesis and purification. We thank the Service for Mass Spectrometry of ETH Zurich for support with MS analysis. This study was supported by a Swiss National Science Foundation project grant to M.J. (SNSF 31003A_149393) and by funding from the Swiss National Competence Center for Research (NCCR) ‘RNA & Disease’ (to M.J. and J.H.). M.J. is an International Research Scholar of the Howard Hughes Medical Institute and Vallee Scholar of the Bert L & N Kuggie Vallee Foundation. C.G.-D. was supported by a Long-Term Fellowship from the European Molecular Biology Organization. J.T.R. was supported by a Boehringer Ingelheim Fonds PhD fellowship. L.A.M. is supported by the Rita Allen Scholars Program, a Burroughs Wellcome Fund PATH award, a National Institutes of Health Director’s New Innovator Award (1DP2AI104556-01), and a Howard Hughes Medical Institute-Simons Faculty Scholar Award.

Author information

Author notes

    • Ole Niewoehner
    •  & Carmela Garcia-Doval

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

    • Ole Niewoehner
    • , Carmela Garcia-Doval
    •  & Martin Jinek
  2. Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065-6399, USA

    • Jakob T. Rostøl
    •  & Luciano A. Marraffini
  3. Department of Chemistry and Applied Biosciences, Institute for Pharmaceutical Sciences, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland

    • Christian Berk
    •  & Jonathan Hall
  4. BIOLOG Life Science Institute GmbH, Flughafendamm 9a, D-28199 Bremen, Germany

    • Frank Schwede
  5. Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

    • Laurent Bigler

Authors

  1. Search for Ole Niewoehner in:

  2. Search for Carmela Garcia-Doval in:

  3. Search for Jakob T. Rostøl in:

  4. Search for Christian Berk in:

  5. Search for Frank Schwede in:

  6. Search for Laurent Bigler in:

  7. Search for Jonathan Hall in:

  8. Search for Luciano A. Marraffini in:

  9. Search for Martin Jinek in:

Contributions

O.N., C.G.-D., and M.J. conceived the study. O.N., C.G.-D., J.T.R., L.M., and M.J. designed experiments. O.N. expressed and purified recombinant Csm6 proteins, performed oligoA activation and ATPase assays, and performed enzymatic probing of the cyclic oligoA product. C.G.-D. expressed and purified recombinant EiCsm(1–5) complexes, performed oligoA activation assays, and assisted with LC–MS analysis. J.T.R. performed phage infection assays under supervision of L.M. C.B. synthesized 2′,3′-cyclic phosphate-terminated nucleotides and performed LC–MS analysis under supervision of J.H. F.S. synthesized 2′,3′-cyclic phosphate-terminated A4 nucleotide and advised on nucleotide chemistry. L.B. performed additional LC–MS analyses of Csm6 activators. O.N., C.G.-D., and M.J. wrote the manuscript, with input from the remaining authors.

Competing interests

F.S. is an employee of BIOLOG Life Science Institute GmbH, which markets synthetic nucleotides. The other authors declare no competing financial interests.

Corresponding author

Correspondence to Martin Jinek.

Reviewer Information Nature thanks S. Bailey, P. Kranzusch and R. Staals for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure

    This file contains the uncropped gels.

  2. 2.

    Reporting Summary

Excel files

  1. 1.

    Supplementary Data

    This file contains Supplementary Table 1

  2. 2.

    Supplementary Data

    This file contains Supplementary Table 2.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature23467

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.