Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Accession codes

Primary accessions

Protein Data Bank

References

  1. 1

    Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3

    Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4

    van der Oost, J., Jore, M.M., Westra, E.R., Lundgren, M. & Brouns, S.J. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem. Sci. 34, 401–407 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    Karginov, F.V. & Hannon, G.J. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol. Cell 37, 7–19 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Marraffini, L.A. & Sontheimer, E.J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11, 181–190 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Pougach, K. et al. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol. 77, 1367–1379 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8

    Brouns, S.J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9

    Tang, T.H. et al. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. USA 99, 7536–7541 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    Tang, T.H. et al. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus. Mol. Microbiol. 55, 469–481 (2005).

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Hale, C., Kleppe, K., Terns, R.M. & Terns, M.P. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14, 2572–2579 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Lillestøl, R.K. et al. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol. Microbiol. 72, 259–272 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Carte, J., Wang, R., Li, H., Terns, R.M. & Terns, M.P. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22, 3489–3496 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Hale, C.R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Haurwitz, R.E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J.A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Jansen, R., Embden, J.D., Gaastra, W. & Schouls, L.M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Jore, M.M. et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat. Struct. Mol. Biol. 10.1038/nsmb.2019 (3 April 2011).

  19. 19

    Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Marraffini, L.A. & Sontheimer, E.J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Kunin, V., Sorek, R. & Hugenholtz, P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 8, R61 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Carte, J., Pfister, N.T., Compton, M.M., Terns, R.M. & Terns, M.P. Binding and cleavage of CRISPR RNA by Cas6. RNA 16, 2181–2188 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Godde, J.S. & Bickerton, A. The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes. J. Mol. Evol. 62, 718–729 (2006).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Ebihara, A. et al. Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain. Protein Sci. 15, 1494–1499 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25

    Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Leslie, A.G.W. Recent changes to MOSFLM package for processing film and image plate data. Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography Vol. 26 (Daresbury Laboratory, Warrington, UK, 1992).

  27. 27

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Langer, G., Cohen, S.X., Lamzin, V.S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding author

Correspondence to Jennifer A Doudna.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Methods (PDF 5543 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing