Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum

Abstract

Type IV CRISPR–Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR–Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5′-repeat tags and stable 3′ hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR–ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein–protein and protein–RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR–Cas systems and demonstrate that type IV CRISPR–Cas systems employ crRNA-guided effector complexes.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Maturation of A. aromaticum crRNAs.
Fig. 2: Csf5 is a crRNA processing enzyme.
Fig. 3: Crystal structures of Csf5.
Fig. 4: A. aromaticum type IV Cas protein production in E. coli.
Fig. 5: type IV crRNP complex protein–protein crosslinking.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Crystallographic data and models have been deposited at the protein data bank (PDB) under accession codes 6H9H and 6H9I.

References

  1. 1.

    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  Google Scholar 

  2. 2.

    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  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  Google Scholar 

  4. 4.

    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  Google Scholar 

  5. 5.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Westra, E. R. et al. The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311–339 (2012).

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

    CAS  Article  Google Scholar 

  8. 8.

    Nunez, J. K. et al. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nat. Struct. Mol. Biol. 21, 528–534 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Swarts, D. C., Mosterd, C., van Passel, M. W. & Brouns, S. J. CRISPR interference directs strand specific spacer acquisition. PLoS ONE 7, e35888 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Pul, U. et al. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol. Microbiol. 75, 1495–1512 (2010).

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    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 

  13. 13.

    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 

  14. 14.

    Jore, M. M. et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat. Struct. Mol. Biol. 18, 529–536 (2011).

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    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 

  17. 17.

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

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Westra, E. R. et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell 46, 595–605 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

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

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Kazlauskiene, M., Tamulaitis, G., Kostiuk, G., Venclovas, C. & Siksnys, V. Spatiotemporal control of Type III-A CRISPR-Cas immunity: coupling DNA degradation with the target RNA recognition. Mol. Cell 62, 295–306 (2016).

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Huo, Y. et al. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nat. Struct. Mol. Biol. 21, 771–777 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Sashital, D. G., Wiedenheft, B. & Doudna, J. A. Mechanism of foreign DNA selection in a bacterial adaptive immune system. Mol. Cell 46, 606–615 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Voloshin, O. N. & Camerini-Otero, R. D. The DinG protein from Escherichia coli is a structure-specific helicase. J. Biol. Chem. 282, 18437–18447 (2007).

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Gleditzsch, D. et al. Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system. Nucleic Acids Res. 44, 5872–5882 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Pausch, P. et al. Structural variation of Type I-F CRISPR RNA guided DNA surveillance. Mol. Cell 67, 622–632 (2017).

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Nam, K. H. et al. Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure 20, 1574–1584 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Hochstrasser, M. L., Taylor, D. W., Kornfeld, J. E., Nogales, E. & Doudna, J. A. DNA targeting by a minimal CRISPR RNA-guided cascade. Mol. Cell 63, 840–851 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Garside, E. L. et al. Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases. RNA 18, 2020–2028 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Hochstrasser, M. L. & Doudna, J. A. Cutting it close: CRISPR-associated endoribonuclease structure and function. Trends Biochem. Sci. 40, 58–66 (2015).

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Plagens, A. et al. In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex. Nucleic Acids Res. 42, 5125–5138 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Reeks, J. et al. Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing. Biochem. J. 452, 223–230 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Shmakov, S. A. et al. The CRISPR spacer space is dominated by sequences from species-specific mobilomes. mBio 8, e01397-17 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Rabus, R. & Widdel, F. Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Arch. Microbiol. 163, 96–103 (1995).

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Grant, T., Rohou, A. & Grigorieff, N. cisTEM, user-friendly software for single-particle image processing. elife 7, e35383 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Miyatake, H., Hasegawa, T. & Yamano, A. New methods to prepare iodinated derivatives by vaporizing iodine labelling (VIL) and hydrogen peroxide VIL (HYPER-VIL). Acta Crystallogr. D 62, 280–289 (2006).

    Article  PubMed  Google Scholar 

  38. 38.

    Gabadinho, J. et al. MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments. J. Synchrotron. Radiat. 17, 700–707 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

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

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

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

    Article  PubMed  Google Scholar 

  42. 42.

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

    CAS  Article  PubMed  Google Scholar 

  43. 43.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Kramer, K. et al. Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins. Nat. Methods 11, 1064–1070 (2014).

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012).

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Combe, C. W., Fischer, L. & Rappsilber, J. xiNET: cross-link network maps with residue resolution. Mol. Cell Proteomics 14, 1137–1147 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

G.B thanks the LOEWE excellence initiative for financial support. G.B. and P.P. acknowledge the always excellent support by the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. This work was supported by the DFG (FOR1680 to L.R.), SPP2141 (to L.R. and G.B.), TÜBİTAK (to A.Ö.) and the Max Planck Society (to L.R.).

Author information

Affiliations

Authors

Contributions

A.Ö. and P.P. purified proteins. P.P. determined the crystal structure. A.L., A.W. and H.U. performed mass spectrometry analyses. K.S. and J.H. cultured A. aromatoleum strains. T.H. performed transmission electron microscopy analyses. L.R and A.Ö. conceived the experiments. L.R. wrote the manuscript with support from A.Ö., P.P., G.B., J.H., T.H. and H.U.

Corresponding author

Correspondence to Lennart Randau.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figures 1–6, Supplementary Tables 1, 2, 4 and 5.

Reporting Summary

Supplementary Table 3

Overview of crRNP protein–protein cross-links. Contains a structured representation of all identified protein–protein cross-links (cross-linked peptide spectrum matches (CSM)) of the recombinant type IV crRNP of Aromatoleum aromaticum. A detailed legend is included as a separate worksheet.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Özcan, A., Pausch, P., Linden, A. et al. Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum. Nat Microbiol 4, 89–96 (2019). https://doi.org/10.1038/s41564-018-0274-8

Download citation

Further reading