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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.

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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.

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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.).

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Authors and Affiliations

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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.

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Correspondence to Lennart Randau.

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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.

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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

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  • DOI: https://doi.org/10.1038/s41564-018-0274-8

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