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The host-encoded RNase E endonuclease as the crRNA maturation enzyme in a CRISPR–Cas subtype III-Bv system

Nature Microbiology volume 3, pages 367–377 (2018)Cite this article

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Abstract

Specialized RNA endonucleases for the maturation of clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs) are critical in CRISPR–CRISPR-associated protein (Cas) defence mechanisms. The Cas6 and Cas5d enzymes are the RNA endonucleases in many class 1 CRISPR–Cas systems. In some class 2 systems, maturation and effector functions are combined within a single enzyme or maturation proceeds through the combined actions of RNase III and trans-activating CRISPR RNAs (tracrRNAs). Three separate CRISPR–Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Whereas Cas6-type enzymes act in two of these systems, the third, which is classified as subtype III-B variant (III-Bv), lacks cas6 homologues. Instead, the maturation of crRNAs proceeds through the activity of endoribonuclease E, leaving unusual 13- and 14-nucleotide-long 5′-handles. Overexpression of RNase E leads to overaccumulation and knock-down to the reduced accumulation of crRNAs in vivo, suggesting that RNase E is the limiting factor for CRISPR complex formation. Recognition by RNase E depends on a stem-loop in the CRISPR repeat, whereas base substitutions at the cleavage site trigger the appearance of secondary products, consistent with a two-step recognition and cleavage mechanism. These results suggest the adaptation of an otherwise very conserved housekeeping enzyme to accommodate new substrates and illuminate the impressive plasticity of CRISPR–Cas systems that enables them to function in particular genomic environments.

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Fig. 1: Organization of Synechocystis 6803 CRISPR3 cas genes, repeat-spacer array and interference assay.
Fig. 2: Mapping of in vivo processing sites within CRISPR3 pre-crRNA by primer extension.
Fig. 3: RNase E substrates and cleavage sites in Synechocystis 6803.
Fig. 4: Manipulation of RNase E expression affects CRISPR3 crRNA accumulation.
Fig. 5: Identification and analysis of RNase E amino acids interacting with CRISPR3 repeat RNA.
Fig. 6: Model for RNase-E-mediated crRNA recognition and processing.

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References

  1. Bhaya, D., Davison, M. & Barrangou, R. CRISPR–Cas systems in Bacteria and Archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45, 273–297 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Grissa, I., Vergnaud, G. & Pourcel, C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinforma. 8, 172 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Lange, S. J., Alkhnbashi, O. S., Rose, D., Will, S. & Backofen, R. CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems. Nucleic Acids Res. 41, 8034–8044 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Westra, E. R., Dowling, A. J., Broniewski, J. M. & van Houte, S. Evolution and ecology of CRISPR. Annu. Rev. Ecol. Evol. Syst. 47, 307–331 (2016).

    Article  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Charpentier, E., Richter, H., van der Oost, J. & White, M. F. Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR–Cas adaptive immunity. FEMS Microbiol. Rev. 39, 428–441 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Przybilski, R. et al. Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum. RNA Biol. 8, 517–528 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Punetha, A., Sivathanu, R. & Anand, B. Active site plasticity enables metal-dependent tuning of Cas5d nuclease activity in CRISPR–Cas type I-C system. Nucleic Acids Res. 42, 3846–3856 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR–Cas systems. Mol. Cell. 60, 385–397 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Koonin, E. V. et al. Diversity, classification and evolution of CRISPR–Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).

  20. Fonfara, I., Richter, H., Bratovič, M., Le Rhun, A. & Charpentier, E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature 532, 517–521 (2016).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Scholz, I., Lange, S. J., Hein, S., Hess, W. R. & Backofen, R. CRISPR–Cas systems in the cyanobacterium Synechocystis sp. PCC6803 exhibit distinct processing pathways involving at least two Cas6 and a Cmr2 protein. PLoS. ONE 8, e56470 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Reimann, V. et al. Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs. Nucleic Acids Res. 45, 915–925 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wang, R., Preamplume, G., Terns, M. P., Terns, R. M. & Li, H. Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage. Structure 19, 257–264 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gesner, E. M., Schellenberg, M. J., Garside, E. L., George, M. M. & MacMillan, A. M. Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat. Struct. Mol. Biol. 18, 688–692 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Schein, A., Sheffy-Levin, S., Glaser, F. & Schuster, G. The RNase E/G-type endoribonuclease of higher plants is located in the chloroplast and cleaves RNA similarly to the E. coli enzyme. RNA 14, 1057–1068 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Horie, Y. et al. Dark-induced mRNA instability involves RNase E/G-type endoribonuclease cleavage at the AU-box and SD sequences in cyanobacteria. Mol. Genet. Genom. 278, 331–346 (2007).

    Article  CAS  Google Scholar 

  30. Mohanty, B. K., Petree, J. R. & Kushner, S. R. Endonucleolytic cleavages by RNase E generate the mature 3′ termini of the three proline tRNAs in Escherichia coli. Nucleic Acids Res. 44, 6350–6362 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jiang, X., Diwa, A. & Belasco, J. G. Regions of RNase E important for 5′-end-dependent RNA cleavage and autoregulated synthesis. J. Bacteriol. 182, 2468–2475 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chao, Y. et al. In vivo cleavage map illuminates the central role of RNase E in coding and non-coding RNA pathways. Mol. Cell. 65, 39–51 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, J., Graham, S., Tello, A., Liu, H. & White, M. F. Multiple nucleic acid cleavage modes in divergent type III CRISPR systems. Nucleic Acids Res. 44, 1789–1799 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Rott, R., Zipor, G., Portnoy, V., Liveanu, V. & Schuster, G. RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from Escherichia coli. J. Biol. Chem. 278, 15771–15777 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Sharma, K. et al. Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry. Methods 89, 138–148 (2015).

    Article  CAS  PubMed  Google Scholar 

  36. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhang, J.-Y. et al. RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region. RNA 20, 568–579 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hein, S., Scholz, I., Voß, B. & Hess, W. R. Adaptation and modification of three CRISPR loci in two closely related cyanobacteria. RNA Biol. 10, 852–864 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Loedige, I. et al. The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation. Genes. Dev. 28, 749–764 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Schultz, A., Nottrott, S., Watkins, N. J. & Lührmann, R. Protein–protein and protein–RNA contacts both contribute to the 15.5K-mediated assembly of the U4/U6 snRNP and the box C/D snoRNPs. Mol. Cell. Biol. 26, 5146–5154 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Tamulaitis, G., Venclovas, Č. & Siksnys, V. Type III CRISPR–Cas immunity: major differences brushed aside. Trends Microbiol. 25, 49–61 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Kazlauskiene, M., Tamulaitis, G., Kostiuk, G., Venclovas, Č. & 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kopf, M. et al. Comparative genome analysis of the closely related Synechocystis strains PCC 6714 and PCC 6803. DNA Res. 21, 255–266 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Karvelis, T. et al. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 10, 841–851 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Li, H. Structural principles of CRISPR RNA processing. Structure 23, 13–20 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Sesto, N. et al. A PNPase dependent CRISPR system in Listeria. PLoS. Genet. 10, e1004065 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Walker, F. C., Chou-Zheng, L., Dunkle, J. A. & Hatoum-Aslan, A. Molecular determinants for CRISPR RNA maturation in the Cas10–Csm complex and roles for non-Cas nucleases. Nucleic Acids Res. 45, 2112–2123 (2016).

    PubMed Central  Google Scholar 

  51. Stazic, D., Lindell, D. & Steglich, C. Antisense RNA protects mRNA from RNase E degradation by RNA–RNA duplex formation during phage infection. Nucleic Acids Res. 39, 4890–4899 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Sakurai, I. et al. Positive regulation of psbA gene expression by cis-encoded antisense RNAs in Synechocystis sp. PCC 6803. Plant Physiol. 160, 1000–1010 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kuchmina, E., Wallner, T., Kryazhov, S., Zinchenko, V. V. & Wilde, A. An expression system for regulated protein production in Synechocystis sp. PCC 6803 and its application for construction of a conditional knockout of the ferrochelatase enzyme. J. Biotechnol. 162, 75–80 (2012).

    Article  CAS  PubMed  Google Scholar 

  54. Yao, L., Cengic, I., Anfelt, J. & Hudson, E. P. Multiple gene repression in Cyanobacteria using CRISPRi. ACS Synth. Biol. 5, 207–212 (2016).

    Article  CAS  PubMed  Google Scholar 

  55. Blin, K., Pedersen, L. E., Weber, T. & Lee, S. Y. CRISPy-web: an online resource to design sgRNAs for CRISPR applications. Synth. Syst. Biotechnol. 1, 118–121 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Larson, M. H. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Hofacker, I. L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Darty, K., Denise, A. & Ponty, Y. VARNA: interactive drawing and editing of the RNA secondary structure. Bioinformatics 25, 1974–1975 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank O. Alkhnbashi and C. Steglich for helpful comments, U. Ruppert for technical assistance, M. Asayama and E. P. Hudson for the provided plasmids. Financial support from the German Research Foundation programme FOR1680 'Unravelling the Prokaryotic Immune System' (grants HE 2544/8-2 and UR225/1-2) to W.R.H. and H.U. is greatly acknowledged.

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Authors and Affiliations

  1. Genetics and Experimental Bioinformatics, Faculty of Biology, University of Freiburg, Freiburg, Germany

    Juliane Behler, Viktoria Reimann & Wolfgang R. Hess

  2. Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Centre, Göttingen, Germany

    Kundan Sharma & Henning Urlaub

  3. Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Kundan Sharma & Henning Urlaub

  4. Molecular Genetics, Faculty of Biology, University of Freiburg, Freiburg, Germany

    Annegret Wilde

  5. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Annegret Wilde

  6. Freiburg Institute for Advanced Studies, University of Freiburg, Freiburg, Germany

    Wolfgang R. Hess

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Contributions

J.B. and W.R.H. designed the work. The protein–RNA cross-linking experiments and identification of cross-linked peptide–RNA bonds were performed by K.S. and H.U. The RNase E overexpression and partial deletion strains were constructed by A.W. The ∆C1 and ∆C1∆C2 deletion strains were constructed by V.R. All other experiments were carried out by J.B. J.B., K.S. and W.R.H. analysed the data. J.B. and W.R.H. wrote the paper with contributions from all authors.

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Correspondence to Wolfgang R. Hess.

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Supplementary Figures 1–6, Supplementary Table 1, raw full length gels and blots.

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Behler, J., Sharma, K., Reimann, V. et al. The host-encoded RNase E endonuclease as the crRNA maturation enzyme in a CRISPR–Cas subtype III-Bv system. Nat Microbiol 3, 367–377 (2018). https://doi.org/10.1038/s41564-017-0103-5

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