Discovery of Oligonucleotide Signaling Mediated by CRISPR

Sep 22, 2017 - and nucleotide cyclases and an HD nuclease domain. However, until very recently, the activity of the ... cyclases, and in most cases, a...
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Discovery of Oligonucleotide Signaling Mediated by CRISPRAssociated Polymerases Solves Two Puzzles but Leaves an Enigma Eugene V. Koonin* and Kira S. Makarova National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland 20894, United States ABSTRACT: The signature component of type III CRISPRCas systems is the Cas10 protein that consists of two Palm domains homologous to those of DNA and RNA polymerases and nucleotide cyclases and an HD nuclease domain. However, until very recently, the activity of the Palm domains and their role in CRISPR function have not been experimentally established. Most of the type III CRISPR-Cas systems and some type I systems also encompass proteins containing the CARF (CRISPR-associated Rossmann fold) domain that has been predicted to regulate CRISPR functions via nucleotide binding, but its function in CRISPR-Cas remained obscure. Two independent recent studies show that the Palm domain of Cas10 catalyzes synthesis of oligoadenylates, which bind the CARF domain of the Csm6 protein and activate its RNase domain that cleaves foreign transcripts enabling interference by type III CRISPR-Cas. In one coup, these findings resolved two long-standing puzzles of CRISPR biology and reveal a new regulatory pathway that governs the CRISPR response. However, the full extent of this pathway, and especially the driving forces behind the evolution of this complex mechanism of CRISPR-Cas activation, remains to be uncovered.

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that, despite the undeniable polymerase homology, Cas10 functioned solely as a nuclease.12−14 Another early prediction of Cas protein functions that has been elaborated in a subsequent comprehensive analysis is the regulation of CRISPR function by nucleotides via the CARF (CRISPR-associated Rossmann fold) domain that is present in a variety of type III and type I CRISPR-Cas systems as a fusion with various effector domains including several different nucleases and/or regulatory domains such as helix−turn− helix (HTH).6,15 Given that Rossmann fold domains typically bind various nucleotides, it has been proposed that CARF domain proteins regulate the function of CRISPR-Cas systems by sensing nucleotide-derived signaling molecules.15 Several structures of CARF domain proteins have been solved, supporting the Rossmann fold identification,16−18 but the prediction of the signal transduction role of these proteins had remained untested for years. Remarkably, a more specific scheme, in which the predicted CRISPR-associated polymerases and CARF domain proteins could be functionally linked, with the former providing ligands for the latter, has been proposed in the course of a comprehensive analysis of nucleotide signaling involvement in microbial defense functions.19

RISPR-Cas are the adaptive immunity systems of prokaryotes that consist of repeat arrays with unique spacers and the multiple Cas proteins that are encoded in the vicinity of the arrays and form adaptation and effector complexes which mediate the different stages of the immune response.1−3 The molecular functions of the common Cas proteins have been predicted by in depth computational sequence analysis even before CRISPR-Cas was demonstrated to function as an adaptive immunity system.4−6 One of the prominent early predictions was a “CRISPR polymerase,” a large protein that contains a domain homologous to the Palm domain of family B DNA polymerases, reverse transcriptases, viral RNA-dependent RNA polymerases, and nucleotide cyclases, and in most cases, also an HD family nuclease domain. The “CRISPR polymerase,” later denoted Cas10, is the hallmark of type III CRISPR-Cas systems, in which it functions as the large subunit of the effector complex.7,8 All the characteristic catalytic amino acids of polymerases and cyclases are conserved in the Palm domain of Cas10, which led to the prediction that this protein is indeed an active polymerase or cyclase with a role in the CRISPR function.4,6,9 The crystal structure of Cas10 confirmed the presence of the Palm domain and also revealed a second, inactivated copy of this domain that is too diverged to be recognized from sequences alone.10,11 However, the function of Cas10 remained a vexing problem for 15 years after the initial prediction. Experiments have failed to demonstrate apolymerase or cyclase activity but instead have shown that the catalytic residues of the Palm domain were required for target cleavage, leading to the uneasy conclusion © XXXX American Chemical Society

Special Issue: Chemical Biology of CRISPR Received: August 16, 2017 Accepted: September 22, 2017 Published: September 22, 2017 A

DOI: 10.1021/acschembio.7b00713 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. COA-mediated signal transduction pathway and evolution of CRISPR-Cas systems. (A) General organization of second messenger signaling pathways. Proteins are shown by colored shapes according to the four main roles in the pathway. (B) The most common (predicted) components of the cOA-mediated signal transduction pathway in type III CRISPR-Cas systems. Functionally uncharacterized components are shown by shades of gray and denoted by Pfam identifiers or by locus-tag of a representative protein. Genes’ names shown in parentheses follow the current nomenclature of cas genes.8 (C) A hypothetical scenario for the origin and evolution of class 1 CRISPR-Cas systems from a cOA-producing signaling, stress-response module.

protein and allosterically stimulate the RNase activity of the second, HEPN domain of Csm6, which cleaves the RNA target. In type III-A and III-B CRISPR-Cas systems, cleavage of virus transcripts by Csm6 is an essential step of the CRISPR response along with the DNA cleavage catalyzed by the HD nuclease domain of Cas10.22−24 Site-directed mutagenesis of the predicted polymerase catalytic site of Cas10, the nucleotidebinding loop of the CARF domain, and the active site of the HEPN domain of Csm6 all abolished the target RNA cleavage, supporting the existence of a distinct pathway of CRISPR-Cas

In a stunning development, the two CRISPR puzzles, those of the functions of the putative Cas10 polymerases and the CARF domains, have been solved in two independent, compelling experimental studies.20,21 For type III CRISPRCas systems from the bacteria Streptococcus thermophilus, Staphylococcus epidermidis, and Enterococcus italicus and the archaeon Methanothermobacter thermoautotrophicus, it has been shown that binding of the cognate target RNA by the CRISPR RNA-containing effector complex stimulates the synthesis of cyclic oligoadenylates (cOAn, n = 2−6) by Cas10. The produced cOA are bound by the CARF domain of the Csm6 B

DOI: 10.1021/acschembio.7b00713 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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VI.28,29 In the absence of an obvious functional explanation for the presence of the cOA regulatory pathway in type III systems, a historical, evolutionary one can be envisaged. The origin of the effector complex of CRISPR-Cas systems remains unclear.30 The discovery of the cOA pathway suggests the possibility that Cas10 and Csm6 originally were components of a stand-alone signaling and/or toxin−antitoxin system that was activated by cOA, which functioned as a second messenger, or alarmone.31 A putative minimal signaling system that consists of a Cas10 homologue (a much smaller protein than CRISPR-associated Cas10 that effectively comprises the palm domain alone) has been described, although the effector protein or the primary signal sensed by this system has not been identified.19 A cOAproducing stress response module could have been the initial stage of evolution of CRISPR-Cas effector modules (Figure 1C). This simple ancestral state could have evolved into the CRISPR-Cas system via recombination with a casposon,32,33 which contributed the Cas1-based adaptation module and the CRISPR array, possibly along with additional nucleases (Figure 1C). In the course of the subsequent evolution, the cyclase domain of Cas10 was inactivated, and by inference, the cOA pathway was lost in numerous type III variants.8 In a more dramatic evolutionary transition, inactivation of Cas10 followed by drastic sequence divergence could have given rise to Cas8, the large subunit of the Cascade-like effector complexes of type I systems.30,34 Thus, under this scenario, the cOA-producing stress response module was at the root of the evolution of all class 1 CRISPR effector complexes (Figure 1C). Further study of the diversity of prokaryotic signaling and toxin−antitoxin systems as well as the functions of diverse CARF and WYL domain proteins in CRISPR-Cas can be expected to elucidate the connection between the diverse networks of stress response and antiparasite defense in microbes and perhaps reveal the intermediate stage in the proposed scenario of CRISPR-Cas evolution.

activation upon infection (henceforth, the cOA pathway, for brevity).20,21 Three key components of the cOA pathway have been discovered, namely, the second messenger synthetase (Cas10), the sensor domain (CARF), and the effector domain (HEPN) (Figure 1A). Most signaling systems also contain a fourth component, a dedicated enzyme that cleaves the messenger molecule and halts the effector activation (Figure 1A). Notably, cyclic dimeric guanosine monophosphate (c-di-GMP) synthases, known as GDDEF domain cyclases, are homologues of Cas10.4,9 The GDDEF cyclases function in conjunction with several distinct c-di-GMP hydrolases, one of which belongs to the HD superfamily.25,26 Most of the Cas10 proteins contain an HD family nuclease domain that is involved in the target DNA cleavage.14,27 The possibility remains that the HD domain of Cas10 has a dual activity and also cleaves cOA; alternatively, this function might be performed by other, less broadly conserved proteins that comprise the ancillary Cas protein subset8 or by a hydrolase that is not directly associated with CRISPR-Cas systems. The combination of Cas10 with proteins combining CARF and HEPN domains is common to most type III-A and type IIIB CRISPR-Cas systems. However, some Cas10-containing type III loci lack any CARF domain proteins, and many CRISPRCas loci encompass CARF domain proteins but not Cas10; most of the CARF domain proteins that are not associated with Cas10 lack effector domains. Furthermore, the CARF domain proteins display a variety of different architectures.15 Although the CARF-HEPN combination is the most common one, other CARF domain proteins contain distinct nucleases, such as PIN or RelE domains with predicted RNase activity and restriction endonuclease (also known as PD-DExK) family DNases (Figure 1B). Many CARF domain proteins lack any nucleases and contain only a helix−turn−helix domain, suggestive of regulatory rather than effector functions (Figure 1B). The predicted CARF-domain DNases are represented in many subtype III-A and III-B loci, where their activity is most likely stimulated by cOA synthesized by Cas10, whereas the predicted CARF domain-containing transcription regulators are typical of subtype I-A.15 This diversity of CARF domain protein architectures and genomic contexts implies that the cOA pathway is part of a larger network of (oligo)nucleotidemediated regulation of CRISPR-Cas (Figure 1B). Most likely, all CARF domains transmit a signal through a conformational change caused by (oligo)nucleotide-binding, but in CRISPRCas variants that lack Cas10, the ligands evidently come from other sources that remain to be identified. In addition to the CARF domains, some CRISPR-Cas loci encode proteins containing another predicted ligand-binding domain known as WYL (after tryptophan, tyrosine, and leucine that are conserved in a subset of these domains).15 The WYL domains are likely to be key components of yet another network regulating CRISPR-Cas activity. The discovery of the cOA pathway has solved, in a single, clean sweep, two major puzzles of CRISPR-Cas biology, namely, the functions of the polymerase-cyclase domain of Cas10 and the CARF domains. However, a bigger biological enigma remains: why this regulatory circuit? Apart from type III, CRISPR-Cas systems appear to attack their targets directly, without the intermediate regulatory step.1,2 In other words, target recognition itself is sufficient to activate the effector nucleases. In most cases, the target is DNA, but direct, unregulated RNA cleavage has been demonstrated in type



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Eugene V. Koonin: 0000-0003-3943-8299 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS K.S.M. and E.V.K. are supported by intramural funds of the U.S. Department of Health and Human Services (to the National Library of Medicine).



ABBREVIATIONS OA, oligoadenylates; CARF, CRISPR-associated Rossmann fold; WYL, predicted ligand-binding domain associated with many CRISPR-Cas systems (named after the respective amino acids that are partly conserved in the family); HEPN, PIN, RelE, ribonucleases of the respective families; HTH, helix− turn−helix DNA-binding domain; HD, PD-DExK, nuclease (or phosphatases) of the respective superfamilies



REFERENCES

(1) Marraffini, L. A. (2015) CRISPR-Cas immunity in prokaryotes. Nature 526, 55−61.

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DOI: 10.1021/acschembio.7b00713 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology (2) Mohanraju, P., Makarova, K. S., Zetsche, B., Zhang, F., Koonin, E. V., and van der Oost, J. (2016) Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 353, aad5147. (3) Barrangou, R., and Horvath, P. (2017) A decade of discovery: CRISPR functions and applications. Nat. Microbiol 2, 17092. (4) Makarova, K. S., Aravind, L., Grishin, N. V., Rogozin, I. B., and Koonin, E. V. (2002) A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res. 30, 482−496. (5) Haft, D. H., Selengut, J., Mongodin, E. F., and Nelson, K. E. (2005) A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, e60. (6) Makarova, K. S., Grishin, N. V., Shabalina, S. A., Wolf, Y. I., and Koonin, E. V. (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 1, 7. (7) Rouillon, C., Zhou, M., Zhang, J., Politis, A., Beilsten-Edmands, V., Cannone, G., Graham, S., Robinson, C. V., Spagnolo, L., and White, M. F. (2013) Structure of the CRISPR interference complex CSM reveals key similarities with cascade. Mol. Cell 52, 124−134. (8) Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., van der Oost, J., Backofen, R., and Koonin, E. V. (2015) An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722− 736. (9) Pei, J., and Grishin, N. V. (2001) GGDEF domain is homologous to adenylyl cyclase. Proteins: Struct., Funct., Genet. 42, 210−216. (10) Zhu, X., and Ye, K. (2012) Crystal structure of Cmr2 suggests a nucleotide cyclase-related enzyme in type III CRISPR-Cas systems. FEBS Lett. 586, 939−945. (11) Jung, T. Y., An, Y., Park, K. H., Lee, M. H., Oh, B. H., and Woo, E. (2015) Crystal structure of the Csm1 subunit of the Csm complex and its single-stranded DNA-specific nuclease activity. Structure 23, 782−790. (12) Ramia, N. F., Tang, L., Cocozaki, A. I., and Li, H. (2014) Staphylococcus epidermidis Csm1 is a 3′-5′ exonuclease. Nucleic Acids Res. 42, 1129−1138. (13) Elmore, J. R., Sheppard, N. F., Ramia, N., Deighan, T., Li, H., Terns, R. M., and Terns, M. P. (2016) Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system. Genes Dev. 30, 447−459. (14) Zhang, J., Graham, S., Tello, A., Liu, H., and White, M. F. (2016) Multiple nucleic acid cleavage modes in divergent type III CRISPR systems. Nucleic Acids Res. 44, 1789−1799. (15) Makarova, K. S., Anantharaman, V., Grishin, N. V., Koonin, E. V., and Aravind, L. (2014) CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems. Front. Genet. 5, 102. (16) Lintner, N. G., Frankel, K. A., Tsutakawa, S. E., Alsbury, D. L., Copie, V., Young, M. J., Tainer, J. A., and Lawrence, C. M. (2011) The structure of the CRISPR-associated protein Csa3 provides insight into the regulation of the CRISPR/Cas system. J. Mol. Biol. 405, 939−955. (17) Kim, Y. K., Kim, Y. G., and Oh, B. H. (2013) Crystal structure and nucleic acid-binding activity of the CRISPR-associated protein Csx1 of Pyrococcus furiosus. Proteins: Struct., Funct., Genet. 81, 261− 270. (18) Niewoehner, O., and Jinek, M. (2016) Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6. RNA 22, 318−329. (19) Burroughs, A. M., Zhang, D., Schaffer, D. E., Iyer, L. M., and Aravind, L. (2015) Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling. Nucleic Acids Res. 43, 10633−10654.

(20) Kazlauskiene, M., Kostiuk, G., Venclovas, C., Tamulaitis, G., and Siksnys, V. (2017) A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605−609. (21) Niewoehner, O., Garcia-Doval, C., Rostol, J. T., Berk, C., Schwede, F., Bigler, L., Hall, J., Marraffini, L. A., and Jinek, M. (2017) Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543. (22) Samai, P., Pyenson, N., Jiang, W., Goldberg, G. W., HatoumAslan, A., and Marraffini, L. A. (2015) Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity. Cell 161, 1164−1174. (23) Jiang, W., Samai, P., and Marraffini, L. A. (2016) Degradation of Phage Transcripts by CRISPR-Associated RNases Enables Type III CRISPR-Cas Immunity. Cell 164, 710−721. (24) Tamulaitis, G., Venclovas, C., and Siksnys, V. (2017) Type III CRISPR-Cas Immunity: Major Differences Brushed Aside. Trends Microbiol. 25, 49−61. (25) Galperin, M. Y., Nikolskaya, A. N., and Koonin, E. V. (2001) Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol. Lett. 203, 11−21. (26) Hengge, R. (2016) Trigger phosphodiesterases as a novel class of c-di-GMP effector proteins. Philos. Trans. R. Soc., B 371, 20150498. (27) Kazlauskiene, M., Tamulaitis, G., Kostiuk, G., Venclovas, C., and Siksnys, V. (2016) Spatiotemporal Control of Type III-A CRISPR-Cas Immunity: Coupling DNA Degradation with the Target RNA Recognition. Mol. Cell 62, 295−306. (28) Abudayyeh, O. O., Gootenberg, J. S., Konermann, S., Joung, J., Slaymaker, I. M., Cox, D. B., Shmakov, S., Makarova, K. S., Semenova, E., Minakhin, L., Severinov, K., Regev, A., Lander, E. S., Koonin, E. V., and Zhang, F. (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573. (29) Smargon, A. A., Cox, D. B., Pyzocha, N. K., Zheng, K., Slaymaker, I. M., Gootenberg, J. S., Abudayyeh, O. A., Essletzbichler, P., Shmakov, S., Makarova, K. S., Koonin, E. V., and Zhang, F. (2017) Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28. Mol. Cell 65, 618. (30) Makarova, K. S., Wolf, Y. I., and Koonin, E. V. (2013) The basic building blocks and evolution of CRISPR-cas systems. Biochem. Soc. Trans. 41, 1392−1400. (31) Pesavento, C., and Hengge, R. (2009) Bacterial nucleotidebased second messengers. Curr. Opin. Microbiol. 12, 170−176. (32) Krupovic, M., Beguin, P., and Koonin, E. V. (2017) Casposons: the mobile elements that gave rise to the adaptation module of CRISPR-Cas systems. Curr. Opin. Microbiol. 38, 36. (33) Krupovic, M., Makarova, K. S., Forterre, P., Prangishvili, D., and Koonin, E. V. (2014) Casposons: a new superfamily of selfsynthesizing DNA transposons at the origin of prokaryotic CRISPRCas immunity. BMC Biol. 12, 36. (34) Makarova, K. S., Aravind, L., Wolf, Y. I., and Koonin, E. V. (2011) Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol. Direct 6, 38.

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DOI: 10.1021/acschembio.7b00713 ACS Chem. Biol. XXXX, XXX, XXX−XXX