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Biochemical Society Transactions (2013) Volume 41, part 6
The Cmr complex: an RNA-guided endoribonuclease Scott Bailey*1 *Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, U.S.A.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system protects prokaryotes from infection by viruses and other potential genome invaders. This system represents an inheritable and adaptable immune system that is mediated by large ribonucleoprotein complexes, the CRISPR–Cas effector complexes. The Cmr complex is unique among CRISPR–Cas effector complexes in that it destroys invading RNA and not DNA. To date, the Cmr complexes from two species have been characterized in vitro and, strikingly, they degrade RNA via distinct mechanisms. The possible in vivo targets, as well as our current knowledge of the Cmr complex, is reviewed in the present paper.
Introduction Immune pathways protect all organisms from infection by mobile genetic elements such as viruses. It was recently discovered that prokaryotes control such infection via an RNA-based adaptive immune system, called the CRISPR (clustered regularly interspaced short palindromic repeats)– Cas (CRISPR-associated) system [1]. This system incorporates short fragments of the invading DNA between the repeat sequences of CRISPRs. The resulting CRISPR transcripts are processed into individual crRNAs (CRISPR RNAs) that then guide Cas complexes to destroy the nucleic acids of the invader. CRISPR–Cas systems have been classified into three types, each type characterized by the presence of a signature gene [2]. The Type III systems, which frequently occur in archaea [2,3], are characterized by the presence of the cas10 gene. Cas10 is a large multidomain protein containing an N-terminal HD (histidine–aspartate) domain followed by a zinc finger and a C-terminal domain with sequence similarity to the palm domain of polymerases and cyclases [4]. This system is further divided into subtype III-A and subtype IIIB modules [2]. Subtype III-A modules contain five additional proteins (Csm2–Csm6) and target plasmid DNA in vivo [5]. Subtype III-B modules (the topic of the present mini-review) contain five or six additional proteins, Cmr1 and Cmr3– Cmr7. Four of the Cmr proteins (Cmr1, Cmr3, Cmr4 and Cmr6) are RNA-binding proteins belonging to the RAMP (repeat-associated mysterious proteins) family [6]. Cmr5, the smallest subunit of the complex, and Cmr7, a dimeric protein only found in Sulfolobales [7], have no known or predicted function. In contrast with all other characterized CRISPR– Cas systems, which target DNA, the subtype III-B system targets RNA both in vitro [7,8] and in vivo [9].
Key words: clustered regularly interspaced short palindromic repeats (CRISPR), Cmr, repeatassociated mysterious protein (RAMP), RNA interference, metal ion, nuclease. Abbreviations used: Cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR RNA; RAMP, repeat-associated mysterious protein. 1 email [email protected].
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Identification and activity of the Cmr complex The Cmr complex was first identified in Pyrococcus furiosus by fractionation of a cellular extract, guided by Northern blot analysis of an individual crRNA [8]. MS revealed the complex to contain all six Cmr proteins of the subtype III-B module in P. furiosus. The stoichiometry of the subunits in the P. furiosus complex is currently unknown. The only other Cmr complex to have been characterized was purified from Sulfolobus solfataricus extracts on the basis of Western blots using antibodies raised against Cmr7 [7]. Gel analysis showed that the complex contained all seven Cmr proteins of the S. solfataricus subtype III-B module. Densitometry of the gel suggested that the complex contained one copy of subunits Cmr1–Cmr6 and six copies of Cmr7 [7]. This predicts that the size of the S. solfataricus complex, including the crRNA, is ∼430 kDa, consistent with the elution volume of the complex from a size-exclusion column. In the subtype III-B system, mature crRNAs (also previously referred to as prokaryotic silencing RNAs or psiRNAs) are generated from CRISPR transcripts in two distinct steps: primary processing and maturation. During primary processing the precursor transcripts are cleaved by the Cas6 endoribonuclease within the repeat region to yield crRNA intermediates [10]. During maturation, the 3 end of these intermediates is degraded to yield mature crRNAs [10]. The nuclease responsible for this degradation is not yet known. Once processing is complete, mature crRNAs consist of a unique guide sequence, typically 30–40 nt, flanked by 8 nt of repeat sequence on the 5 end (the 5 tag). A role for the Cmr proteins in crRNA processing has not been reported. However, genetic studies suggest that several Csm proteins bind and therefore protect crRNA against degradation or overprocessing [11]. As the Csm proteins share some sequence similarity with the Cmr proteins [12], it is likely that the Cmr proteins might function similarly during processing. In vitro, the Cmr complexes from P. furiosus and S. solfactaricus specifically cleave ssRNA that is complementary Biochem. Soc. Trans. (2013) 41, 1464–1467; doi:10.1042/BST20130216
CRISPR: Evolution, Mechanisms and Infection
Figure 1 RNA cleavage mechanisms of the Cmr complexes Cartoons explaining how the Cmr complexes from (a) P. furiosus and (b) S. solfataricus cleave RNA targets.
to the crRNA in the complex [7,8]. dsRNA and dsDNA or ssDNA are not cleaved. Cleavage is metal-ion-dependent, requiring Mn2 + ions. Additionally, CMR from P. furiosus, but not S. solfataricus, can use Mg2 + ions. An intact 5 tag on the crRNA is also required. Despite these similarities in activity, there are striking differences in the mechanism of cleavage by the two complexes [7,8]. First, the P. furiosus Cmr complex cleaves the RNA target, in a sequence-independent manner, via a ruler machanism (Figure 1). The S. solfataricus Cmr complex, however, utilizes a sequence-dependent mechanism to cleave the RNA target at ‘UA’ sites (Figure 1). Secondly, whereas cleavage by P. furiosus Cmr complex generates 3 - (or 2 ,3 -cyclic) phosphate and 5 -OH, cleavage by S. solfataricus Cmr complex generates 3 -OH and 5 -phosphate. This different chemical nature of the cleavage products suggests that the two complexes employ different cleavage chemistry. Does the Cmr complex cleave complementary RNA in vivo? Although of unknown function, antisense RNA derived from CRISPR loci has been found in P. furiosus [9] and multiple Sulfolobales [13–15]. Deep sequencing analysis of P. furious RNA reveals that these antisense RNAs are cleared at a position consistent with the activity of the Cmr complex. This suggests that the Cmr complex does cleave endogenous complementary RNAs in vivo [9]. What other targets could the Cmr complex cleave in vivo? Although still a major question in the field, existing evidence suggests several possibilities. The complex could target mRNA, thus functioning in gene regulation in a manner analogous to eukaryotic RNAi mechanisms. Although appealing, there is as yet no direct experimental evidence in support of this theory. Cmr complexes could also target the genomes of RNA viruses. A putative RNA virus that infects archaea has recently been reported, and several sequences complementary to this viral genome have been identified in CRISPR loci from Sulfolobus [16]. If the subtype III-B system, as found in Sulfolobus, protects against infection by RNA viruses, then there must be a mechanism that incorporates DNA copies of fragments from the RNA viral genome into the CRISPR loci of the host DNA genome. To date, such a mechanism has not been
reported. Cmr complexes could also silence transcripts of the invading DNA bacteriophage, strengthening the immune response. This hypothesis is supported by the observation that subtype III-B modules are only, with few exceptions, found in genomes that contain other CRISPR–Cas modules, suggesting either an auxiliary role or a dependence on the other systems for function. Finally, a Sulfolobus islandicus Cmr complex has been putatively implicated in transcriptiondependent DNA targeting [17]. Targeting requires the Csx1 gene, which is often linked with subtype III-B modules, and transcripts that are complementary to the crRNA in the Cmr complex. Further functional studies will be needed to understand the mechanism and significance of this DNA targeting.
Structural analysis of the Cmr complex Currently there is no crystal structure of an entire Cmr complex, but significant progress is being made on the structures of the individual subunits (reviewed in [18]). Crystal structures of four of the seven subunits found in Cmr complexes have been determined: Cas10dHD (PDB codes 3UNG and 4DOZ; a truncated Cas10 subunit lacking the N-terminal HD domain) [19–21], Cmr3 (PDB code 4H4K; in the context of a complex with Cas10dHD ) [21], Cmr5 (PDB code 2ZOP) [22] and Cmr7 (PDB codes 2XVO and 2X5Q) [7]. In addition, a low-resolution EM structure of the S. solfataricus Cmr complex has also been determined [7] revealing a clamp-like structure containing a deep cleft that could accommodate dsRNA. The crystal structure of Cas10dHD revealed that it consists of two adenylate cyclase domains (denoted D1 and D3) and two α-helical domains (denoted D2 and D4). The significance of the similarity between Cas10 and adenylate cyclase is currently unclear. The D2 domain remotely resembles the thumb domain of A-family DNA polymerases [20]. In polymerases, thumb domains bind the primer–template duplex [23]. Although the role of D2 is unknown, this similarity may suggest an interaction with the crRNA–RNA target duplex. D4 is structurally homologous with Cmr5 [20] C The
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and the N-terminal domain of Cse1, the small subunit of the effector complex from the subtype I-E CRISPR–Cas system [18]. The biological implications of this similarity are unclear, but it could indicate an interaction between D4 and Cmr5 in Cmr complexes, and a similar function for Cmr5 and Cse1 in their respective complexes. Cmr3 has significant structural similarity to Cas6. Both proteins contain two RAMP domains, which form ferredoxin-like folds similar to the RRM (RNA-recognition motif). Predictions from sequence analysis suggest that the other RAMP subunits of the Cmr complex (Cmr1, Cmr4 and Cmr6) will have structural similarity to Cmr3 [12]. The function of all the RAMP subunits probably involves interactions with the crRNA and/or the RNA target. The crystal structure of a Cmr complex is a high priority for the structural biology community. However, even in the absence of this, it is reasonable to assume that fitting crystal structures into higher-resolution EM maps will produce pseudo-atomic models of the complex in the near future. Such models will provide a valuable framework that will drive biochemical studies dissecting the mechanism of the Cmr complex and the role of its individual subunits.
The catalytic subunit of the Cmr complex The catalytic subunit of the Cmr complex is currently unknown. Initial predictions suggested that Cas10 would be the catalytic subunit since it contained an HD domain that in the Type I system is the active nuclease [24–26]. However, a Cmr complex formed with Cas10dHD cleaved RNA target as efficiently as the wild-type complex [19]. Mutation of the active site of the cyclase domain of Cas10 also failed to inhibit the nuclease activity of the complex [19]. Thus it appears unlikely that Cas10 is the catalytic subunit. Cmr5 can also be ruled out, as it is not required for activity [8], as can the Sulfolobales-specific subunit Cmr7 [7]. This leaves the four RAMP subunits, Cmr1, Cmr3, Cmr4 and Cmr6, as potential catalytic subunits. Of these, Cmr4 is perhaps the top candidate as sequence analysis reveals a conserved histidine residue that is in an equivalent position to the catalytic histidine residue of the known RAMP nuclease, Cas6 [12]. Although Cmr6 also contains a conserved histidine residue, its location is not consistent with the catalytic histidine residue of Cas6 [12]. Based on analysis of both sequence and structure, Cmr3 is unlikely to have nuclease activity [12,21], and sequence analysis also does not predict nuclease activity for Cmr1. It should also be noted that the identification of the catalytic subunit could be complicated by the nuclease active site being formed by more than one subunit and that given the difference in cleavage mechanism between the P. furiosus and S. solfataricus Cmr complexes, different subunits may be catalytic in different Cmr complexes.
Acknowledgements I thank Jennifer M. Kavran for a careful reading of the paper prior to submission.
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References 1 Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A. and Horvath, P. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 2 Makarova, K.S., Haft, D.H., Barrangou, R., Brouns, S.J. J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F.J.M., Wolf, Y.I., Yakunin, A.F. et al. (2011) Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467–477 3 Garrett, R.A., Shah, S.A., Vestergaard, G., Deng, L., Gudbergsdottir, S., Kenchappa, C.S., Erdmann, S. and She, Q. (2011) CRISPR-based immune systems of the Sulfolobales: complexity and diversity. Biochem. Soc. Trans. 39, 51–57 4 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 5 Marraffini, L.A. and Sontheimer, E.J. (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 6 Wang, R. and Li, H. (2012) The mysterious RAMP proteins and their roles in small RNA-based immunity. Protein Sci. 21, 463–470 7 Zhang, J., Rouillon, C., Kerou, M., Reeks, J., Brugger, K., Graham, S., Reimann, J., Cannone, G., Liu, H., Albers, S.-V. et al. (2012) Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol. Cell 45, 303–313 8 Hale, C.R., Zhao, P., Olson, S., Duff, M.O., Graveley, B.R., Wells, L., Terns, R.M. and Terns, M.P. (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 9 Hale, C.R., Majumdar, S., Elmore, J., Pfister, N., Compton, M., Olson, S., Resch, A.M., Glover, C.V.C., Graveley, B.R., Terns, R.M. et al. (2012) Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol. Cell 45, 292–302 10 Hale, C., Kleppe, K., Terns, R.M. and Terns, M.P. (2008) Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14, 2572–2579 11 Hatoum-Aslan, A., Maniv, I. and Marraffini, L.A. (2011) Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc. Natl. Acad. Sci. U.S.A. 108, 21218–21222 12 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 13 Deng, L., Kenchappa, C.S., Peng, X., She, Q. and Garrett, R.A. (2012) Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus. Nucleic Acids Res. 40, 2470–2480 14 Lillestøl, R.K., Shah, S.A., Brugger, K., Redder, P., Phan, H., Christiansen, J. and Garrett, R.A. (2009) CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol. Microbiol. 72, 259–272 15 Lillestøl, R.K., Redder, P., Garrett, R.A. and Brugger, K. (2006) A putative viral defence mechanism in archaeal cells. Archaea 2, 59–72 16 Bolduc, B., Shaughnessy, D.P., Wolf, Y.I., Koonin, E.V., Roberto, F.F. and Young, M. (2012) Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone hot springs. J. Virol. 86, 5562–5573 17 Deng, L., Garrett, R.A., Shah, S.A., Peng, X. and She, Q. (2013) A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus. Mol. Microbiol. 87, 1088–1099 18 Reeks, J., Naismith, J.H. and White, M.F. (2013) CRISPR interference: a structural perspective. Biochem. J. 453, 155–166 19 Cocozaki, A.I., Ramia, N.F., Shao, Y., Hale, C.R., Terns, R.M., Terns, M.P. and Li, H. (2012) Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex. Structure 20, 545–553 20 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 21 Shao, Y., Cocozaki, A.I., Ramia, N.F., Terns, R.M., Terns, M.P. and Li, H. (2013) Structure of the Cmr2-Cmr3 subcomplex of the Cmr RNA silencing complex. Structure 21, 376–384 22 Sakamoto, K., Agari, Y., Agari, K., Yokoyama, S., Kuramitsu, S. and Shinkai, A. (2009) X-ray crystal structure of a CRISPR-associated RAMP superfamily protein, Cmr5, from Thermus thermophilus HB8. Proteins 75, 528–532 23 Steitz, T.A. (1999) DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395–17398
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24 Mulepati, S. and Bailey, S. (2011) Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J. Biol. Chem. 286, 31896–31903 25 Sinkunas, T., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P. and Siksnys, V. (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 30, 1335–1342
26 Beloglazova, N., Petit, P., Flick, R., Brown, G., Savchenko, A. and Yakunin, A.F. (2011) Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J. 30, 4616–4627
Received 5 September 2013 doi:10.1042/BST20130216
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Biochemical Society Transactions (2013) Volume 41, part 6
The Cmr complex: an RNA-guided endoribonuclease Scott Bailey*1 *Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, U.S.A.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system protects prokaryotes from infection by viruses and other potential genome invaders. This system represents an inheritable and adaptable immune system that is mediated by large ribonucleoprotein complexes, the CRISPR–Cas effector complexes. The Cmr complex is unique among CRISPR–Cas effector complexes in that it destroys invading RNA and not DNA. To date, the Cmr complexes from two species have been characterized in vitro and, strikingly, they degrade RNA via distinct mechanisms. The possible in vivo targets, as well as our current knowledge of the Cmr complex, is reviewed in the present paper.
Introduction Immune pathways protect all organisms from infection by mobile genetic elements such as viruses. It was recently discovered that prokaryotes control such infection via an RNA-based adaptive immune system, called the CRISPR (clustered regularly interspaced short palindromic repeats)– Cas (CRISPR-associated) system [1]. This system incorporates short fragments of the invading DNA between the repeat sequences of CRISPRs. The resulting CRISPR transcripts are processed into individual crRNAs (CRISPR RNAs) that then guide Cas complexes to destroy the nucleic acids of the invader. CRISPR–Cas systems have been classified into three types, each type characterized by the presence of a signature gene [2]. The Type III systems, which frequently occur in archaea [2,3], are characterized by the presence of the cas10 gene. Cas10 is a large multidomain protein containing an N-terminal HD (histidine–aspartate) domain followed by a zinc finger and a C-terminal domain with sequence similarity to the palm domain of polymerases and cyclases [4]. This system is further divided into subtype III-A and subtype IIIB modules [2]. Subtype III-A modules contain five additional proteins (Csm2–Csm6) and target plasmid DNA in vivo [5]. Subtype III-B modules (the topic of the present mini-review) contain five or six additional proteins, Cmr1 and Cmr3– Cmr7. Four of the Cmr proteins (Cmr1, Cmr3, Cmr4 and Cmr6) are RNA-binding proteins belonging to the RAMP (repeat-associated mysterious proteins) family [6]. Cmr5, the smallest subunit of the complex, and Cmr7, a dimeric protein only found in Sulfolobales [7], have no known or predicted function. In contrast with all other characterized CRISPR– Cas systems, which target DNA, the subtype III-B system targets RNA both in vitro [7,8] and in vivo [9].
Key words: clustered regularly interspaced short palindromic repeats (CRISPR), Cmr, repeatassociated mysterious protein (RAMP), RNA interference, metal ion, nuclease. Abbreviations used: Cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR RNA; RAMP, repeat-associated mysterious protein. 1 email [email protected].
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Identification and activity of the Cmr complex The Cmr complex was first identified in Pyrococcus furiosus by fractionation of a cellular extract, guided by Northern blot analysis of an individual crRNA [8]. MS revealed the complex to contain all six Cmr proteins of the subtype III-B module in P. furiosus. The stoichiometry of the subunits in the P. furiosus complex is currently unknown. The only other Cmr complex to have been characterized was purified from Sulfolobus solfataricus extracts on the basis of Western blots using antibodies raised against Cmr7 [7]. Gel analysis showed that the complex contained all seven Cmr proteins of the S. solfataricus subtype III-B module. Densitometry of the gel suggested that the complex contained one copy of subunits Cmr1–Cmr6 and six copies of Cmr7 [7]. This predicts that the size of the S. solfataricus complex, including the crRNA, is ∼430 kDa, consistent with the elution volume of the complex from a size-exclusion column. In the subtype III-B system, mature crRNAs (also previously referred to as prokaryotic silencing RNAs or psiRNAs) are generated from CRISPR transcripts in two distinct steps: primary processing and maturation. During primary processing the precursor transcripts are cleaved by the Cas6 endoribonuclease within the repeat region to yield crRNA intermediates [10]. During maturation, the 3 end of these intermediates is degraded to yield mature crRNAs [10]. The nuclease responsible for this degradation is not yet known. Once processing is complete, mature crRNAs consist of a unique guide sequence, typically 30–40 nt, flanked by 8 nt of repeat sequence on the 5 end (the 5 tag). A role for the Cmr proteins in crRNA processing has not been reported. However, genetic studies suggest that several Csm proteins bind and therefore protect crRNA against degradation or overprocessing [11]. As the Csm proteins share some sequence similarity with the Cmr proteins [12], it is likely that the Cmr proteins might function similarly during processing. In vitro, the Cmr complexes from P. furiosus and S. solfactaricus specifically cleave ssRNA that is complementary Biochem. Soc. Trans. (2013) 41, 1464–1467; doi:10.1042/BST20130216
CRISPR: Evolution, Mechanisms and Infection
Figure 1 RNA cleavage mechanisms of the Cmr complexes Cartoons explaining how the Cmr complexes from (a) P. furiosus and (b) S. solfataricus cleave RNA targets.
to the crRNA in the complex [7,8]. dsRNA and dsDNA or ssDNA are not cleaved. Cleavage is metal-ion-dependent, requiring Mn2 + ions. Additionally, CMR from P. furiosus, but not S. solfataricus, can use Mg2 + ions. An intact 5 tag on the crRNA is also required. Despite these similarities in activity, there are striking differences in the mechanism of cleavage by the two complexes [7,8]. First, the P. furiosus Cmr complex cleaves the RNA target, in a sequence-independent manner, via a ruler machanism (Figure 1). The S. solfataricus Cmr complex, however, utilizes a sequence-dependent mechanism to cleave the RNA target at ‘UA’ sites (Figure 1). Secondly, whereas cleavage by P. furiosus Cmr complex generates 3 - (or 2 ,3 -cyclic) phosphate and 5 -OH, cleavage by S. solfataricus Cmr complex generates 3 -OH and 5 -phosphate. This different chemical nature of the cleavage products suggests that the two complexes employ different cleavage chemistry. Does the Cmr complex cleave complementary RNA in vivo? Although of unknown function, antisense RNA derived from CRISPR loci has been found in P. furiosus [9] and multiple Sulfolobales [13–15]. Deep sequencing analysis of P. furious RNA reveals that these antisense RNAs are cleared at a position consistent with the activity of the Cmr complex. This suggests that the Cmr complex does cleave endogenous complementary RNAs in vivo [9]. What other targets could the Cmr complex cleave in vivo? Although still a major question in the field, existing evidence suggests several possibilities. The complex could target mRNA, thus functioning in gene regulation in a manner analogous to eukaryotic RNAi mechanisms. Although appealing, there is as yet no direct experimental evidence in support of this theory. Cmr complexes could also target the genomes of RNA viruses. A putative RNA virus that infects archaea has recently been reported, and several sequences complementary to this viral genome have been identified in CRISPR loci from Sulfolobus [16]. If the subtype III-B system, as found in Sulfolobus, protects against infection by RNA viruses, then there must be a mechanism that incorporates DNA copies of fragments from the RNA viral genome into the CRISPR loci of the host DNA genome. To date, such a mechanism has not been
reported. Cmr complexes could also silence transcripts of the invading DNA bacteriophage, strengthening the immune response. This hypothesis is supported by the observation that subtype III-B modules are only, with few exceptions, found in genomes that contain other CRISPR–Cas modules, suggesting either an auxiliary role or a dependence on the other systems for function. Finally, a Sulfolobus islandicus Cmr complex has been putatively implicated in transcriptiondependent DNA targeting [17]. Targeting requires the Csx1 gene, which is often linked with subtype III-B modules, and transcripts that are complementary to the crRNA in the Cmr complex. Further functional studies will be needed to understand the mechanism and significance of this DNA targeting.
Structural analysis of the Cmr complex Currently there is no crystal structure of an entire Cmr complex, but significant progress is being made on the structures of the individual subunits (reviewed in [18]). Crystal structures of four of the seven subunits found in Cmr complexes have been determined: Cas10dHD (PDB codes 3UNG and 4DOZ; a truncated Cas10 subunit lacking the N-terminal HD domain) [19–21], Cmr3 (PDB code 4H4K; in the context of a complex with Cas10dHD ) [21], Cmr5 (PDB code 2ZOP) [22] and Cmr7 (PDB codes 2XVO and 2X5Q) [7]. In addition, a low-resolution EM structure of the S. solfataricus Cmr complex has also been determined [7] revealing a clamp-like structure containing a deep cleft that could accommodate dsRNA. The crystal structure of Cas10dHD revealed that it consists of two adenylate cyclase domains (denoted D1 and D3) and two α-helical domains (denoted D2 and D4). The significance of the similarity between Cas10 and adenylate cyclase is currently unclear. The D2 domain remotely resembles the thumb domain of A-family DNA polymerases [20]. In polymerases, thumb domains bind the primer–template duplex [23]. Although the role of D2 is unknown, this similarity may suggest an interaction with the crRNA–RNA target duplex. D4 is structurally homologous with Cmr5 [20] C The
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and the N-terminal domain of Cse1, the small subunit of the effector complex from the subtype I-E CRISPR–Cas system [18]. The biological implications of this similarity are unclear, but it could indicate an interaction between D4 and Cmr5 in Cmr complexes, and a similar function for Cmr5 and Cse1 in their respective complexes. Cmr3 has significant structural similarity to Cas6. Both proteins contain two RAMP domains, which form ferredoxin-like folds similar to the RRM (RNA-recognition motif). Predictions from sequence analysis suggest that the other RAMP subunits of the Cmr complex (Cmr1, Cmr4 and Cmr6) will have structural similarity to Cmr3 [12]. The function of all the RAMP subunits probably involves interactions with the crRNA and/or the RNA target. The crystal structure of a Cmr complex is a high priority for the structural biology community. However, even in the absence of this, it is reasonable to assume that fitting crystal structures into higher-resolution EM maps will produce pseudo-atomic models of the complex in the near future. Such models will provide a valuable framework that will drive biochemical studies dissecting the mechanism of the Cmr complex and the role of its individual subunits.
The catalytic subunit of the Cmr complex The catalytic subunit of the Cmr complex is currently unknown. Initial predictions suggested that Cas10 would be the catalytic subunit since it contained an HD domain that in the Type I system is the active nuclease [24–26]. However, a Cmr complex formed with Cas10dHD cleaved RNA target as efficiently as the wild-type complex [19]. Mutation of the active site of the cyclase domain of Cas10 also failed to inhibit the nuclease activity of the complex [19]. Thus it appears unlikely that Cas10 is the catalytic subunit. Cmr5 can also be ruled out, as it is not required for activity [8], as can the Sulfolobales-specific subunit Cmr7 [7]. This leaves the four RAMP subunits, Cmr1, Cmr3, Cmr4 and Cmr6, as potential catalytic subunits. Of these, Cmr4 is perhaps the top candidate as sequence analysis reveals a conserved histidine residue that is in an equivalent position to the catalytic histidine residue of the known RAMP nuclease, Cas6 [12]. Although Cmr6 also contains a conserved histidine residue, its location is not consistent with the catalytic histidine residue of Cas6 [12]. Based on analysis of both sequence and structure, Cmr3 is unlikely to have nuclease activity [12,21], and sequence analysis also does not predict nuclease activity for Cmr1. It should also be noted that the identification of the catalytic subunit could be complicated by the nuclease active site being formed by more than one subunit and that given the difference in cleavage mechanism between the P. furiosus and S. solfataricus Cmr complexes, different subunits may be catalytic in different Cmr complexes.
Acknowledgements I thank Jennifer M. Kavran for a careful reading of the paper prior to submission.
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26 Beloglazova, N., Petit, P., Flick, R., Brown, G., Savchenko, A. and Yakunin, A.F. (2011) Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J. 30, 4616–4627
Received 5 September 2013 doi:10.1042/BST20130216
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