Type III CRISPR-Cas systems which are widespread in nature, are less useful as a genetic tool but do also protect microbes from viruses. In the prokaryotic type III systems, multiple Cas proteins assemble with crRNA into Csm (type III-A/D) or Cmr (type III-B/C) effector complexes that provide interference against invading nucleic acids through transcription-dependent DNA silencing. In addition to targeting DNA, type III CRISPR targets the related RNA molecules from viruses. When it encounters RNA from a virus, the type III CRISPR produces a small molecule called cyclic oligoadenylate (cOA). The cOA molecule activates Csm6 RNases known as non-specific ribonucleases, which can destroy all the RNA in the cell. This defence is a less subtle than that provided type II CRISPR and can also damage the cell by destroying other RNA molecules that the microbes use to survive.
The interference complex of type III-A CRISPR-Cas systems is not only a crRNA-guided RNase and DNase, but also a cyclic oligoadenylate synthetase. Related studies have shown that the type III CRISPR interference complex has three enzymatic activities: (i) a crRNA-guided endoribonuclease activity against target RNA harboured by the Csm3 subunits, (ii) target RNA-stimulated DNase activity harboured by the HD domain of Cas10 (Csm1 in type III-A/D or Cmr2 in type III-B/C systems), (iii) target RNA-stimulated cyclic oligoadenylate synthetase activity harboured by the Palm domain of Cas10.
After infection, the transcription of phage DNA is initiated to establish and maintain the infection cycle. In bacteria, the crRNA-guided Csm/Cmr complex scans for the complementary target sequence in the invader's RNA. Tethering of the Csm/Cmr complex to the transcript by crRNA triggers RNA cleavage by Csm3/Cmr4 subunits and simultaneously activates the ssDNase activity of the Cas10 subunit for coupled degradation of the ssDNA in the transcription bubble. Csm/Cmr complexes avoid autoimmunity by checking the complementarity between the crRNA 5′-handle, which originates from the CRISPR repeat, and the 3′-sequence flanking the target sequence in RNA. The cyclase domain of Cas10 can synthesise cOA from ATP, when activated by target RNA binding. cOA in turn binds to and activates Csm6 and Csx1, enhancing their ribonuclease activity to degrade invader RNA transcripts, providing an additional interference mechanism.
Fig 1. Mechanism of Type Ⅲ-A CRISPR-Cas interference Systems.
Fig 2. Cryo-EM structure and functional organization of Cmr/Csm complex.
Csm/Cmr complex: In type III systems, the interference complex (known as Csm complex in type III-A/D systems and Cmr complex in type III-B/C systems) is assembled from the signature multidomain protein Cas10 (Csm1 in type III-A/D or Cmr2 in type III-B/C systems) and additional Cas proteins (Csm2-5 in type III-A/D, or Cmr1 and Cmr3-6 in type III-B/C). In these systems, interference is transcription-dependent and is initiated by the crRNA-guided interference complex binding to the nascent transcript of the target gene. The target RNA-bound complex functions as a sequence-specific endoribonuclease (RNase) to cleave the bound target RNA and in addition has a target RNA-stimulated non-specific deoxyribonuclease (DNase) activity that cleaves single-stranded DNA. Native Cmr complexes associate with multiple size forms of crRNAs and cleave complementary target RNAs at multiple sites in the region recognized by the crRNA.
The type III-B Cmr immune effector complex is a multi-subunit noncoding RNP that carries out RNA-guided cleavage of invader target RNAs in diverse bacterial and archaeal organisms. crRNA binding requires four Cmr proteins. The 5' tag is the defining feature of the crRNAs, is essential for Cmr complex formation and function. Cmr3 cross-links with the signature 5' tag sequence of the crRNAs. The interaction of Cmr3 with crRNAs also requires Cmr2, Cmr4, and Cmr5. Cmr2-5 form a complex with a crRNA interaction pattern and tag sequence dependence. Each of the four proteins is required for formation of the complex. Remarkably, efficient target RNA capture depends on the additional presence of Cmr1 and Cmr6. Despite the presence of the crRNA, which includes a 37-nt guide region that is complementary to the target RNA, the crRNP formed by Cmr2-5 does not substantially interact with the target RNA.
Cas10 protein: The Cas10 subunit (called Csm1 and Cmr2 in the type III-A/D and III-B/C systems, respectively) harbors an N-terminal HD domain, two small a-helical domains, and two Palm domains that share a ferredoxin-like fold with the core domain of nucleic acid polymerases and nucleotide cyclases. The HD domain of Cas10 is responsible for ssDNA degradation in vitro. When crRNA guides the Csm/Cmr complex to cleave the transcript, the single-stranded deoxyribonuclease (ssDNase) activity of the Cas10 subunit is activated for coupled degradation of the ssDNA in the transcription bubble. The conserved GGDD motif in one of the two Palm domains can synthesise cyclic oligoadenylate (cOA) molecules from ATP, when activated by target RNA binding. cOA in turn binds to and activates Csm6 and Csx1, enhancing their ribonuclease activity to degrade invader RNA transcripts, providing an additional interference mechanism. cOA thus represents a new type of second messenger, generated by type III CRISPR effector complexes, that sculpts the cellular response to invasion by MGE.
Csm6 protein: The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts. Members of the Csm6 or the related Csx1 protein families are frequently encoded within type III CRISPR-Cas systems. The proteins have an N-terminal CARF (CRISPR-associated Rossmann fold) domain and a C-terminal HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain. Csm6 proteins function as RNases to non-specifically degrade invader-derived RNA transcripts, providing an additional interference mechanism that complements the nuclease activities of the type III interference complex. Studies have shown that target RNA binding by the Csm effector complex of Streptococcus thermophilus triggers Cas10 to synthesize cyclic oligoadenylates by means of the Palm domains. Acting as signaling molecules, cyclic oligoadenylates bind Csm6 to activate its nonspecific RNA degradation. This cyclic oligoadenylate-based signaling pathway coordinates different components of CRISPR-Cas to prevent phage infection and propagation.
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