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dcyphr | Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas

Abstract

Cmr-β is a kind of CRISPR-Cas complex that recognizes RNA and executes a multicomponent immune response against genetic elements related to infection. In this study, the researchers explain how the structure of Cmr-β relates to its function, revealing how binding RNA regulates the Cmr2 subunit of the complex. Using cryoelectron microscopy (cryo-EM), the researchers were able to take pictures of the subunit’s unique structure and captured it in different stages of its immune response. Binding RNA induces a conformational change in Cmr2, along with the 5’ tag of CRISPR RNAs complementing the 3’ tag of target RNA, triggers different conformations of the Cmr3 subunit, which allows Cmr2’s immune response to recognize what RNA/DNA is its own and what is not. These results show the diverse defense strategies of CRISPR-Cas complexes.

Aims

The researchers wanted to understand how the structure of CMR-β allowed it to function, cleaving genetic elements that are not its own.

Introduction

Prokaryotes use RNA-guided immune systems called CRISPR-Cas against mobile genetic elements (MEGs), such as viruses and plasmids, which can infect prokaryotes and alter their genome. CRISPR-Cas immunity is acquired when short DNA sequences (protospaces), are recognized and integrated into a part of the host’s genome called the CRISPR locus. When the CRISPR loci are transcribed, they produce CRISPR RNAs (crRNA) which assembles with Cas proteins to form a complex. This complex is guided by the crRNA to recognize the same sequence in MEGs and degrade it, which can prevent infection.


CRISPR-Cas systems are divided into two classes and six types. Type III, separated into III-A through III-F, are especially interesting to researchers because they can degrade both RNA and DNA. III-A and III-B complexes have been the most well-studied.

When the host transcribes the foreign DNA sequences, the mRNA produced by transcription is recognized through base pairing with complementary crRNA and cleaved into single-stranded RNA by Cmr4/Csm3 subunits. When Cmr4 and Csm3 bind, the CRISPR-Cas complex is bound to the transcription bubble. Cmr2/Csm1 then recognize the mRNA as foreign, based on complementation between the cRNA 5’ tag and the 3’ tag of the target foreign DNA. This complementation triggers the prevention of an autoimmune response, blocking CRISPR-Cas from attacking itself. In contrast, lack of this complementation leads to host RNA degradation, which can lead to cell dormancy or death.

Recent studies of type III-A Csm CRISPR-Cas have revealed how CRISPR-Cas distinguishes its own RNA/DNA from that of foreign invaders. However, the different types of CRISPR-Cas are very different, and full understanding is yet to be achieved. In this study, the researchers characterize Cmr-β using cryo-EM and show how it differentiates between self and foreign mRNA.

Results

Cmr-β Cleaves Target RNA, Generates cOA, and Cute Non-complementary ssDNA and ssRNA

The bacterial species studied, S. islandicus, has two III-B complexes: SisCmr-α and Sis-Cmr-β. The researchers isolated SisCmr-β from the bacteria, and analyzed their sequences. The analysis suggested that catalytic residues in SisCmr-β are conserved, which target RNA degradation. The researchers tested the cleavage of target RNA by SisCmr-β, all which resulted in four separate fragments. They found that target RNA is cleaved independently of whether or not 5’ tag and 3’ tag are hybridized. In addition, ssDNA cleavage and cOA synthesis were only dependent on CTR binding. In addition, they observed that mismatches between the 5’ and 3’ tags triggered ssRNA cleavage. The researchers concluded that SisCmr-β cleaves target ssRNA by a mechanism independent of 5’ and 3’ tag hydribization.


Overall Structure of the Cmr-β Complex Revealed by Cryo-EM

To fully understand the structure of Cmr-β, the researchers created 7 cryo-EM structures of the complex bound to different DNA/RNA sequences (Figures S2-S4). Detailed descriptions of the chemical interactions between subunits can be found in this section.


The Autoimmunity Protection Mechanism is Revealed by the NTR-Bound Structure

The researchers determined the structure of the non-cognate target RNA (NTR)-bound SisCmr-β to understand how it discriminates between its own and foreign RNA transcripts. They did so by using cryo-EM and inactivating cleavage, which showed that the 5’ tag and 3’ tag are hybridized (Figure 2) and that a conformational change occurs.


In the Csm complexes, Csm1 is responsible for discriminating its own RNA from foreign RNA. But, these regions are not conserved in Cmr complexes, such that the recognition mechanism must be different. The researchers’ analysis showed that self-recognition involves a unique loop in the Cmr3 subunit, which changes conformation upon binding target RNA. This promotes the displacement of Cmr2, but additional activities are required to trigger Cmr2 catalytic activity. Taken together, these structures suggest that binding NTR results in a Cmr2-inactive state, which allows avoidance of autoimmunity.


The Immunity Response Mechanism Is Revealed by a Series of CTR-Bound Structures

To investigate how Cmr2 catalytic activity is triggered, the researchers determined the structure of a cognate target RNA (CTR)-bound SisCmr-β complex. They found that this binding induces two different conformational changes, one with a stalk loop in Cmr3 and one without.

The researchers completed another analysis to reach full understanding of the CTR-bound conformational changes. They found that in the CTR-bound state, the stalk loop alternates between an extended and contracted conformation based on how well the 5’ tag and 3’ tag are bound together. The dynamic fluctuations between the two conformations control Cmr2 activities and the immune response by SisCmr-β.


Target RNA Cleavage by Cmr4 and Its Effect on Cmr2 Activity

Further analysis of the CTR-bound state showed that Cmr4 are brought close to target-RNA-bound structures. In addition, analysis showed that cleavage of target RNA is not impacted by blocking cOA and ssDNA active sites. This shows that the absence of target RNA cleavage leads to constant activation of Cmr2 activities, and that cleavage of invading RNA is necessary to switch off Cmr2 activities.


Cmr2 cOA Catalytic Site

Synthesis of cOA by SisCmr-β occurs in the presence of Mn2+, and less so when Co2+ is present, and is dependent on the conformation of the Cmr3 stalk loop. The interplay between the 5’ and 3’ tags control the entrance and exit to and from the coA catalytic site through the stalk loop conformation.


The Cmr2 HD Catalytic Center

The HD catalytic center of Cmr2 is located inside of a cavity on the surface of the subunit, which is surrounded by an electron-rich area allowing interaction with the phosphate backbone (Figures 5A and 5B). The region around this center interacts with the rest of Cmr2. The HD domain undergoes conformational changes that allow it to drive catalysis.


The presence of ATP stimulated catalysis in the HD domain

The researchers investigated the relationship between the HD and cOA active sites, and suggested that the HD domain cleaving phosphodiesters decreases coA production. Taken together, these results show that ATP binding for cOA synthesis enhances the catalytic activity in the HD domain, but phosphodiester cleavage by the HD domain decreases the production of cOA. Further studies are necessary to understand how this relationship is linked to the immune response.


Allosteric Coordination between Target RNA Cleavage and Cmr2 Activation

To understand whether there was any relationship between target RNA cleavage and Cmr2 activation, the researchers created target RNAs of different lengths and tested whether their cleavage was linked to Cmr2 activity. They found that target RNA was independently cleaved by Cmr4 regardless of Cmr2.


Cmr7 Is a Modulator of SisCmr-β Activities

The researchers investigated the role of Cmr7, and found that it regulates the target RNA cleavage activity of the Cmr4 subunits. However, additional studies are necessary to clarify how Cmr7 associate with other subunits, such as Cmr2 and Cmr3.

Discussion

The presence of multiple CRISPR-Cas systems, SisCmr-β and SisCmr-α, suggest that they are complementary in achieving a similar immune response. SisCmr-β is more active in cleaving phosphodiester bonds in DNA/RNA, while SisCmr-α generates a larger amount of CoA. Cmr 7 modulates the activites of  SisCmr-β.


Autoimmunity in SisCmr-β Effector Complexes Is Avoided by Locking the Stalk Loop

In the NTR-bound SisCmr-β, the complementation between 5’ and 3’ tags induces the retracted conformation of the stalk loop, which enables new associations with Cmr2. All conformational changes associated with the entry of target RNA into the complex seems to drive Cmr2 towards cOA synthesis and ssDNA and UA ssRNA degradation. In addition, the structures showed that complementation of crRNA and target RNA is dynamic. The researchers propose an immunity model for Cmr complexes in which 5’ and 3’ tag complementation triggers cOA synthesis and phosphodiester cleavage in ssDNA and UA ssRNA.