Abstract
Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.
Highlights
The CRISPR system provides adaptive immunity against viruses and other Mobile Genetic Elements (MGE) in bacteria and archaea
The Csx3 protein from A. fulgidus was originally crystallised in the absence and presence of a pseudo-symmetric RNA tetranucleotide, and shown to have RNA deadenylase activity in vitro (Yan et al, 2015)
It has previously been suggested that the binding site for the RNA tetranucleotide could be compatible with binding of a symmetric cyclic oligonucleotide (Topuzlu and Lawrence, 2016), similar to those observed in other CRISPR Associated Rossman Fold (CARF) family proteins
Summary
The CRISPR system provides adaptive immunity against viruses and other Mobile Genetic Elements (MGE) in bacteria and archaea (reviewed in Wright et al, 2016; Koonin and Makarova, 2017). This allows detection of the RNA encoded by invading MGE via base-pairing to the crRNA Binding of this ‘target RNA’ results in subtle conformational changes in the effector complex, activating the catalytic Cas subunit which uses its HD-nuclease domain to degrade DNA (Elmore et al, 2016; Estrella et al, 2016; Kazlauskiene et al, 2016; Jung et al, 2015; Han et al, 2017b) and its cyclase domain to synthesise cyclic oligoadenylate (cOA) molecules by polymerisation of ATP (Kazlauskiene et al, 2017; Niewoehner et al, 2017; Rouillon et al, 2018). A wide variety of alternative CARF-domain proteins associated with type III CRISPR loci have been identified but not yet described (Shmakov et al, 2018; Shah et al, 2019), and recent work
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