Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage

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.

Analysis of a photosynthetic cyanobacterium rich in internal membrane systems via gradient profiling by sequencing (Grad-seq)

“…CARF proteins sense ligands such as cyclic oligoadenylate (cOA) signaling molecules that can be synthesized by the polymerase domain of Cas10 upon recognition of its target (Kazlauskiene et al, 2017;Niewoehner et al, 2017;Han et al, 2018). The Archaeoglobus fulgidus Csx3 protein AfCsx3 is a ring nuclease that degrades the CRISPR signaling molecule cOA4 (Athukoralage et al, 2020). However, Slr7080/Csx3 is substantially larger than AfCsx3 (310 compared to 110 amino acids) and possesses an additional AAA ATPase domain in the C-terminal region (Shah et al, 2019).…”

The Cyclic Oligoadenylate Signaling Pathway of Type III CRISPR-Cas Systems

“…Notably, very recently, it was found the HEPN domain of Csm6 can also degrade cOA to self-regulate its RNase activity ( Smalakyte et al, 2020 ). Moreover, recent studies report that the widespread CRISPR associated protein Csx3 is a novel ring nuclease, named Crn3 (CRISPR associated ring nuclease 3; Athukoralage et al, 2020a ; Brown et al, 2020 ). Interestingly, an unusual cooperative catalytic mechanism was found in which an active site of Csx3 tetramer is formed by two dimers sandwiching a cOA 4 substrate ( Athukoralage et al, 2020a ).…”

“…Moreover, recent studies report that the widespread CRISPR associated protein Csx3 is a novel ring nuclease, named Crn3 (CRISPR associated ring nuclease 3; Athukoralage et al, 2020a ; Brown et al, 2020 ). Interestingly, an unusual cooperative catalytic mechanism was found in which an active site of Csx3 tetramer is formed by two dimers sandwiching a cOA 4 substrate ( Athukoralage et al, 2020a ). In addition, a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) from Sulfolobus islandicus has recently been identified as a novel cOA-degrading enzyme ( Zhao et al, 2020 ).…”

“…Indeed, a new family of viral anti-CRISPR (Acr) protein, AcrIII-1, was recently identified as a ring nuclease that specifically degrades cOA 4 , suggesting that it functions as Acr protein against cOA 4 -triggered type III CRISPR-Cas immunity ( Athukoralage et al, 2020c ). AcrIII-1 has a higher activity for cOA 4 degradation than Crn1 and Crn3, and is unrelated to the CARF family proteins ( Athukoralage et al, 2020a , c ). AcrIII-1 homologs are widespread in various prokaryotes, where AcrIII-1 homologs may function as host-encoded ring nuclease like Crn1 and Crn3, thus named Crn2 ( Athukoralage et al, 2020c ).…”

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

“…Only recently, a new protein family, the ring nucleases Crn1 (CRISPR-associated ring nuclease 1), was discovered in the archaeon Saccharolobus solfataricus [144] and shown to specifically linearize and therefore inactivate cOA molecules, resetting the cell to a ground state once the virus is defeated by type III systems. While Crn1 ring nucleases seem to be limited to crenarchaeota, a new type, Crn3 (former Csx3) was recently biochemically characterized in the euryarchaeon A. fulgidus and found to be widespread in prokaryotes [145]. Furthermore, Csx1/Csm6 ribonucleases, or even Crn-Csx fusion proteins exist which intrinsically degrade the cOA substrates themselves, thus representing self-limiting enzymes independent of a trans-acting ring nuclease [146][147][148][149].…”