Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Abstract

9Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to 11 signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This 12 can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell 13once a viral infection has been defeated. Enzymes specialised for this task are known as ring 14nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR 15 associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that 16rapidly degrades cyclic tetra-adenylate (cA 4 ). The enzyme has an unusual cooperative reaction 17 mechanism involving an active site that spans the interface between two dimers, sandwiching the 18 cA 4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 19family. 20 Keywords: 21 CRISPR, Csx3, ring nuclease, cyclic tetra-adenylate, CARF, cooperative enzyme 22 103 terminal Csx3 domain fused to a C-terminal kinase/transferase domain of unknown function. B. Phosphor 104 images of native gel electrophoresis visualising cA 4 (20 nM) or RNA oligonucleotide 49-9A (50 nM) binding 105 by A. fulgidus Csx3. Csx3 binds to cA 4 with high affinity (apparent K D ~ 50 nM) and binds the RNA 49-9A 106 with significantly lower affinity (apparent K D ~ 10 µM). Images are representative of three technical 107 replicates. 108 109 Figure 1figure supplement 1. Multiple sequence alignment of Csx3 proteins showing conserved 110 residues. Csx3 proteins are shown from Archaeoglobus fulgidus; Methanosarcinia mazei; candidatus 111 Bathyarchaeota; Thermococcus celericrescens; Methylacidiphilum fumariolicum; Aquifex aeolicus; 112 Dictyoglomus turgidum; Spirulina major; Oscillatoria nigro-viridis. Absolutely conserved residues are shaded. 113 Conserved residues H60 and D69 (Afu numbering) are indicated by asterisks. 114 131 Figure 2. Csx3 is a potent ring nuclease. A. TLC analysis of the reaction products of radiolabelled cA 4 132 (200 nM) incubated with A. fulgidus Csx3 (8 µM dimer) in reaction buffer at 50 °C (time points every 5 s from 133 10-60 s then 1.5, 2, 3, 4, 5 and 10 min, n = 6 technical replicates). The cA 4 was rapidly converted into A 2 -P, 134 with a small amount of A 2 >P (linear A 2 with a cyclic 2',3' terminal phosphate). B. Denaturing polyacrylamide 135 gel electrophoresis visualising cleavage of 5'-end radiolabelled RNA 49-9A (50 nM) by Csx3 (8 µM dimer) at 136 50 °C (time points every 5 min from 5-40 min then 50, 60 and 90 min, n = 3 technical replicates). C. Plot 137 comparing single-turnover kinetics of cA 4 (blue) and RNA 49-9A (green) cleavage by AfCsx3 at 50 °C. Data 138 points are the average of three technical replicates and error bars represent the standard deviation of the 139 mean. D. Liquid-chromatography high-resolution mass spectrometry analysis of reaction products when cA 4

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23

“…The ribose moieties facilitate the formation of this distorted, and presumably high energy, conformation of cA4, by adopting an unusual 2 3T twist conformation with C2'-endo/C3'-exo pucker. The conformation of cA4 bound to Csx23 CTD is distinct to that observed in complex with other cA4 binding proteins such as AcrIII-1 [24], Crn3 [23], Can1 [13], Can2/Card1 [14,15] and Csx1 [10]. The angle between each C2'-hydroxyl group on the ribose and adjacent oxygen and phosphate atoms in cA4 is 156°.…”

“…Furthermore, the distribution of cOA species produced by type III systems can differ in vivo compared to in vitro [34]. A range of CRISPR effector proteins bind cA4, but they tend to utilize a conserved CARF/SAVED/Csx3 domain [10,12,13,15,23], which is a member of the Rossman fold superfamily [35] or the unrelated Crn2/AcrIII-1 domain [24]. The cA4 binding CTD of Csx23 was predicted to be completely unrelated to either family and could thus represent a new class of cA4 recognition domain.…”

“…Type III CRISPR systems have been used as the basis for new diagnostic applications due to their ability to directly detect any desired RNA and generate an amplified cOA signal that in turn activates a reporter nuclease [19][20][21]. In some situations, cellular enzymes known as ring nucleases degrade the second messengers to revert cells to a noninfected ground state [22,23]. Viruses also encode ring nucleases to subvert type III CRISPR immunity [24].…”