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