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

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.Publisher PDFPeer reviewe

The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence

Funding: Biotechnology and Biological Sciences Research Council [BB/S000313/1 to M.F.W., BB/T004789/1 to M.F.W. and T.M.G.]; Wellcome Trust Institutional Strategic Support Funding [204821/Z/16/Z to M.F.W. and T.M.G.]; China Scholarship Council [201703780015 to W.Z.]. Funding for open access charge: RCUK block grant.Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.Publisher PDFPeer reviewe

Activation of Csm6 ribonuclease by cyclic nucleotide by cyclic nucleotide binding : in an emergency twist to open

Biotechnology and Biological Sciences Research Council [BB/T004789/1 to M.F.W. and T.M.G.]; European Research Council [101018608 to M.F.W.]; equipment was funded by BBSRC [BB/R013780/1 and BB/T017740/1]; T.M.G. is a recipient of a Royal Society Leverhulme Trust Senior Research Fellowship [SRF\R1\221056].Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6’ ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6’ in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the ‘jaws’ of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.Publisher PDFPeer reviewe

Rapid Oligocene to Early Miocene Extension Along the Grant Range Detachment System, Nevada, USA: Insights From Multipart Cooling Histories of Footwall Rocks

In Nevada and Utah, the Cordilleran orogen underwent a protracted Cenozoic transition to an extensional setting. However, the geodynamic processes that controlled this transition are poorly understood, in part because the space-time patterns of extension are not known in many areas. Localities of pre-Neogene extension have the potential to elucidate the dynamics of the Cordilleran crust during the final stages of subduction. Here we present data that constrain the timing of extension in the Grant Range in eastern Nevada, which was deformed by a low-angle normal fault system. We present temperature-time histories of eight granite samples exhumed by this fault system, constrained by muscovite Ar-40/Ar-39 multi-diffusion domain modeling and fission track and (U-Th)/He ages from zircon and apatite. These data demonstrate rapid cooling (20-35 degrees C/Myr) from 350-425 to 25-50 degrees C between 28-31 and 15-19Ma. The fault system accommodated 24km of extension (115%), and exhumed the granite samples from 7-9km depths to the near-surface. Rapid Oligocene-early Miocene cooling is interpreted to date the duration of motion on the fault system, and defines an extension rate of 1.5-2.6km/Myr. This was one of the most significant fault systems active during an episode of spatially distributed late Eocene-Oligocene extension, which overlaps temporally with volcanism generated by slab rollback. Reduced interplate coupling that accompanied slab rollback is interpreted as the primary driver of extension of the Cordilleran plateau during the final stages of subduction. This supports a scenario of orogenic collapse that proceeded in distinct episodes that were initiated by external geodynamic events.Makoil, Inc.6 month embargo; published online: 15 October 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]