Spacer acquisition by Type III CRISPR-Cas system during bacteriophage infection of Thermus thermophilus.

Type III CRISPR-Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.

Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence.

Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.

Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants.

PIWI-interacting RNAs (piRNAs) promote fertility in many animals. However, whether this is due to their conserved role in repressing repetitive elements (REs) remains unclear. Here, we show that the progressive loss of fertility in Caenorhabditis elegans lacking piRNAs is not caused by derepression of REs or other piRNA targets but, rather, is mediated by epigenetic silencing of all of the replicative histone genes. In the absence of piRNAs, downstream components of the piRNA pathway relocalize from germ granules and piRNA targets to histone mRNAs to synthesize antisense small RNAs (sRNAs) and induce transgenerational silencing. Removal of the downstream components of the piRNA pathway restores histone mRNA expression and fertility in piRNA mutants, and the inheritance of histone sRNAs in wild-type worms adversely affects their fertility for multiple generations. We conclude that sRNA-mediated silencing of histone genes impairs the fertility of piRNA mutants and may serve to maintain piRNAs across evolution.