Anti-viral defense by an ADP-ribosyltransferase that targets mRNA to block translation (RIP-Seq in vitro)
Host-pathogen conflicts are crucibles of molecular innovation. Selection for immunity to pathogens has driven the evolution of sophisticated immunity mechanisms throughout biology, including in bacteria that must evade their viral predators known as bacteriophages. Here, we characterize a toxin-antitoxin-chaperone system, CmdTAC, in Escherichia coli that provides robust defense against infection by T4 phage. During infection, newly synthesized capsid protein triggers dissociation of the chaperone CmdC from the CmdTAC complex, leading to destabilization and degradation of the antitoxin CmdA, with consequent liberation of the toxin CmdT, an ADP-ribosyltransferase. Strikingly, CmdT does not target a protein, DNA, or structured RNA, the known targets of other ADP-ribosyltransferases. Instead, CmdT modifies the N6 position of adenine in GA dinucleotides within single-stranded RNAs to robustly block mRNA translation and viral replication. Our work reveals both a new mechanism of anti-phage defense and a new class of ADP-ribosyltransferases that targets mRNA. Overall design: The goal of this experiment is to identify transcripts modified by the ADP-ribosyltransferase CmdT with an in vitro approach. CmdT was incubated with RNA extracted from E. coli infected with the bacteriophage T4 and supplied biotinylated NAD+ and then a streptavidin pulldown was performed on the RNA to identify modified transcripts.