Molecular determinants of nucleic acid recognition by an RNA-targeting ADP-ribosyltransferase toxin

ADP-ribosyltransferases use NAD⁺ to catalyze ADP-ribosylation reactions that regulate diverse cellular pathways in eukaryotes or function as toxins delivered by bacteria to kill competitor or host cells. Although most characterized ARTs target proteins, we recently identified RhsP2 as an antibacterial ART toxin that modifies the 2'-OH groups of structured RNAs during bacterial competition. However, the molecular basis for RhsP2's unique specificity toward RNA remains poorly understood. Here, we show that RhsP2 is a divergent member of the ART superfamily that recognizes nucleic acid substrates via a positively charged RNA-binding surface adjacent to its catalytic site. Mutations within this surface disrupt both RNA binding and ADP-ribosylation activity, abolishing RhsP2's antibacterial function. We further demonstrate that RhsP2 binds distinct small regulatory RNAs with varying affinities, suggesting that both electrostatic interactions and shape complementarity contribute to RNA target selection. Together, our findings define the molecular determinants of nucleic acid recognition by an unusual RNA-targeting ART toxin.