Identification of Novel Enzymes Regulating ADP-Ribosylation of Uracil and Thymine in Nucleic Acids.

ADP-ribosylation (ADPr) is a conserved modification that regulates a wide range of cellular processes. Although proteins are the best-characterized substrates of ADPr, recent studies have demonstrated that nucleic acids can also be modified. However, the enzymes responsible for installing and removing nucleic acid ADPr, as well as its biological functions, remain poorly understood. Here, we identify human PARP15 as an ADP-ribosyltransferase that robustly modifies uracil bases in single-stranded RNA (ssRNA) and confirm that human TARG1 reverses PARP15-catalysed ADPr. We further identify the first phage-encoded hydrolase capable of efficiently removing both uracil- and thymine-linked ADP-ribose from nucleic acids. Biochemical analyses provide proof-of-principle evidence that uracil ADP-ribosylation (U-ADPr) may modulate vertebrate antiviral responses, whereas we provide new evidence that thymine ADP-ribosylation (T-ADPr) functions as a molecular signal in bacterial-phage warfare. Together, our findings expand the known repertoire of nucleic acid ADP-ribosylation, define key enzymes regulating pyrimidine ADPr dynamics, and suggest a conserved enzymatic framework for pyrimidine ADPr in immunity across evolution.