Toll/interleukin-1 receptor (TIR) domain-containing proteins exhibit deNAMing activity towards NAD-capped RNAs

The occurrence of NAD+ as a non-canonical RNA cap has been demonstrated in diverse prokaryotic and eukaryotic organisms. The Toll/interleukin-1 receptor (TIR) domain present in all kingdoms of life acts in biotic defense responses and can have NADase activity. Here, we show that the TIR domain-containing proteins from several bacterial and one archaeal species can remove the nicotinamide (NAM) moiety from NAD-capped RNAs (NAD-RNAs). By employing mass spectrometry-based analysis, we demonstrate that the deNAMing activity of AbTir (from Acinetobacter baumannii) specifically produces cyclic ADPR-RNA. We further show that this cyclic ADPR-RNA can be decapped in vitro by enzymes such as E. coli NudC and eukaryotic DXO, which suggests that bacterial TIR domains could lead to the removal of the RNA NAD cap in vivo in a stepwise decapping mode. Heterologous expression of the wild-type but not a catalytic mutant AbTir in E. coli cells suppressed cell propagation and reduced the levels of NAD-RNAs from a small number of genes before cellular NAD+ levels are impacted. Collectively, both the in vitro reconstitution and the in vivo analysis demonstrate that TIR domain-containing proteins can function as a novel deNAMing enzyme of NAD-RNAs, which implicates NAD-RNA decapping as a mechanism in pathogen-host interactions.