Structural and biochemical studies define Nudt12 as a new class of RNA deNADding enzyme in mammalian cells

We recently demonstrated mammalian cells harbor NAD-capped mRNAs that are hydrolyzed by the DXO deNADding enzyme. Here we report the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targets different RNAs. Crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 Angstrom resolution provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to metabolic stress manifests changes in NAD-capped RNA levels indicating an association between NAD-capped RNAs and cellular metabolism. Lastly, we show that the bacterial RppH protein also possesses deNADding activity toward NAD-capped RNA but not free NAD, revealing a third class of deNADding enzymes.