5' End Nicotinamide Adenine Dinucleotide Cap in Human Cells Promotes RNA Decay through DXO-Mediated deNADding

Eukaryotic mRNAs generally possess a 5' end N7 methyl guanosine (m⁷G) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD⁺) cap that, in contrast to the m⁷G cap, does not support translation but instead promotes mRNA decay. The mammalian and Fungal noncanonical DXO/Rai1 decapping Enzymes efficiently remove NAD⁺ caps, and cocrystal structures of DXO/Rai1 with 3'-NADP⁺ illuminate the molecular mechanism for how the "deNADding" reaction produces NAD⁺ and 5' phosphate RNA. Removal of DXO from cells increases NAD⁺-capped mRNA levels and enables detection of NAD⁺-capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD⁺ caps can be added to 5'-processed termini. Our findings establish NAD⁺ as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD⁺-capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5' end modification distinct from the classical m⁷G cap that promotes rather than inhibits RNA decay.