Presence of NAD+-capped RNA in human cells: function and removal by the DXO deNADing Protein

Eukaryotic mRNAs generally possess an N7 methyl guanosine cap at their 5? end to promote their translation and stability. Here we demonstrate mammalian mRNAs can carry a 5'-end nicotinamide adenine dinucleotide (NAD+) cap. We further demonstrate fungal and mammalian noncanonical DXO family of decapping enzymes can efficiently remove NAD+ caps from mRNAs in vitro and cocrystal structures of DXO with 3´ phosphate NAD+ illuminates the molecular mechanism for the “deNADing” reaction. An NAD+ cap promotes mRNA decay in wild type mammalian cells and confers mRNA stability in the absence of DXO. Importantly, mammalian cells possess a capping mechanism that NAD+ caps a subset of intronic small nucleolar RNAs that are selectively enriched in DXO deficient cells. Our data establish NAD+ as a bona fide mammalian RNA cap and identifies the DXO proteins as potent deNADing enzymes that modulate the levels of NAD+-capped RNAs in cells. Two replicates of HEK293T cell RNA were prepared and sequenced using standard methods. Three replicates each of HEK293T (Wt) and HEK293T-DXO-KO (DXO-KO) cell RNA were prepared and enriched by NAD-Capture for sequencing library preparation and seqeuencing.