GSE110801
Nudt12 is an mRNA DeNADding Enzyme
The 5´-end 7-methylguanosine cap structure has long been known as a signature feature of eukaryotic cellular and viral mRNAs that confers mRNA stability and efficient translation.  Recent findings in diverse organisms have demonstrated that RNAs can additionally possess a non-canonical cap structure consisting of a nicotinamide adenosine dinucleotide (NAD+) at their 5´ end in place of m7G.  It has been shown that 5´ end-NAD+ cap promotes rapid decay of the RNA at least in part by the DXO family of proteins in mammalian cells.  This observation led to the hypothesis that mammalian cells harbor additional deNADding enzymes that may function in distinct pathways.  Here we report Nudt12 efficiently removes NAD+ caps and functions as alternate cellular deNADding enzyme that targets NAD+-capped RNAs distinct from DXO.  Importantly, with the use of an NAD-Cap Detection (NAD-CapD) approach that utilizes enzymatic properties to release intact NAD+/NADH from the 5´ end of NAD-capped cellular RNAs and a colorimetric NAD Quantitation to detect released NAD+/NADH, we can follow total cellular NAD+ cap levels.  Removal of Nudt12 or DXO deNADding enzymes from cells significantly increased levels of NAD+-capped cellular RNAs.  Moreover, fungal Rai1 and Dxo1, previously demonstrated to possess deNADding activity in vitro, can also function as deNADding enzymes in yeast cells.  Double disruption of Rai1 and Dxo1 in yeast cells lead to accumulation of NAD+-capped RNAs, indicating that both enzymes function to clear NAD+ from the 5´ end of RNAs.  Finally, our findings established that alterations in cellular NAD+ levels impact NAD+-capped RNA levels implying NAD+ capping is a modulated process that may be linked to the metabolic state of the cell. Overall design: Three replicates each of HEK293T-Nudt12-KO (N12-KO) cell RNA were prepared and enriched by NAD-Capture for sequencing library preparation and seqeuencing.  Results were compared with previously-published samples of HEK293T-WT and HEK293T-DXO-KO prepared by identical methods.