FaNUDT19 contributes to 5′ cap quality control by modulating NAD+ capped RNA dynamics during strawberry fruit ripening

Nicotinamide adenine dinucleotide (NAD+)-capped RNAs (NAD-RNAs) are widely present in prokaryotic and eukaryotic transcriptomes. However, their regulation during development remains unclear. Here, we investigated NAD-RNA regulation during receptacle ripening in strawberry (Fragaria × ananassa, Fa), a model for non-climacteric fruit ripening. We performed "click reaction"-based sequencing of NAD-RNAs (SPAAC-NAD-seq) to globally profile NAD-RNAs during ripening. This identified 9,109 NAD-RNAs produced from protein-coding genes (NAD-mRNAs), 6,479 from transposable elements (NAD-TE-RNAs), 4 from the mitochondrial genome, and 11 from the chloroplast genome. The number of NAD-RNAs tended to decrease, corresponding with lower abundance of the NAD+ cap during ripening. In addition, we characterized the function of the NAD-RNA scavenger Nudix (Nucleoside diphosphate linked moiety X)-Type Motif 19 (FaNUDT19). FaNUDT19 expression increased ∼3.5-fold during ripening. Biochemically, FaNUDT19 could hydrolyze multiple 5' caps, including NAD+, ATP, AMP, and m7G caps, with the strongest activity on NAD-RNAs. Indeed, FaNUDT19 overexpression in strawberry selectively affected a subset of NAD-mRNAs, allowing for the persistence of stable levels of ripening-related genes, such as PEROXIDASE (FaPOD). These results suggest that FaNUDT19 affects strawberry ripening by stabilizing mRNAs that produce proteins promoting coloration. Overall, our findings highlight the importance of dynamic NAD+ decapping by FaNUDT19 in strawberry ripening.