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

NAD+ capped RNAs (NAD-RNAs) are widely present in prokaryote and eukaryote
transcriptomes. However, their regulation during developmental processes
are largely unknown. In this study, we investigated the regulation of
NAD-RNAs during receptacle ripening in the strawberry (Fragaria ×
ananassa, Fa), a model for non-climacteric ripening. To do this, we
performed “click reaction”-based method to sequence NAD-RNAs
(SPAAC-NAD-seq) to globally profile NAD-RNAs in the context of strawberry
ripening. From this analysis, we 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. We also found that the number of NAD-RNAs displayed an
overall decreasing trend that corresponded with lower abundance of NAD+
cap throughout the course of ripening. Concurrently, we characterized the
function of FaNUDT19, a potent NAD-RNA scavenger, in strawberry fruit
ripening. Corresponding to the decreased abundance of NAD+ cap, we found
that the expression of FaNUDT19 increased ~3.5-fold during the course of
ripening. We found that FaNUDT19 could hydrolyze multiple 5′ caps,
including NAD+, ATP, AMP, and m7G caps. However, it has the most
pronounced activity on NAD-RNAs. Likely due to this specific 5′-cap
activity, we found that 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 through the
stabilization of mRNAs producing proteins that promote coloration.
Overall, our findings highlight the importance of dynamic NAD+ decapping
by FaNUDT19 in strawberry ripening.