NAD-capQ detection and quantitation of RNA generated in vivo. (A) Quantitation of cellular NAD-capped RNA levels in E. coli, S. cerevisiae, or human HEK293T cells plotted on an NADH standard curve (orange data-point, E. coli; purple data-point, S. cerevisiae; blue data-point, human HEK293T; black data-points, NADH standard). n ≥ 3. (B) Values of NAD-RNA fmol/µg were calculated by dividing the calculated value of NAD-capped RNA detected by NAD-capQ in A by the amount of total cellular RNA analyzed. Quantitative values of NAD-capped RNA represented as RNA molecules/cell were calculated using total RNA concentrations per cell in E. coli (0.06 pg; Neidhardt et al. 1990), S. cerevisiae (0.7 pg; von der Haar 2008), and mammals (20 pg; Palazzo and Lee 2015), respectively. (C) Effects of disrupting deNADding enzymes on NAD-capped RNA levels in E. coli, S. cerevisiae, and human HEK293T cells. Graphs plot the fold-change in NAD-capped RNA values in the indicated mutant cells relative to the value observed in wild-type cells. (Error bars represent ±SD, n ≥ 3; [*] P < 0.05; [***] P < 0.001).
