"NAD-capQ" detection and quantitation of NAD caps

Abstract

RNA 5' cap structures comprising the metabolic effector nicotinamide adenine dinucleotide (NAD) have been identified in diverse organisms. Here we report a simple, two-step procedure to detect and quantitate NAD-capped RNA, termed "NAD-capQ." By use of NAD-capQ we quantitate NAD-capped RNA levels in Escherichia coli, Saccharomyces cerevisiae, and human cells, and we measure increases in NAD-capped RNA levels in cells from all three organisms harboring disruptions in their respective "deNADding" enzymes. We further show that NAD-capped RNA levels in human cells respond to changes in cellular NAD concentrations, indicating that NAD capping provides a mechanism for human cells to directly sense and respond to alterations in NAD metabolism. Our findings establish NAD-capQ as a versatile, rapid, and accessible methodology to detect and quantitate 5'-NAD caps on endogenous RNA in any organism.

Keywords: NAD cap; NAD-capQ; mRNA deNADding; mRNA decapping.

FIGURE 1.

NAD-cap detection and quantitation assay. (A) Steps in NAD-capQ. (B) NAD-capQ analysis of RNA generated in vitro. (Red) NAD-RNA; (green) Gppp-RNA; (gray) background signal detected without nuclease P1. Error bars represent ±SD, n ≥ 3. (C) Schematic of 5′-NAD cap removal from NAD-capped RNA using pretreatment with Rai1. (D) NAD-capQ analysis with or without pretreatment of NAD-RNA with Rai1. (Dark red) Background corrected absorbance without Rai1; (light red) background corrected absorbance with Rai1. Significance as in B. (E) Thin-layer chromatography analysis revealing 80% efficiency of Rai1 hydrolysis of ³²P-5′ end labeled NAD-capped RNA spiked into 50 µg total RNA.

FIGURE 2.

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).

FIGURE 3.

Cellular NAD concentrations can influence NAD cap levels. (A) The synthesis of NAD from NAM or NR. NAM is converted to NMN by the rate-limiting NAMPT, using PRPP as a cosubstrate. NMN is also the product of NR phosphorylation by NR kinases. NMN is converted to NAD by the NMNAT enzyme. FK866 is a small molecule inhibitor of NAMPT. (NAMPT) Nicotinamide phosphoribosyltransferase; (NMNAT) NMN adenylyltransferase; (NRK) NR kinase; (PRPP) phosphoribosyl pyrophosphate. (B) Quantitation of cellular total NAD following 48 h treatment with 5 nM FK866 or 5 mM NAM to decrease and increase cellular NAD levels, respectively. (C) Quantification of NAD-caps, detected with NAD-CapQ following 48 h treatment with 5 nM FK866 or 5 mM NAM. Mean values are plotted. Error bars represent ±SD. Statistical significance was calculated by one-way ANOVA with a Dunnett's multiple comparison post-test. P-values are denoted by asterisks. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.