LC/MS analysis of cellular RNA reveals NAD-linked RNA

Abstract

We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule. This technique revealed NAD-linked RNA in Escherichia coli and Streptomyces venezuelae. Subsequent characterization showed that NAD is a 5' modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs and is present at a surprisingly high abundance of approximately 3,000 copies per cell.

Figure 1

Discovery of a small molecule-linked nucleotide of [M-H]^- m/z = 540.0533 from E. coli and S. venezuelae RNA. (a) The general method for biological small molecule-RNA conjugate discovery developed in this work. Paired samples for comparative LC/MS analysis are generated by treatment of cellular RNA with active nuclease P1 versus treatment with heat-inactivated nuclease P1 under otherwise identical conditions. (b) The extracted ion chromatograms (EICs) for [M-H]^- m/z = 540.0533 from E. coli RNA exposed to active nuclease P1 (cleavage conditions) or to heat-inactivated nuclease P1 (control conditions). (c) Same as (b), but using S. venezuelae RNA. (d) Isotope-labeled S. venezuelae total RNA reveals that the [M-H]^- m/z = 540.0533 ion contains five nitrogen atoms and 15 carbon atoms. (e) The EICs for [M-H]^- m/z = 662.1018 from E. coli RNA or (f) from S. venezuelae RNA digested with nuclease P1 or control conditions (heat-inactivated nuclease incubation). (g) One possible structure of NAD-linked cellular RNA. We note that our data is also consistent with RNA attachment to the 2’ hydroxyl of NAD, or to either of the nicotinamide-linked ribose hydroxyl groups.

Figure 2

Characterization of NAD-RNA. (a) Spiking large quantities of NAD or NADH into E. coli cell lysate before RNA isolation and treatment with active nuclease P1 does not change the observed ion counts of these species. Error bars represent the standard deviation of three independent trials. (b) Total E. coli RNA was separated into RNAs of length greater than 200 nucleotides (fraction 1) and RNAs of length less than 200 nucleotides (fraction II) using a silica column (Qiagen RNeasy). Each fraction was subjected to nuclease P1 digestion and analyzed by LC/MS. The presence of NAD in fraction II, similar to that of Phe-AMP, suggests that the NAD-linked RNA(s) are primarily less than 200 nucleotides in length.

Figure 3