Zoe Wardle, “The Ability of Metabolic Enzymes to Utilize Nicotinamide Adenine Dinucleotide Covalently Attached to RNA 5’-ends”
Mentor: David Frick, Chemistry & Biochemistry
Poster #206

Nicotinamide adenine dinucleotide (NAD), and similar coenzymes, are widely known to be attached to the 5’ ends of many cellular RNAs, both in prokaryotes and eukaryotes. The current paradigm assumes that such unusual caps primarily exist to modulate RNA function, but an alternate explanation might be that RNA instead modulates the activity of NAD. Most enzymes that use NAD catalyze either redox reactions using oxidized NAD to accept electrons or catalyze the transfer AMP, or ADP-ribose, from NAD to other compounds. Here we examine the ability of such enzymes to utilize NAD-capped RNA that was in vitro synthesized using T7 RNA polymerase. Oxidation or cleavage of NAD was monitored using novel assays for authentic NAD with picomole sensitivity. First, the following redox enzymes previously shown to bind RNA were tested: glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase, pyruvate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, alanine dehydrogenase, glucose-6-phosphate dehydrogenase, and dihydrofolate reductase. Second, the following transferases that use NAD were tested: DNA ligase, mono-ADP ribosyltransferase, poly (ADP-ribose) polymerase, and various NAD-dependent deacetylases (sirtuins). All enzymes tested showed a clear preference for free NAD over NAD-capped RNA. Importantly, while some appeared to consume NAD (e.g., GAPDH), their apparent activity was dramatically lower after further enzyme purification. Similarly, longer RNAs synthesized in the presence of NAD, sometimes tested positive, suggesting that prior studies examining the activity of enzymes on NAD-capped RNA might have been confounded by NAD copurifying with other macromolecules. While still preliminary, these results do not support the contention that key metabolic enzymes use NAD-capped RNA. Instead, NAD installation on the 5’ end of RNA might act to sequester or block the action of this important coenzyme. Supported by NIH grant 1R15GM144859.