# RNA-coupled Coenzymes

> **NIH NIH R15** · UNIVERSITY OF WISCONSIN MILWAUKEE · 2021 · $442,896

## Abstract

Nicotinamide adenine dinucleotide (NAD) is a widely studied, important
coenzyme, which is involved in many critical biological redox reactions. Oxidized NAD
(NAD+) accepts two electrons while its reduced form (NADH) donates two electrons to a
variety of substrates. Recently, NAD has been identified as a non-canonical initiating
nucleotide (NCIN) in both prokaryotic and eukaryotic RNA. Since a pyrophosphate links
the adenine and nicotinamide nucleotides in NAD, such an NCIN resembles the 7-
methylguanylate cap found on the 5’ end of most eukaryotic messenger RNAs. The
reason that RNA starts with NAD is not clear, but the current paradigm in the field
assumes the NCINs primarily exist to modulate RNA function (i.e. they are
“epitranscriptomic modifications”). This proposal examines another hypothesis that
contends the opposite is true, i.e., that the RNA component is needed to modulate
coenzyme activity. If the RNA influences coenzyme activity then reactions catalyzed by
the NAD-utilizing enzymes should proceed at different rates or extents, and the
sequence of the RNA should influence the reaction. The first prediction will be tested by
comparing the ability of various enzymes to use NAD+-, NADP+-, NADH-, NADPH-
capped RNA to their ability to use free dinucleotides. The enzymes chosen for analysis
are glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase (LDH),
pyruvate dehydrogenase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase;
malate dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate
dehydrogenase, dihydrofolate reductase, DNA ligase, mono-ADP ribosyltransferase,
poly (ADP-ribose) polymerase, and NAD-dependent deacetylase. Each enzyme that
reacts with capped-RNA will be examined in more detail to estimate the RNA affinity and
RNA specificity. Affinity will be monitored using enzyme kinetics and direct binding
assays. Specificity will be investigated using a library of previously reported cellular
NAD-capped RNAs, and by sequencing RNAs that co-immuno-precipitate with each
enzyme. Preliminary results show LDH uses NAD-capped RNA as a coenzyme,
suggesting that this could be the first study to show NCINs can participate in cellular
chemical reactions, and possibly form a new class of key ribonucleoproteins or
ribozymes.

## Key facts

- **NIH application ID:** 10359222
- **Project number:** 1R15GM144859-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN MILWAUKEE
- **Principal Investigator:** David N Frick
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $442,896
- **Award type:** 1
- **Project period:** 2021-09-20 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10359222

## Citation

> US National Institutes of Health, RePORTER application 10359222, RNA-coupled Coenzymes (1R15GM144859-01). Retrieved via AI Analytics 2026-06-10 from https://api.ai-analytics.org/grant/nih/10359222. Licensed CC0.

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