Date of Award

December 2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

David N Frick

Committee Members

Madhusudan Dey, Shama Mirza, Xiaohua Peng, Nicholas R Silvaggi

Abstract

Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds (x). These enzymes have catalytic activity on a wide range of substrates such as dNTPs (both canonical and their oxidized forms), nucleotide sugars, alcohols, dinucleoside polyphosphates, dinucleotide cofactors, and nucleoside diphosphates linked to RNA. The bacterial genome encodes around 13, while the human genome encodes for 22 such nudix proteins.

The E. coli genome encodes for a mutT mutator gene, the progenitor of the family expressing the MutT pyrophosphohydrolase (NudA) protein (Treffers et al., 1954; Bhatnagar et al., 1988). The enzyme was found to hydrolyze both canonical and mutagenic nucleoside triphosphates, most commonly the toxic form of dGTP, 8-oxo-dGTP, preventing its misincorporation during DNA replication (Maki et al., 1992). Nudt1 is its functional homologue in humans and plays a vital role in removing mutagenic dNTPs to prevent A:T to C:G transversion, by hydrolyzing the 8-oxo dGTP to 8-oxo dGMP (Nakabeppu, 2001). The E. coli NudC and its functional homolog in humans, Nudt12 are members of the NADH diphosphatases of the Nudix hydrolase superfamily, cleaving the pyrophosphate bond in their nucleotide substrates, such as such as NADH, NADPH, NADP+, ADP-ribose and AppA to yield two nucleoside monophosphates. Isomeric forms of NADH were discovered as a substrate for the enzyme, renalase (Beaupre et al., 2015). For the first time, experiments in this thesis tests the ‘housecleaning’ activity of NudC and Nudt12 on these toxic isomers of their nascent substrate.

Since the early discovery of the Nudix hydrolase class of proteins, several members have been reported to have mRNA decapping activity. The E. coli RppH (NudH) nudix enzyme removes pyrophosphate from the 5’-end of RNA while the yeast Dcp2 (Nudt20) nudix protein was the first eukaryotic mRNA decapping enzyme discovered to remove m7GDP from canonically capped mRNA (Wang et al., 2002). Nudt3 has also been demonstrated as an mRNA decapping enzyme (McLennan et al., 2006; Song et al., 2013; Kiledjian et al., 2016). Nudt16 has been reported to have 5’-end U8 snoRNA decapping activity (Grzela et al., 2018). With the recent discovery of the cofactor nicotinamide adenine dinucleotide (NAD+) as a transcriptional modification of prokaryotic mRNA (Chen et al., 2009), it has been suggested that NudC is involved in RNA decapping, eventually triggering its decay (Cahová et al., 2014). Its functional homolog in mouse, Nudt12 has also been shown to decap both canonical and ncinRNA (non-canonical initiating nucleotide) in vitro (Grudzien-Nogalska et al., 2017). Evidence for direct interaction between these decapping Nudix hydrolases and their oligonucleotide substrates is yet to be shown. For the first time, we report binding between DNA/RNA and Nudix decapping proteins, and serendipitously discover that the human Nudt12 protein exclusively binds RNA and not DNA, preferring to bind in vitro transcribed NAD+-capped RNA.

Although the decapping activity of NudC (Cahová et al., 2014) and Nudt12 (Grudzien-Nogalska et al., 2019) on NAD+-capped RNA has previously been shown using TLC, we used a more analytical approach to confirm these results using LC/MS. The preference of these proteins to cleave NADH-capped RNA over NADH was also observed during these experiments.

It has been hypothesized that the NAD+ on the terminal end of both prokaryotic and eukaryotic RNA stabilizes the nucleic acids as the 7mG cap does for eukaryotic mRNAs. NAD+/NADH are widely studied, important coenzymes, 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. Kinetics of NAD+/NADH redox reactions can easily be measured because NADH absorbs light at 340 nm but NAD+ does not. In this thesis, we explored the hypothesis that ncinRNA might play a more obvious chemical role in the cell, by participating in cellular redox reactions. To test if ncinRNA can be functional in redox reactions, T7 RNA polymerase was used to synthesize RNA in vitro, initiating with the ncin: NAD+, NADH, NADP+ and NADPH. After analyzing the nature of the RNA using agarose gel electrophoresis, gel extraction and LC/MS, we compared the ability of the RNAs to reduce pyruvate and oxidize lactate in reactions catalyzed by the E. coli, rabbit muscle and bovine heart L-lactate dehydrogenase (LDH). LDH is a well-characterized redox enzyme, with strict substrate specificity, discriminating between NADH and the very similar NADPH. As suspected, LDH did not oxidize either NADPH or NADPH-capped RNA. However, LDH oxidized both NADH and NADH-capped RNA and reduced NAD+ and NAD+-capped RNA. This is the first study to show a clear biochemical role for ncinRNA, suggesting that ncinRNAs may be participants in cellular chemical reactions. ncinRNAs might therefore form a new and interesting class of key ribonucleoproteins or even ribozymes capable of catalyzing redox reactions.

This thesis aims to the address the elusive physiological role of NADH-cleaving Nudix hydrolases. For the first time, we report a distinct biochemical purpose for the existence of ncinRNAs in vivo involved in redox reactions.

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