Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs.

Ewa Grudzien-Nogalska,Jun Yang,Keith Hamilton,Liang Tong,Megerditch Kiledjian,Sunny Sharma,Xinfu Jiao

doi:10.1093/nar/gkaa402

Ewa Grudzien-Nogalska, Jun Yang + Show 5 more

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https://doi.org/10.1093/nar/gkaa402

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Abstract

We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5′caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins—Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.

Highlights

Cellular metabolism and gene expression are two fundamental biological processes that share an intricate relationship, coordination of which is essential for the survival of all living organisms (1)
The recent identification of 5 end nicotinamide adenine dinucleotide (NAD) caps on mRNA in bacteria, yeast, plants and mammals has further expanded our understanding of the relationship between nucleotide metabolites and a potential direct role in gene expression (2–5)
We show an increase of flavin adenine dinucleotide (FAD)-capped RNA in cells depleted of Nudt[16], implicating its role as a deFADding enzyme in cells and report the crystal structure of Nudt[16] in the complex with FAD to reveal the structural details of the deFADding activity

Summary

Introduction

Cellular metabolism and gene expression are two fundamental biological processes that share an intricate relationship, coordination of which is essential for the survival of all living organisms (1). The recent identification of 5 end NAD caps on mRNA in bacteria, yeast, plants and mammals has further expanded our understanding of the relationship between nucleotide metabolites and a potential direct role in gene expression (2–5). Studies aimed at understanding the function of these non-canonical caps, both in bacteria and mammalian cells, have led to the identification of NAD-cap decapping (deNADding) enzymes––NudC in bacteria, its homolog Nudt[12] and the non-canonical decapping enzyme, DXO in mammals (2– 3,7–8). Similar to canonical decapping enzymes that each preferentially target a subset of mRNAs (9), Nudt[12] and DXO deNAD distinct subsets of NAD-capped mRNAs. Nudt[12] functions on a class of nuclear-encoded mitochondrial protein mRNAs containing an NAD-cap (8) and possesses canonical m7G-cap decapping activity on circadian clock mRNAs (10). Detection of NAD-capped intronic snoRNAs in cells devoid of

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