Structural and mechanistic basis of mammalian Nudt12 RNA deNADding

Abstract

We recently demonstrated that mammalian cells harbor nicotinamide adenine dinucleotide (NAD)-capped messenger RNAs that are hydrolyzed by the DXO deNADding enzyme. Here, we report that the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. The crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 Å resolution provides insights into the molecular basis of the deNADding activity in the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells, and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO, respectively, indicating an association of deNADding to cellular metabolism.

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Fig. 1: Mammalian Nudt12 possesses robust deNADding activity in vitro.
Fig. 2: RppH has RNA deNADding activity in vitro.
Fig. 3: Crystal structure of mouse Nudt12 in complex with AMP and three Mg2+ ions.
Fig. 4: Mammalian Nudt12 is a deNADding enzyme in cells.
Fig. 5: Nudt12 preferentially targets a subset of mRNAs for deNADding.
Fig. 6: Cellular exposure to stress impacts NAD capping.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Sequencing data have been deposited in the Gene Expression Omnibus (GEO) database (accession nos. GSE90884 (DXO-KO) and GSE110801 (N12-KO)). The atomic coordinates have been deposited at the Protein Data Bank (PDB entry 6O3P).

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Acknowledgements

This work was supported by National Institutes of Health (NIH) grant Nos. GM118093 and S10OD012018 (L.T.) and GM126488 (M.K.). We thank B. E. Nickels for helpful discussions and providing recombinant NudC. We thank K. Perry and R. Rajashankar for access to the NE-CAT 24-C beamline at the Advanced Photon Source. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, funded by the NIH (grant No. P41 GM103403). The Pilatus 6 M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (No. S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Computational resources were provided by the Office of Advanced Research Computing (OARC) at Rutgers, The State University of New Jersey, under the National Institutes of Health Grant No. S10OD012346.

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M.K., E.G.N. and L.T. designed the experiments. E.G.N. carried out all experiments unless otherwise indicated. X.J. and H.C. created N12 and N12:DXO CRISPR knockout cell lines. X.J. carried out the initial NAD captureSeq and the assays in Fig. 2. Y.W. and L.T. carried out the structural analysis and interpretations. M.K.M. carried out experiments in Supplementary Fig. 4. R.P.H. carried out all bioinformatics analyses. E.G.N. M.K., L.T., Y.W. and R.P.H. wrote the manuscript.

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Correspondence to Liang Tong or Megerditch Kiledjian.

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Grudzien-Nogalska, E., Wu, Y., Jiao, X. et al. Structural and mechanistic basis of mammalian Nudt12 RNA deNADding. Nat Chem Biol 15, 575–582 (2019). https://doi.org/10.1038/s41589-019-0293-7

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