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Structural and mechanistic basis of mammalian Nudt12 RNA deNADding

Nature Chemical Biology volume 15, pages 575–582 (2019)Cite this article

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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).

References

  1. Chen, Y. G., Kowtoniuk, W. E., Agarwal, I., Shen, Y. & Liu, D. R. LC/MS analysis of cellular RNA reveals NAD-linked RNA. Nat. Chem. Biol. 5, 879–881 (2009).

    Article  CAS  Google Scholar 

  2. Cahova, H., Winz, M. L., Hofer, K., Nubel, G. & Jaschke, A. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs. Nature 519, 374–377 (2015).

    Article  CAS  Google Scholar 

  3. Bird, J. G. et al. The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA. Nature 535, 444–447 (2016).

    Article  CAS  Google Scholar 

  4. Malygin, A. G. & Shemyakin, M. F. Adenosine, NAD and FAD can initiate template-dependent RNA synthesis catalyzed by Escherichia coli RNA polymerase. FEBS Lett. 102, 51–54 (1979).

    Article  CAS  Google Scholar 

  5. Julius, C. & Yuzenkova, Y. Bacterial RNA polymerase caps RNA with various cofactors and cell wall precursors. Nucleic Acids Res. 45, 8282–8290 (2017).

    Article  CAS  Google Scholar 

  6. Walters, R. W. et al. Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 114, 480–485 (2017).

    Article  CAS  Google Scholar 

  7. Jiao, X. et al. 5′ end nicotinamide adenine dinucleotide cap in human cells promotes RNA decay through DXO-mediated deNADding. Cell 168, 1015–1027.e10 (2017).

    Article  CAS  Google Scholar 

  8. Frick, D. N. & Bessman, M. J. Cloning, purification, and properties of a novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic domain in MutT-like enzymes. J. Biol. Chem. 270, 1529–1534 (1995).

    Article  CAS  Google Scholar 

  9. Luciano, D. J., Vasilyev, N., Richards, J., Serganov, A. & Belasco, J. G. Importance of a diphosphorylated intermediate for RppH-dependent RNA degradation. RNA Biol. 15, 703–706 (2018).

    PubMed  Google Scholar 

  10. Mackie, G. A. Ribonuclease E is a 5′-end-dependent endonuclease. Nature 395, 720–723 (1998).

    Article  CAS  Google Scholar 

  11. Zhang, D. et al. Structural basis of prokaryotic NAD-RNA decapping by NudC. Cell Res. 26, 1062–1066 (2016).

    Article  CAS  Google Scholar 

  12. Srouji, J. R., Xu, A., Park, A., Kirsch, J. F. & Brenner, S. E. The evolution of function within the Nudix homology clan. Proteins 85, 775–811 (2017).

    Article  CAS  Google Scholar 

  13. Abdelraheim, S. R., Spiller, D. G. & McLennan, A. G. Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein. Biochem. J. 374, 329–335 (2003).

    Article  CAS  Google Scholar 

  14. Carreras-Puigvert, J. et al. A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family. Nat. Commun. 8, 1541 (2017).

    Article  Google Scholar 

  15. Jiao, X., Chang, J. H., Kilic, T., Tong, L. & Kiledjian, M. A mammalian pre-mRNA 5′ end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing. Mol. Cell 50, 104–115 (2013).

    Article  CAS  Google Scholar 

  16. Kiledjian, M. Eukaryotic RNA 5′-end NAD+ capping and deNADding. Trends Cell Biol. 28, 454–464 (2018).

    Article  CAS  Google Scholar 

  17. Grudzien-Nogalska, E. & Kiledjian, M. New insights into decapping enzymes and selective mRNA decay. Wiley Interdiscip. Rev. RNA 8, e1379 (2017).

    Article  Google Scholar 

  18. Grudzien-Nogalska, E., Bird, J. G., Nickels, B. E. & Kiledjian, M. ‘NAD-capQ’ detection and quantitation of NAD caps. RNA 24, 1418–1425 (2018).

    Article  CAS  Google Scholar 

  19. Abdelraheim, S. R., Spiller, D. G. & McLennan, A. G. Mouse Nudt13 is a mitochondrial Nudix hydrolase with NAD(P)H pyrophosphohydrolase activity. Protein J. 36, 425–432 (2017).

    Article  CAS  Google Scholar 

  20. Piccirillo, C., Khanna, R. & Kiledjian, M. Functional characterization of the mammalian mRNA decapping enzyme hDcp2. RNA 9, 1138–1147 (2003).

    Article  CAS  Google Scholar 

  21. Song, M. G., Bail, S. & Kiledjian, M. Multiple Nudix family proteins possess mRNA decapping activity. RNA 19, 390–399 (2013).

    Article  CAS  Google Scholar 

  22. Mildvan, A. S. et al. Structures and mechanisms of Nudix hydrolases. Arch. Biochem. Biophys. 433, 129–143 (2005).

    Article  CAS  Google Scholar 

  23. Walters, R. W., Shumilin, I. A., Yoon, J. H., Minor, W. & Parker, R. Edc3 function in yeast and mammals is modulated by interaction with NAD-related compounds. G3 4, 613–622 (2014).

    Article  CAS  Google Scholar 

  24. Hofer, K. et al. Structure and function of the bacterial decapping enzyme NudC. Nat. Chem. Biol. 12, 730–734 (2016).

    Article  Google Scholar 

  25. Wang, Z. & Kiledjian, M. Functional link between the mammalian exosome and mRNA decapping. Cell 107, 751–762 (2001).

    Article  CAS  Google Scholar 

  26. Song, M. G., Li, Y. & Kiledjian, M. Multiple mRNA decapping enzymes in mammalian cells. Mol. Cell. 40, 423–432 (2010).

    Article  CAS  Google Scholar 

  27. Winz, M. L. et al. Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq. Nat. Protoc. 12, 122–149 (2017).

    Article  CAS  Google Scholar 

  28. Fulco, M. et al. Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt. Dev. Cell 14, 661–673 (2008).

    Article  CAS  Google Scholar 

  29. Canto, C. et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 458, 1056–1060 (2009).

    Article  CAS  Google Scholar 

  30. Canto, C. et al. Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle. Cell Metab. 11, 213–219 (2010).

    Article  CAS  Google Scholar 

  31. Chen, D. et al. Tissue-specific regulation of SIRT1 by calorie restriction. Genes Dev. 22, 1753–1757 (2008).

    Article  CAS  Google Scholar 

  32. Raynes, R. et al. The SIRT1 modulators AROS and DBC1 regulate HSF1 activity and the heat shock response. PLoS ONE 8, e54364 (2013).

    Article  CAS  Google Scholar 

  33. Li, Y., Ho, E. S., Gunderson, S. I. & Kiledjian, M. Mutational analysis of a Dcp2-binding element reveals general enhancement of decapping by 5′-end stem-loop structures. Nucleic Acids Res. 37, 2227–2237 (2009).

    Article  CAS  Google Scholar 

  34. Li, Y., Song, M. G. & Kiledjian, M. Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein. Mol. Cell Biol. 28, 939–948 (2008).

    Article  CAS  Google Scholar 

  35. Arribas-Layton, M., Wu, D., Lykke-Andersen, J. & Song, H. Structural and functional control of the eukaryotic mRNA decapping machinery. Biochim. Biophys. Acta 1829, 580–589 (2013).

    Article  CAS  Google Scholar 

  36. Li, Y. & Kiledjian, M. Regulation of mRNA decapping. Wiley Interdiscip. Rev. RNA 1, 253–265 (2010).

    Article  Google Scholar 

  37. Frindert, J. et al. Identification, biosynthesis, and decapping of NAD-capped RNAs in B. subtilis. Cell Rep. 24, 1890–1901.e8 (2018).

    Article  CAS  Google Scholar 

  38. Coleman, T. M., Wang, G. & Huang, F. Superior 5′ homogeneity of RNA from ATP-initiated transcription under the T7 phi 2.5 promoter. Nucleic Acids Res. 32, e14 (2004).

    Article  Google Scholar 

  39. Jiao, X. et al. Identification of a quality-control mechanism for mRNA 5′-end capping. Nature 467, 608–611 (2010).

    Article  CAS  Google Scholar 

  40. Liu, S. W., Jiao, X., Welch, S. & Kiledjian, M. Analysis of mRNA decapping. Methods Enzymol. 448, 3–21 (2008).

    Article  CAS  Google Scholar 

  41. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

    Article  CAS  Google Scholar 

  42. Erijman, A., Dantes, A., Bernheim, R., Shifman, J. M. & Peleg, Y. Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J. Struct. Biol. 175, 171–177 (2011).

    Article  CAS  Google Scholar 

  43. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010).

    Article  CAS  Google Scholar 

  44. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  45. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002).

    Article  Google Scholar 

  46. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  47. Andersen, J., VanScoy, S., Cheng, T. F., Gomez, D. & Reich, N. C. IRF-3-dependent and augmented target genes during viral infection. Genes Immun. 9, 168–175 (2008).

    Article  CAS  Google Scholar 

  48. Dudoit, S., Gentleman, R. C. & Quackenbush, J. Open source software for the analysis of microarray data. Biotechniques 34, S45–S51 (2003).

    Article  Google Scholar 

  49. Gentleman, R. C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

    Article  Google Scholar 

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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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  1. These authors contributed equally: E. Grudzien-Nogalska, Y. Wu.

Authors and Affiliations

  1. Department Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA

    Ewa Grudzien-Nogalska, Xinfu Jiao, Huijuan Cui, Maria K. Mateyak, Ronald P. Hart & Megerditch Kiledjian

  2. Department Biological Sciences, Columbia University, New York, NY, USA

    Yixuan Wu & Liang Tong

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Contributions

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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