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Nuclear RNR-α antagonizes cell proliferation by directly inhibiting ZRANB3

Abstract

Since the origins of DNA-based life, the enzyme ribonucleotide reductase (RNR) has spurred proliferation because of its rate-limiting role in de novo deoxynucleoside-triphosphate (dNTP) biosynthesis. Paradoxically, the large subunit, RNR-α, of this obligatory two-component complex in mammals plays a context-specific antiproliferative role. There is little explanation for this dichotomy. Here, we show that RNR-α has a previously unrecognized DNA-replication inhibition function, leading to growth retardation. This underappreciated biological activity functions in the nucleus, where RNR-α interacts with ZRANB3. This process suppresses ZRANB3’s function in unstressed cells, which we show to promote DNA synthesis. This nonreductase function of RNR-α is promoted by RNR-α hexamerization—induced by a natural and synthetic nucleotide of dA/ClF/CLA/FLU—which elicits rapid RNR-α nuclear import. The newly discovered nuclear signaling axis is a primary defense against elevated or imbalanced dNTP pools that can exert mutagenic effects irrespective of the cell cycle.

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Fig. 1: Functional interaction of RNR-α with nuclear protein ZRANB3.
Fig. 2: Nuclear RNR-α suppresses DNA replication by eliciting loss of function of ZRANB3.
Fig. 3: dA-Mimetics drive partial RNR-α nuclear translocation that saturates rapidly, independent of cell cycle, DNA damage, RNR-reductase activity, or RNR-β/-p53β.
Fig. 4: RNR-α nuclear translocation is functionally linked to RNR-α6RD hexamerization driven by dATP and its mimetics.
Fig. 5: IRBIT regulates nucleus:cytosol levels of endogenous RNR-α through cytosol anchoring of RNR-α6RD hexamers.
Fig. 6: Schematic model illustrating the double-agent role discovered for the enzyme RNR.

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Acknowledgements

Thanks to members of the individual labs who generously provided plasmids and shRNAs as indicated in on-line methods; J. Page for contributing to the creation of RNR-α(D57N) knock-in mice; A. Arnaoutov (Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH) for pfastbac-GST-IRBIT; V. Gorbunova (University of Rochester) for NHEJ-reporter plasmids; J. Yuan (Columbia University) for the plasmids SFB-ZRANB3, SFB-ZRANB3-Δ PIP and SFBZRANB3(Q519A); D. Ahel (Oxford University) for the plasmids YFP-ZRANB3 and Flag-ZRANB3; Z. Zhang (University of Delaware) for the plasmid pet15b-His5-PCNA; A. Grimson (Cornell University) for the shRNA plasmids for RNR-α, RNR-β and IRBIT. Research: Pershing Square Sohn Cancer Research Alliance grant (to Y.A.); Meyer Cancer Center grant (Weill Cornell Medicine) (to Y.A. and R.S.W.); and the Canadian Institutes of Health Research grant (MOP-82930) (to J.O.). Instrumentation and shared supplies: NIH DP2 New Innovator (1DP2GM114850); NSF CAREER (CHE-1351400); Office of Naval Research (ONR) Young Investigator (N00014-17-1-2529); Beckman Young Investigator; Sloan Fellowship (FG-2016-6379) (to Y.A.); Cornell NMR facility (NSF MRI: CHE-1531632; PI: Y.A.) and Cornell Imaging Center (NIH 1S10RR025502; PI: R.M. Williams).

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Y.F., M.J.C.L. and Y.A. designed the experiments. Y.F. and M.J.C.L. performed the experiments. S.W. synthesized ClF, CLA and FLU nucleotides. H.I. and J.O. performed electron microscopy analysis. I.M.E. assisted M.J.C.L. with targeted mutagenesis for binding-site analysis. M.J.C.L., T.M.P., J.C.B. and R.S.W. generated mouse embryonic fibroblast cultures. Y.F., M.J.C.L. and Y.A. analyzed and interpreted the data. Y.F., M.J.C.L. and Y.A. wrote the paper with proof-editing contributions from R.S.W. and J.O.

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Correspondence to Yimon Aye.

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Fu, Y., Long, M.J.C., Wisitpitthaya, S. et al. Nuclear RNR-α antagonizes cell proliferation by directly inhibiting ZRANB3. Nat Chem Biol 14, 943–954 (2018). https://doi.org/10.1038/s41589-018-0113-5

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