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Structural basis for the activity of a cytoplasmic RNA terminal uridylyl transferase

Nature Structural & Molecular Biology volume 19, pages 782–787 (2012)Cite this article

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Abstract

Cytoplasmic terminal uridylyl transferases comprise a conserved family of enzymes that negatively regulate the stability or biological activity of a variety of eukaryotic RNAs, including mRNAs and tumor-suppressor let-7 microRNAs. Here we describe crystal structures of the Schizosaccharomyces pombe cytoplasmic terminal uridylyl transferase Cid1 in two apo conformers and bound to UTP. We demonstrate that a single histidine residue, conserved in mammalian Cid1 orthologs, is responsible for discrimination between UTP and ATP. We also describe a new high-affinity RNA substrate–binding mechanism of Cid1, which is essential for enzymatic activity and is mediated by three basic patches across the surface of the enzyme. Overall, our structures provide a basis for understanding the activity of Cid1 and a mechanism of UTP selectivity conserved in its human orthologs, suggesting potential implications for anticancer drug design.

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Figure 1: The crystal structure of tCid1.
Figure 2: The UTP specificity of Cid1 is conferred by a conserved histidine residue.
Figure 3: Cid1 binds RNA via an extended basic surface.

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References

  1. Hagan, J.P., Piskounova, E. & Gregory, R.I. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 16, 1021–1025 (2009).

    Article  CAS  Google Scholar 

  2. Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696–708 (2009).

    Article  CAS  Google Scholar 

  3. Jones, M.R. et al. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat. Cell Biol. 11, 1157–1163 (2009).

    Article  CAS  Google Scholar 

  4. Lehrbach, N.J. et al. LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 16, 1016–1020 (2009).

    Article  CAS  Google Scholar 

  5. Mullen, T.E. & Marzluff, W.F. Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation of the mRNA both 5′ to 3′ and 3′ to 5′. Genes Dev. 22, 50–65 (2008).

    Article  CAS  Google Scholar 

  6. Schmidt, M.-J., West, S. & Norbury, C. The human cytoplasmic RNA terminal U-transferase ZCCHC11 targets histone mRNAs for degradation. RNA 17, 39–44 (2011).

    Article  CAS  Google Scholar 

  7. Shen, B. & Goodman, H.M. Uridine addition after microRNA-directed cleavage. Science 306, 997 (2004).

    Article  CAS  Google Scholar 

  8. Rissland, O.S., Mikulasova, A. & Norbury, C.J. Efficient RNA polyuridylation by noncanonical poly(A) polymerases. Mol. Cell. Biol. 27, 3612–3624 (2007).

    Article  CAS  Google Scholar 

  9. Rissland, O.S. & Norbury, C.J. Decapping is preceded by 3′ uridylation in a novel pathway of bulk mRNA turnover. Nat. Struct. Mol. Biol. 16, 616–623 (2009).

    Article  CAS  Google Scholar 

  10. Bard, J. et al. Structure of yeast poly(A) polymerase alone and in complex with 3′-dATP. Science 289, 1346–1349 (2000).

    Article  CAS  Google Scholar 

  11. Martin, G., Möglich, A., Keller, W. & Doublié, S. Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase. J. Mol. Biol. 341, 911–925 (2004).

    Article  CAS  Google Scholar 

  12. Piskounova, E. et al. Lin28A and Lin28B inhibit let-7 MicroRNA biogenesis by distinct mechanisms. Cell 147, 1066–1079 (2011).

    Article  CAS  Google Scholar 

  13. Sawaya, M.R., Pelletier, H., Kumar, A., Wilson, S.H. & Kraut, J. Crystal structure of rat DNA polymerase β: evidence for a common polymerase mechanism. Science 264, 1930–1935 (1994).

    Article  CAS  Google Scholar 

  14. Stagno, J., Aphasizheva, I., Rosengarth, A., Luecke, H. & Aphasizhev, R. UTP-bound and Apo structures of a minimal RNA uridylyltransferase. J. Mol. Biol. 366, 882–899 (2007).

    Article  CAS  Google Scholar 

  15. Riffel, N. et al. Atomic resolution structure of Moloney murine leukemia virus matrix protein and its relationship to other retroviral matrix proteins. Structure 10, 1627–1636 (2002).

    Article  CAS  Google Scholar 

  16. Stuart, D.I., Levine, M., Muirhead, H. & Stammers, D.K. Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 A. J. Mol. Biol. 134, 109–142 (1979).

    Article  CAS  Google Scholar 

  17. Stagno, J., Aphasizheva, I., Aphasizhev, R. & Luecke, H. Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases. Proc. Natl. Acad. Sci. USA 104, 14634–14639 (2007).

    Article  CAS  Google Scholar 

  18. Martin, G. & Keller, W. RNA-specific ribonucleotidyl transferases. RNA 13, 1834–1849 (2007).

    Article  CAS  Google Scholar 

  19. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  20. Deng, J., Ernst, N.L., Turley, S., Stuart, K.D. & Hol, W.G. Structural basis for UTP specificity of RNA editing TUTases from Trypanosoma brucei. EMBO J. 24, 4007–4017 (2005).

    Article  CAS  Google Scholar 

  21. Hamill, S., Wolin, S.L. & Reinisch, K.M. Structure and function of the polymerase core of TRAMP, a RNA surveillance complex. Proc. Natl. Acad. Sci. USA 107, 15045–15050 (2010).

    Article  CAS  Google Scholar 

  22. LaCava, J. et al. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121, 713–724 (2005).

    Article  CAS  Google Scholar 

  23. Vaňáčová, S. et al. A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol. 3, e189 (2005).

    Article  Google Scholar 

  24. Read, R.L., Martinho, R.G., Wang, S.W., Carr, A.M. & Norbury, C.J. Cytoplasmic poly(A) polymerases mediate cellular responses to S phase arrest. Proc. Natl. Acad. Sci. USA 99, 12079–12084 (2002).

    Article  CAS  Google Scholar 

  25. Deo, R.C., Bonanno, J.B., Sonenberg, N. & Burley, S.K. Recognition of polyadenylate RNA by the poly(A)-binding protein. Cell 98, 835–845 (1999).

    Article  CAS  Google Scholar 

  26. Bai, Y., Srivastava, S.K., Chang, J.H., Manley, J.L. & Tong, L. Structural basis for dimerization and activity of human PAPD1, a noncanonical poly(A) polymerase. Mol. Cell 41, 311–320 (2011).

    Article  CAS  Google Scholar 

  27. Hayward, S. & Berendsen, H.J. Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme. Proteins 30, 144–154 (1998).

    Article  CAS  Google Scholar 

  28. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  29. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  Google Scholar 

  30. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

  31. Leslie, A.G.W. & Powell, H.R. in Evolving Methods for Macromolecular Crystallography, Nato Science Series II Vol. 245 (eds Read, R.J. & Sussman, J.L.) 41–51 (Springer, 2007).

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

    Article  CAS  Google Scholar 

  33. Stein, N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641–643 (2008).

    Article  CAS  Google Scholar 

  34. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D Biol. Crystallogr. 60, 2210–2221 (2004).

    Article  CAS  Google Scholar 

  35. Zhang, K.Y., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Methods Enzymol. 277, 53–64 (1997).

    Article  CAS  Google Scholar 

  36. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).

    Article  Google Scholar 

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

    Article  Google Scholar 

  38. Painter, J. & Merritt, E.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62, 439–450 (2006).

    Article  Google Scholar 

  39. Kleywegt, G.J. Crystallographic refinement of ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 63, 94–100 (2007).

    Article  CAS  Google Scholar 

  40. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  Google Scholar 

  41. Pettersen, E.F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank N. Proudfoot, A. van der Merwe, F. Esashi and T. Schiffner for discussions and comments on the manuscript, A. Watson and J. Endicott for their input in the early stages of this project, and Cancer Research UK (C.J.N.), The Royal Society (R.J.C.G.), the Wellcome Trust (core-Centre grant 090532/Z/09/Z), the Medical Research Council (L.A.Y. and K.H.) and the EP Abraham Research Fund (C.J.N.) for financial support.

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Author notes

  1. Olivia S Rissland

    Present address: Present address: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.,

  2. Luke A Yates and Sophie Fleurdépine: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Luke A Yates, Luigi De Colibus, Karl Harlos & Robert J C Gilbert

  2. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Sophie Fleurdépine, Olivia S Rissland & Chris J Norbury

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  1. Luke A Yates
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Contributions

The project was initiated by O.S.R., L.A.Y., C.J.N. and R.J.C.G.; C.J.N and R.J.C.G. designed and supervised the project. O.S.R. designed the tCid1 expression construct. S.F. characterized the RNA-binding capacity of Cid1, designed and purified the tCid1 mutants and designed and performed EMSA, TUTase assays and SPR. L.A.Y. purified, crystallized tCid1 and optimized crystals for data collection. K.H. soaked and handled crystals for data collection. L.A.Y. collected, processed and analyzed the X-ray diffraction data. L.D.C. solved the initial structure; L.A.Y. solved subsequent structures and built and refined all tCid1 structures. L.A.Y. and R.J.C.G. performed the structural and phylogenetic analysis. L.A.Y., S.F., C.J.N. and R.J.C.G. wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Chris J Norbury or Robert J C Gilbert.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–4 and Supplementary Note (PDF 4937 kb)

Supplementary Video 1

Morphing between the Apo I, UTP-bound and Apo II structures (salmon pink) superimposed on the UTP-bound structure (yellow, UTP in green), viewed into active site cleft. (AVI 2118 kb)

Supplementary Video 2

As Supplementary Video 1, viewed from side. (AVI 1924 kb)

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Yates, L., Fleurdépine, S., Rissland, O. et al. Structural basis for the activity of a cytoplasmic RNA terminal uridylyl transferase. Nat Struct Mol Biol 19, 782–787 (2012). https://doi.org/10.1038/nsmb.2329

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