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Structural basis for the recruitment of the human CCR4–NOT deadenylase complex by tristetraprolin

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Abstract

Tristetraprolin (TTP) is an RNA-binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3′ untranslated regions of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multisubunit CCR4–NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds a central domain of CNOT1, a core subunit of the CCR4–NOT complex. A high-resolution crystal structure of the TTP–CNOT1 complex was determined, providing the first structural insight, to our knowledge, into an ARE-binding protein bound to the CCR4–NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.

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Figure 1: Human TTP directly binds a central fragment of CNOT1.
Figure 2: Crystal structure of the TTP–CNOT1 complex.
Figure 3: Analysis of TTP-CNOT1 interaction.
Figure 4: Disruption of the TTP-CNOT1 interaction impairs mRNA deadenylation in vitro and mRNA stability in vivo.

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References

  1. Fabian, M.R., Sonenberg, N. & Filipowicz, W. Regulation of mRNA translation and stability by microRNAs. Annu. Rev. Biochem. 79, 351–379 (2010).

    Article  CAS  Google Scholar 

  2. von Roretz, C., Di Marco, S., Mazroui, R. & Gallouzi, I.E. Turnover of AU-rich-containing mRNAs during stress: a matter of survival. Wiley Interdiscip. Rev. RNA 2, 336–347 (2011).

    Article  CAS  Google Scholar 

  3. Sanduja, S., Blanco, F.F. & Dixon, D.A. The roles of TTP and BRF proteins in regulated mRNA decay. Wiley Interdiscip. Rev. RNA 2, 42–57 (2011).

    Article  CAS  Google Scholar 

  4. Blackshear, P.J. Tristetraprolin and other CCCH tandem zinc-finger proteins in the regulation of mRNA turnover. Biochem. Soc. Trans. 30, 945–952 (2002).

    Article  CAS  Google Scholar 

  5. Lai, W.S., Stumpo, D.J. & Blackshear, P.J. Rapid insulin-stimulated accumulation of an mRNA encoding a proline-rich protein. J. Biol. Chem. 265, 16556–16563 (1990).

    CAS  PubMed  Google Scholar 

  6. Sanduja, S., Blanco, F.F., Young, L.E., Kaza, V. & Dixon, D.A. The role of tristetraprolin in cancer and inflammation. Front. Biosci. 17, 174–188 (2012).

    Article  CAS  Google Scholar 

  7. Rounbehler, R.J. et al. Tristetraprolin impairs myc-induced lymphoma and abolishes the malignant state. Cell 150, 563–574 (2012).

    Article  CAS  Google Scholar 

  8. Carballo, E., Lai, W.S. & Blackshear, P.J. Feedback inhibition of macrophage tumor necrosis factor-α production by tristetraprolin. Science 281, 1001–1005 (1998).

    Article  CAS  Google Scholar 

  9. Taylor, G.A. et al. A pathogenetic role for TNFα in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 4, 445–454 (1996).

    Article  CAS  Google Scholar 

  10. Lai, W.S., Carballo, E., Thorn, J.M., Kennington, E.A. & Blackshear, P.J. Interactions of CCCH zinc finger proteins with mRNA: binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA. J. Biol. Chem. 275, 17827–17837 (2000).

    Article  CAS  Google Scholar 

  11. Lykke-Andersen, J. & Wagner, E. Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. Genes Dev. 19, 351–361 (2005).

    Article  CAS  Google Scholar 

  12. Sandler, H., Kreth, J., Timmers, H.T. & Stoecklin, G. Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin. Nucleic Acids Res. 39, 4373–4386 (2011).

    Article  CAS  Google Scholar 

  13. Collart, M.A. & Panasenko, O.O. The Ccr4–Not complex. Gene 492, 42–53 (2012).

    Article  CAS  Google Scholar 

  14. Goldstrohm, A.C. & Wickens, M. Multifunctional deadenylase complexes diversify mRNA control. Nat. Rev. Mol. Cell Biol. 9, 337–344 (2008).

    Article  CAS  Google Scholar 

  15. Schütz, P. et al. Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions. Proc. Natl. Acad. Sci. USA 105, 9564–9569 (2008).

    Article  Google Scholar 

  16. Ponting, C.P. Novel eIF4G domain homologues linking mRNA translation with nonsense-mediated mRNA decay. Trends Biochem. Sci. 25, 423–426 (2000).

    Article  CAS  Google Scholar 

  17. Baron-Benhamou, J., Gehring, N.H., Kulozik, A.E. & Hentze, M.W. Using the λN peptide to tether proteins to RNAs. Methods Mol. Biol. 257, 135–154 (2004).

    CAS  PubMed  Google Scholar 

  18. Fabian, M.R. et al. miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4–NOT. Nat. Struct. Mol. Biol. 18, 1211–1217 (2011).

    Article  CAS  Google Scholar 

  19. Blackshear, P.J. et al. Zfp36l3, a rodent X chromosome gene encoding a placenta-specific member of the Tristetraprolin family of CCCH tandem zinc finger proteins. Biol. Reprod. 73, 297–307 (2005).

    Article  CAS  Google Scholar 

  20. Basquin, J. et al. Architecture of the nuclease module of the yeast CCR4–NOT complex: the Not1-Caf1-Ccr4 interaction. Mol. Cell 48, 207–218 (2012).

    Article  CAS  Google Scholar 

  21. Petit, A.P. et al. The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4–NOT deadenylase complex. Nucleic Acids Res. 40, 11058–11072 (2012).

    Article  CAS  Google Scholar 

  22. Chekulaeva, M. et al. miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs. Nat. Struct. Mol. Biol. 18, 1218–1226 (2011).

    Article  CAS  Google Scholar 

  23. Chrestensen, C.A. et al. MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding. J. Biol. Chem. 279, 10176–10184 (2004).

    Article  CAS  Google Scholar 

  24. Stoecklin, G. et al. MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J. 23, 1313–1324 (2004).

    Article  CAS  Google Scholar 

  25. Marchese, F.P. et al. MAPKAP kinase 2 blocks tristetraprolin-directed mRNA decay by inhibiting CAF1 deadenylase recruitment. J. Biol. Chem. 285, 27590–27600 (2010).

    Article  CAS  Google Scholar 

  26. Clement, S.L., Scheckel, C., Stoecklin, G. & Lykke-Andersen, J. Phosphorylation of tristetraprolin by MK2 impairs AU-rich element mRNA decay by preventing deadenylase recruitment. Mol. Cell Biol. 31, 256–266 (2011).

    Article  CAS  Google Scholar 

  27. Mossessova, E. & Lima, C.D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876 (2000).

    Article  CAS  Google Scholar 

  28. Jinek, M., Fabian, M.R., Coyle, S.M., Sonenberg, N. & Doudna, J.A. Structural insights into the human GW182-PABC interaction in microRNA-mediated deadenylation. Nat. Struct. Mol. Biol. 17, 238–240 (2010).

    Article  CAS  Google Scholar 

  29. Lai, W.S. et al. Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor α mRNA. Mol. Cell Biol. 19, 4311–4323 (1999).

    Article  CAS  Google Scholar 

  30. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).

    Article  Google Scholar 

  31. Schneider, T.R. & Sheldrick, G.M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  Google Scholar 

  32. Usón, I., Stevenson, C.E., Lawson, D.M. & Sheldrick, G.M. Structure determination of the O-methyltransferase NovP using the 'free lunch algorithm' as implemented in SHELXE. Acta Crystallogr. D Biol. Crystallogr. 63, 1069–1074 (2007).

    Article  Google Scholar 

  33. Langer, G., Cohen, S.X., Lamzin, V.S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008).

    Article  CAS  Google Scholar 

  34. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by Canadian Institutes of Health Research grants to N. Sonenberg (MOP-93607) and to B.N. (MOP-82929). N. Sonenberg was also supported by a Howard Hughes Medical Institute Senior International Scholarship. P.J.B. was supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Environmental Health Sciences. N. Siddiqui is supported by a fellowship from the Cole Foundation.

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Authors

Contributions

M.R.F., F.F., C.R. and N. Siddiqui designed experiments. M.R.F. and C.R. performed binding assays and in vitro deadenylation assays. N. Siddiqui performed ITC experiments. F.F. crystallized the CNOT1–TTP complex, and B.N. and F.F. determined its structure. W.S.L. and P.J.B. performed in vivo stability assays. A.K. provided technical support. M.R.F., F.F., B.N. and N. Sonenberg wrote the manuscript.

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Correspondence to Marc R Fabian, Bhushan Nagar or Nahum Sonenberg.

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The authors declare no competing financial interests.

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Fabian, M., Frank, F., Rouya, C. et al. Structural basis for the recruitment of the human CCR4–NOT deadenylase complex by tristetraprolin. Nat Struct Mol Biol 20, 735–739 (2013). https://doi.org/10.1038/nsmb.2572

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