Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment

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Authors

  • D. Bulbrook
  • H. Brazier
  • P. Mahajan
  • M. Kliszczak
  • O. Fedorov
  • F. P. Marchese
  • A. Aubareda
  • R. Chalk
  • S. Picaud
  • C. Strain-Damerell
  • P. Filippakopoulos
  • O. Gileadi
  • W.w. Yue
  • N. A. Burgess-Brown
  • J. L. E. Dean

Colleges, School and Institutes

Abstract

The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3′ untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex “carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT),” which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3′-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay.

Details

Original languageEnglish
JournalJournal of Molecular Biology
Early online date29 Dec 2017
Publication statusE-pub ahead of print - 29 Dec 2017

Keywords

  • deadenylation , inflammatory , mRNA , AU-rich elements , post-translational controls