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

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, A. R. Clark, W.w. Yue, N. A. Burgess-Brown, J. L. E. Dean

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

Keywords

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

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