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

Journal of molecular biology 2018 Mar 02

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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. Copyright © 2018 Elsevier Ltd. All rights reserved.

Citation

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. Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment. Journal of molecular biology. 2018 Mar 02;430(5):722-736