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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 (Trp) residues in TTP that serve as major sites of interaction with two Trp-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 publication

DOI

10.1016/j.jmb.2017.12.018

Type

Journal article

Journal

Journal of molecular biology

Publication Date

29/12/2017

Addresses

Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, United Kingdom.