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

J Mol Biol. 2018 Mar 2;430(5):722-736. doi: 10.1016/j.jmb.2017.12.018. Epub 2017 Dec 29.

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.

Keywords: AU-rich elements; deadenylation; inflammatory; mRNA; post-translational control.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Autoantigens / genetics
  • Autoantigens / metabolism
  • Exoribonucleases / genetics
  • Exoribonucleases / metabolism
  • HeLa Cells
  • Humans
  • Mutagenesis, Site-Directed
  • Protein Interaction Domains and Motifs
  • RNA Stability
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Receptors, CCR4 / genetics
  • Receptors, CCR4 / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Tristetraprolin / genetics
  • Tristetraprolin / metabolism*
  • Tryptophan / genetics
  • Tryptophan / metabolism*

Substances

  • 3' Untranslated Regions
  • Autoantigens
  • CCR4 protein, human
  • CNOT1 protein, human
  • CNOT9 protein, human
  • RNA, Messenger
  • RNA-Binding Proteins
  • Receptors, CCR4
  • TNRC6A protein, human
  • Transcription Factors
  • Tristetraprolin
  • ZFP36 protein, human
  • Tryptophan
  • Exoribonucleases
  • poly(A)-specific ribonuclease