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

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 catabo...

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Bibliographic Details
Published in:	Journal of molecular biology Vol. 430; no. 5; pp. 722 - 736
Main Authors:	Bulbrook, D., Brazier, H., Mahajan, P., Kliszczak, M., Fedorov, O., Marchese, F.P., Aubareda, A., Chalk, R., Picaud, S., Strain-Damerell, C., Filippakopoulos, P., Gileadi, O., Clark, A.R., Yue, W.W., Burgess-Brown, N.A., Dean, J.L.E.
Format:	Journal Article
Language:	English
Published:	England Elsevier Ltd 02-03-2018
Subjects:	3' Untranslated Regions AU-rich elements Autoantigens - genetics Autoantigens - metabolism deadenylation Exoribonucleases - genetics Exoribonucleases - metabolism HeLa Cells Humans inflammatory mRNA Mutagenesis, Site-Directed post-translational control Protein Interaction Domains and Motifs Receptors, CCR4 - genetics Receptors, CCR4 - metabolism RNA Stability RNA, Messenger - metabolism RNA-Binding Proteins - genetics RNA-Binding Proteins - metabolism Transcription Factors - genetics Transcription Factors - metabolism Tristetraprolin - genetics Tristetraprolin - metabolism Tryptophan - genetics Tryptophan - metabolism CXCL ORF IL FL inflammatory UTRs AU-rich elements GST mRNA post-translational control MAPK TNF TTP CTD Trp ARE MK2 CCR4-NOT ZFD deadenylation BLI NTD
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Summary:	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. [Display omitted] •TTP targets inflammatory mRNA for degradation by interacting with CCR4-CNOT complex.•We identified one subunit of the complex, CNOT9, as a novel interactor of TTP.•TTP binds using conserved Trp residues to CNOT9 regions common to other modulators.•Trp-mediated interactions with CNOT9 are essential to TTP-mediated decay.
ISSN:	0022-2836 1089-8638
DOI:	10.1016/j.jmb.2017.12.018