Structural basis for the recruitment of the human CCR4–NOT deadenylase complex by tristetraprolin

Abstract

Tristetraprolin (TTP) is an RNA binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3′UTRs of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multi subunit CCR4–NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds to a central domain of CNOT1, a core subunit of the CCR4–NOT complex. A high-resolution crystal structure of the TTP-CNOT1 complex was determined, providing the first structural insight into an ARE-binding protein bound to the CCR4–NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.

The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies

“…This sequence is not particularly well conserved in the other insects in the alignment shown, but there are neighboring regions of alternating hydrophobic residues that may turn out to be nuclear export sequences. The conserved C-terminal region in human TTP has recently been shown to be responsible for binding to the NOT1 scaffolding protein and appears to be involved in recruiting deadenylases to the protein-RNA complex (33). Outside of these two domains, there was remarkably little sequence conservation between the protein from D. melanogaster and those from the other two insects.…”

“…3E, using a mouse Mlp mRNA protein coding domain fused to a portion of the mouse Tnf mRNA as a target transcript (33), the N-terminal HA-tagged Drosophila Tis11 protein was effective in decreasing the steady-state levels of the target transcript (Fig. 3E, compare lanes 1 and 2).…”

“…The conserved C-terminal motif of nine amino acids, RLP(I/V)FNR(I/L)S, recently implicated in the binding of human TTP to NOT1 (33), is located at 414 -422 of the Tis11 isoform A (GenBank TM accession number NP_ 511141.2). A 5Ј primer containing the Asp718 restriction enzyme site and encoding the Tis11 isoform A residues 1-7, 5Ј-ggtaccATGTCTGCTGATATTCTGCAG-3Ј, was used to create the following expression constructs.…”

“…Structural Models of the Drosophila Tis11 Putative Not1 Binding Domain-To explore further the feasibility of the Tis11 C-terminal domain as a Not1 binding domain, we performed simulation solution modeling of the proposed association between the Tis11 C-terminal peptide and the putative Drosophila Not1 Tis11 binding domain, based on the original coordinates of Fabian et al (33). The C-terminal sequences from human TTP and Drosophila Tis11 are aligned in Fig.…”

“…Human TTP (or Tis11) Bound to Human NOT1 or Drosophila Not1-Using the x-ray crystal structure (Protein Data Bank entry 4J8S) of human TTP bound to human NOT1 (residues 820 -999) (33), an initial model for the conserved Tis11 C-terminal domain bound to the TTP binding domain of human NOT1 was created. Necessary mutations were carried out using the program Coot (37).…”