Recruitment of the 4EHP-GYF2 cap-binding complex to tetraproline motifs of tristetraprolin promotes repression and degradation of mRNAs with AU-rich elements

Abstract

The zinc finger protein tristetraprolin (TTP) promotes translation repression and degradation of mRNAs containing AU-rich elements (AREs). Although much attention has been directed toward understanding the decay process and machinery involved, the translation repression role of TTP has remained poorly understood. Here we identify the cap-binding translation repression 4EHP-GYF2 complex as a cofactor of TTP. Immunoprecipitation and in vitro pull-down assays demonstrate that TTP associates with the 4EHP-GYF2 complex via direct interaction with GYF2, and mutational analyses show that this interaction occurs via conserved tetraproline motifs of TTP. Mutant TTP with diminished 4EHP-GYF2 binding is impaired in its ability to repress a luciferase reporter ARE-mRNA. 4EHP knockout mouse embryonic fibroblasts (MEFs) display increased induction and slower turnover of TTP-target mRNAs as compared to wild-type MEFs. Our work highlights the function of the conserved tetraproline motifs of TTP and identifies 4EHP-GYF2 as a cofactor in translational repression and mRNA decay by TTP.

Keywords: ARE-mediated decay; AU-rich elements; mRNA turnover; translation repression; tristetraprolin.

FIGURE 1.

TTP interacts with the 4EHP-GYF2 complex. (A, top) Western blot for TTP in samples immunoprecipitated with anti-TTP or normal rabbit serum (NRS) from mouse macrophage RAW264.7 cells treated with lipopolysaccharide (LPS) for various lengths of time as indicated. (IgG h.c.) IgG heavy chain. (Bottom) Graph showing the number of peptides for 4EHP, GYF2, and GYF1 detected per 1000 of total detected peptides in LC-MS/MS analyses of the IPs. (B) Western blots of input and anti-Flag IPs from HEK293T cells cotransfected with myc-TTP and Flag-4EHP or Flag-GYF2. Lysates were treated with RNase A prior to IP. (C) Western blots monitoring His₆-4EHP and His₆-GYF2 interaction with GST or GST-TTP in in vitro pull-down assays using glutathione Sepharose beads. GST and GST-TTP were detected using anti-GST; 4EHP and GYF2 were detected using anti-4EHP and anti-GYF2, respectively. (D) Proposed model of interaction between TTP and the 4EHP-GYF2 complex. TTP directly binds GYF2, which is known to interact with 4EHP.

FIGURE 2.

The TTP N-terminal domain is necessary and sufficient for association with 4EHP and GYF2. Western blots for indicated factors in input and anti-myc IP fractions from RNase A-treated extracts of HEK293T cells transiently expressing indicated myc-tagged TTP variants and Flag-tagged 4EHP. A schematic of TTP is shown at the top indicating the N-terminal domain (NTD), the RNA-binding domain (RBD), the C-terminal domain (CTD), and the CNOT-interacting motif (CIM).

FIGURE 3.

TTP interacts with GYF2 through tetraproline motifs 1 and 2. (A) Sequence alignment of the tetraproline motifs of mouse TTP with corresponding regions of TTP from other vertebrates. Amino acids matching the PPPGφ GYF-binding consensus are highlighted in bold. (B) Western blots for proteins indicated on the left in input and anti-Flag IP samples from RNase A-treated extracts of HEK293T cells transiently expressing indicated Flag-tagged TTP variants. The first PPPPGF motif of TTP was mutated to PSSSGF (SSS) or PPPPDE (DE) in the contexts of full-length TTP (FL), the TTP NTD, or the 33 amino acids surrounding the motif (TTP_61–93). (C) Same as panel B, with each of the three tetraproline motifs of Flag-tagged TTP mutated as indicated in the schematic above (1-S, 2-S, 3-S), and all combinations of those mutations. (D) Western blot for an in vitro pull-down assay similar to Figure 1C, with the addition of GST-tagged 12-S mutant of TTP. (E) Western blots of input and anti-Flag IP samples similar to panel C. (F) Western blots of input and anti-myc IP samples from RNase A-treated extracts of HEK293T cells transiently expressing myc-tagged mouse TTP, BRF1, or BRF2. A mutant version of mouse TTP with all prolines of the first tetraproline motif and the immediate downstream four prolines replaced with alanines (Pmut) was also included.

FIGURE 4.

TTP mutated in tetraprolines 1 and 2 is impaired in its ability to repress a luciferase reporter ARE-mRNA. (A) Luciferase luminescence assays from TTP^−/− MEFs transiently coexpressing wild-type or mutant TTP with two reporters, one encoding firefly luciferase (F-Luc) containing in its 3′ UTR the AU-rich element (ARE) from human GM-CSF mRNA, and one encoding Renilla luciferase (R-Luc) without an ARE as an internal control. For each sample, F-Luc activity was normalized to R-Luc and values were normalized to the wild-type TTP-transfected condition, which was set as 1. Error bars represent standard error of the mean (SEM) (n = 4). P-values were calculated using Student's t-test (paired, two-tailed). (B) Representative Western blot of samples used in panel A. PABP was used as the loading control. (C) Luciferase assays in 4EHP^+/+ and 4EHP^−/− MEFs similar to the experiment in panel A except in the absence of cotransfected TTP and the absence or presence of transiently expressed 4EHP and GYF2 as indicated. Error bars represent SEM (n = 3).

FIGURE 5.

4EHP knockout MEFs show increased induction and stability of TTP-target mRNAs. (A) Quantification of Ier3, Cxcl10, Csf2, and Fos mRNA levels using qRT-PCR during serum induction of MEFs from 4EHP^+/+ (blue) and 4EHP^−/− (red) littermates. Gapdh mRNA was used as an internal control for normalization and values were normalized to the values for 4EHP^+/+ MEFs at t = 0. Error bars represent SEM (n = 3). (B, top) Western blots for TTP, 4EHP, and PABP during the serum induction time course described in panel A. (Bottom) qRT-PCR quantification of Ttp mRNA levels during the serum induction time course with error bars as in panel A. (C) Decay assays using actinomycin D transcriptional shutoff. mRNA levels were quantified by qRT-PCR and normalized to Gapdh mRNA, with values at t = 0 set to 1. Error bars represent SEM (n = 3). Calculated half-lives ± SEM of each mRNA species (assuming an infinite Gapdh mRNA half-life) are listed in minutes. P-values were calculated using Student's t-test (paired, two-tailed).

FIGURE 6.

TTP recruits multiple corepressors to repress target mRNAs. Schematic showing TTP motifs and domains contributing to mRNA repression. The CIM recruits the CCR4-NOT deadenylation complex via direct interaction with CNOT1. The NTD of TTP associates with the DCP2 decapping complex. Our work demonstrates the conserved tetraproline motifs of TTP recruiting the 4EHP-GYF2 complex.