Cap-binding protein 4EHP effects translation silencing by microRNAs

Abstract

MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.

Keywords: 4E-T; 4EHP; CCR4–NOT; mRNA translation; microRNA.

Fig. 1.

Proteomic identification of the 4EHP and eIF4E proximal proteins. (A) High-confidence protein interactions discovered by BioID for the indicated baits in HEK293 cells. CT (C-terminal) and NT (N-terminal) indicate the location of BirA* fusion protein in relation to the Bait protein. An average of two independent experiments for each tagged variant is presented. Interacting proteins were categorized according to their known functions. Avg-Spec shows the spectral counts for each indicated prey. BFDR, Bayesian FDR. A complete list of the proximal proteins for each bait is presented in Dataset S1. (B) GO analysis of the BirA*–4EHP proximal proteins. The 10 most significantly enriched biological processes identified by using prohits-viz.lunenfeld.ca/ running g:Profiler (51) software are presented. A complete list of the enriched biological processes and molecular pathways is presented in Dataset S2. (C) HEK293T cells were transiently transfected with control or 3xFlag-4EHP plasmids. Two days later, cytoplasmic cell lysate was immunoprecipitated by using anti-Flag antibody in the presence of RNase A. LE, long exposure; SE, short exposure. (D) Cytoplasmic cell lysate from HEK293T cells was immunoprecipitated by using anti-DDX6 or IgG antibodies. (E) HEK293T cells were transiently transfected with control or HA-PATL-1 plasmid. Two days later, cytoplasmic cell lysate was immunoprecipitated by using anti-HA antibody. Western blot was performed by using the specified antibodies.

Fig. S1.

Analysis of 4EHP and eIF4E-proximal proteins by BioID assay. (A) Summary of workflow used to identify BirA*–4EHP and BirA*–eIF4E proximal proteins through BioID. (B) Lysates from cells used in Fig. 1A were analyzed by Western blot with anti-Flag antibody. CT (C-terminal) and NT (N-terminal) indicate the location of BirA* fusion protein in relation to the bait proteins. Extract from cells transfected by an empty vector (EV) is also shown. The spectral counts for the BirA tag are indicated for each bait. Total protein staining (Ponceau S) was used for loading control. (C and D) Correlation between replicates in BirA*–4EHP and –eIF4E BioID assays. R² indicates Pearson correlation.

Fig. 2.

miRISC/CCR4–NOT-mediated translational silencing is impaired by 4EHP depletion. (A, Upper) Schematic representation of the RL-Hmga2 3′ UTR reporter. (A, Lower) WT and 4EHP-KO MEFs were cotransfected with RL-Hmga2 3′ UTR (WT) or mutant (Mut), along with Firefly luciferase (FL). Luciferase activity was measured 24 h after transfection. Renilla luciferase (RL) values were normalized against FL levels, and repression fold was calculated for the RL-Hmga2 3′ UTR (WT) relative to RL-Hmga2 3′ UTR (Mut) level for each population. The same data are shown as relative RL/FL levels in Fig. S2B. (B, Upper) Schematic representation of the FL-E2f 3′ UTR reporter. (B, Lower) shCTR and sh4EHP U251 cells were cotransfected with pGL3-FL-E2f 3′ UTR (WT) or a variant with mutations disrupting the two miR-17/20a–binding sites (Mut), along with RL. FL values were normalized against RL levels, and repression fold was calculated for the FL-E2f 3′ UTR (WT) relative to FL-E2f 3′ UTR (Mut) level for each population. (C) Diagram of full-length CNOT1 and the N-terminal, middle, and C-terminal fragments used in D. The binding partners in CNOT1 are also depicted and the main domains are shown in green. Amino acid positions at domain boundaries are indicated below the protein outlines. (D, Upper) Vectors expressing V5-CNOT1 and 3xFlag–4EHP (or control plasmid) were transfected into HEK293T cells. IP of V5-CNOT1 from RNase A-treated extracts was performed by using anti-V5 antibody. Purified proteins were analyzed by Western blot. (D, Lower) HEK293T cells were transfected with vectors expressing V5-CNOT1 fragments (as described in C): N, N-terminal region (amino acids 1–690); M, middle repressive module (amino acids 1,030–1,600); and C, C-terminal region (amino acids 1,830–2,376) and 3xFlag-4EHP. Extracts were subjected to anti-V5 IP, and the eluted fractions were analyzed by Western blot. (E) The 4EHP protein mediates translational repression by tethered CNOT1 and GW182(SD). (E, Upper) Schematic representation of the RL-5boxB-A₁₁₄-N₄₀-HhR reporter. Control HEK293T cells (shCTR) or cells depleted of 4EHP (sh4EHP) were cotransfected with vectors expressing either λN-CNOT1, λN-GW182(SD) or λN control, along with RL-5boxB-A₁₁₄-N₄₀-HhR or RL-A₁₁₄-N₄₀-HhR, and FL. RL luminescence was normalized against the FL level. (E, Lower) Fold repression of RL-5boxB-A₁₁₄-N₄₀-HhR relative to RL-A₁₁₄-N₄₀-HhR expression is shown. Repression of RL-5boxB-A₁₁₄-N₄₀-HhR by λN alone in shCTR cells was set as 1. (F) Rescue assay for λN-CNOT1–dependent silencing was performed, as described in E, in cells depleted of 4EHP. shCTR and sh4EHP HEK293T cells were transfected with the indicated plasmids in combination with constructs expressing shRNA-resistant versions of 3xFlag–4EHP (WT), 3xFlag–4EHP^W124A cap-binding mutant (Mut), or empty vector (EV). The experiments illustrated in A, B, E, and F are represented as mean values (±SD) of three independent experiments. The P value was determined by two-tailed Student's t test. ns, nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S2.

The 4EHP-dependent translational repression by the miRISC/CCR4–NOT complex. (A) Lysates from WT and 4EHP-KO MEFs were analyzed by Western blot with the indicated antibodies. (B) Expression of RL-Hmga2 3′ UTR reporter in WT and 4EHP KO MEFs. Data from Fig. 2A are represented as relative RL/FL levels. Values show relative RL activities (RL/FL), with normalized RL-Hmga2 3′ UTR (Mut) levels set equal to 100% for each cell population. (C) Western blot analysis of protein lysates from U251 cells (Fig. 2B) with the indicated antibodies. (D, Upper) Schematic representation of the FL-3xmiR-19a reporter. (D, Lower) Repression of FL-3xmiR-19a reporter in 4EHP-depleted HeLa cells. Cells were transfected by nontargeting siRNAs (siCTR) or by siRNAs targeting 4EHP (si4EHP). Two days after transfection, cells were cotransfected by pmiRGLO-FL-3xmiR-19a (WT) or a variant containing mutations in the three miR-19a–binding sites (Mut). Luciferase activity was measured 24 h after transfection. Firefly values were normalized against Renilla levels, and repression fold was calculated for FL-3xmiR-19a (WT) relative to FL-3xmiR-19a (Mut) level for each population. (E) Extracts from the HeLa cells used in D were analyzed by Western blot with the indicated antibodies. (F) Quantitative PCR analysis of the indicated miRNAs in control and 4EHP-depleted cells. Expression of let-7a, miR-19a, miR-17a, and miR-20a was measured in the indicated cell lines by using TaqMan miRNA quantitative RT-PCR. miRNA levels were normalized to 18S expression values, and the results are presented as fold changes (control cells set as 1). (G) Western blot analysis of the shCTR and sh4EHP lysates from Fig. 2E. A total of 20 µg of extracts were loaded on a 4–12% gradient SDS/PAGE gel, and Western blot was performed by using the specified antibodies. (H, Upper) Schematic depiction of λN-CNOT1 tethered to RL-5boxB-A₁₁₄-N₄₀-HhR reporter mRNA. (H, Lower) HEK293T cells were transfected by nontargeting siRNAs (siCTR) or by siRNAs targeting 4E-T (si4E-T). Two days after transfection, cells were cotransfected by a mixture of plasmids to measure to silencing activity of λN-CNOT1 as described in Fig. 2E. Repression fold of RL-5boxB-A₁₁₄-N₄₀-HhR relative to RL-A₁₁₄-N₄₀-HhR expression is shown. Repression of RL-5boxB-A₁₁₄-N₄₀-HhR by λN alone was set as 1 in each cell population. (I) Lysates from H were analyzed by Western blot. (J) Lysates from Fig. 2F were analyzed by Western blot with the indicated antibodies. The experiments illustrated in B, D, F, and H are represented as mean values (±SD) of three independent experiments. The P value was determined by two-tailed Student's t test. ns, nonsignificant. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

The 4EHP protein competes with eIF4E for binding to 4E-T. (A) HEK293T cells were transfected with vectors expressing HA-tagged WT 4E-T or the eIF4E/4EHP-binding mutant variants and 3xFlag–4EHP (or control vector). Lysates were immunoprecipitated by using the anti-HA antibody. Western blot was performed by using the specified antibodies. (B) Fractionation of endogenous 4E-T, 4EHP, and eIF4E by size-exclusion chromatography. A total of 5 mg of proteins from HEK293T cells was loaded onto a Superose 6 column and run at a flow rate of 0.5 mL/min. Fractions of 0.5 mL were collected, and 50 µL of each fraction was analyzed by Western blot. The elution position of the molecular size markers is shown. A total of 50 µg (1%) of input was used for Western blot. (C) HEK293T cells were transfected with vectors expressing HA–4E-T and increasing amounts of 3xFlag–4EHP. Anti-HA IP and Western blot were performed as described in A. (D) Binding assays with 4E-T/4EHP and 4E-T/eIF4E complexes. Constant amounts of the HA–4E-T–bound beads were incubated with increasing concentrations of recombinant His-4EHP or His-eIF4E (0, 0.03, 0.06, 0.12, 0.25, 0.5, and 1 µM). After washing the beads, the retained complexes were eluted and analyzed by Western blot with anti-His and -HA antibodies. (E) In vitro displacement assay. Preassembled eIF4E/4E-T complexes bound on anti-HA agarose beads were incubated with increasing amounts of 4EHP (0, 0.11, 0.22, 0.44, 0.89, and 1.78 µM). Proteins were eluted and analyzed by Western blot. (F) Bar graph, obtained by densitometry analysis of Western blot data from E, shows quantified intensities of eIF4E and 4EHP signals normalized against 4E-T. The data are expressed as mean values (±SD) of two independent experiments.

Fig. S3.

Analysis of recombinant 4EHP and eIF4E. (A) SDS/PAGE and Coomassie brilliant blue staining of purified His-4EHP and -eIF4E proteins. (B) Quantification of purified His-4EHP and -eIF4E proteins by SDS/PAGE and Coomassie staining. (C) Binding assay with 4E-T/4EHP and 4E-T/eIF4E complexes. Similar to Fig. 3D, constant amounts of the 4E-T–bound beads were incubated with increasing concentrations of recombinant His-4EHP (0, 66, 85, 160, 234, 468, 930, 3,750, and 7,500 nM) or His-eIF4E (0, 234, 468, 930, 1,875, 3,750, 7,500, 15,000, and 30,000 nM). After washing the beads, the retained complexes were eluted and analyzed as described in Fig. 3D. For each point across the titration range, the signal intensity was quantified by densitometry and analyzed by curve fitting using nonlinear regression. The graph reports the quantified intensities of eIF4E and 4EHP signals normalized against 4E-T.

Fig. S4.

Thermodynamic parameters for the interaction of 4EHP and eIF4E with m⁷GTP in presence of 4E-T peptides. (A) Schematic diagram of full-length 4E-T and 4E-T peptides used in ITC experiments. The canonical (C), noncanonical (NC), and Cup-homology domain (CHD) of the 4E-T protein are depicted. We used three different 4E-T peptides harboring either the canonical YTKEELL motif alone (4E-T^28–37), in combination with the noncanonical motif (4E-T^28–71), or covering the entire N-terminal extremity including the eIF4E-binding motifs and the CUP-homology domain (4E-T^1–265). The 4E-T peptides were incubated with recombinant 4EHP or eIF4E proteins and titrated with m⁷GTP. (B–I) ITC profiles of 4EHP + m⁷GTP (B), 4EHP + 4E-T^28–37 + m⁷GTP (C), 4EHP + 4E-T^28–71 + m⁷GTP (D), 4EHP + 4E-T^1–265 + m⁷GTP (E), eIF4E + m⁷GTP (F), eIF4E + 4E-T^28–37 + m⁷GTP (G), eIF4E+ 4E-T^28–71 + m⁷GTP (H), and eIF4E + 4E-T^1–265 + m⁷GTP (I). The reaction cell contained 200 μL of 30 μM protein and was titrated with 19 injections of 2 μL of 300 μM m⁷GTP. B–I, Upper show the raw data (μcal⋅s⁻¹), and B–I, Lower represents the integrated data (kcal⋅mol⁻¹ injectant) of heat changes. The binding isotherm was fit with a binding model that uses a single set of independent sites to determine the thermodynamic binding constants and stoichiometry (see Table 1 for details).

Fig. 4.

Model of 4EHP-mediated translation repression by miRNAs. The recruitment of 4EHP to the miRNA target mRNA through the CCR4–NOT/DDX6/4E-T axis promotes its binding to the cap. The assembly of this complex is likely to initiate the formation of a closed-loop structure (Right) resembling the cap-to-tail closed-loop mRNA conformation involving eIF4G/PABP interaction (Left).