GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

Abstract

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5' cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP-GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity.

Keywords: eIF4E; translational regulation; translational repression.

Figure 1.

GYF1/2 proteins use canonical, noncanonical, and auxiliary sequences to bind to 4EHP. (A) GYF1/2 proteins contain a central GYF domain and an N-terminal 4EHP-binding region (4EHP-BR). The 4EHP-BR includes canonical, noncanonical, and auxiliary motifs (A1–3) connected by linker sequences (nc-L and auxiliary linkers 1–3 [a-L1–3]). The 4E-binding region (4E-BR) of 4E-BP1 contains canonical and noncanonical motifs. (B,C) Western blots showing the interaction between V5-SBP-4EHP (wild type or the indicated mutants) and GFP-4E-BP1 (full-length) or endogenous GYF2. The proteins were pulled down using streptavidin-coated beads. V5-SBP-MBP (maltose-binding protein) served as negative control. The inputs (1.5% for the V5-tagged proteins and 1% for the GFP-tagged proteins) and bound fractions (3%–5% for the V5-tagged proteins and 20% for GYF2 and GFP-4E-BP1) were analyzed by Western blotting using anti-V5, anti-GFP, and anti-GYF2 antibodies. (D,E) Ni-NTA pull-down assays showing the interactions between GYF2 fragments (C+L+NC+A, C+L+NC, and C) and 4EHP-His₆ (M1–F234) (D) or eIF4E-His₆ (E). 4E-BP1 and MBP served as positive and negative controls, respectively. The GYF2 and 4E-BP1 peptides contain an N-terminal MBP tag and a C-terminal GB1 tag. The starting material (SM; 1.3% for MBPs and 6% for 4EHP and purified eIF4E) and bound fractions (7%–10%) were analyzed by SDS-PAGE followed by Coomassie blue staining. (F) The interaction between V5-SBP-eIF4E or 4EHP proteins and endogenous GYF2, 4E-T, and 4E-BP1 was analyzed in HEK293T cell lysates using streptavidin pull-downs. The input (1% for 4E-BP1 and 4E-T and 1.5% for V5-SBP-tagged proteins and GYF2) and bound fractions (20% for 4E-BP1 and 4ET, 30% for GYF2, and 5% for the V5-SBP-tagged proteins) were analyzed by Western blotting using the indicated antibodies. (G) Ni-NTA pull-down assay showing the interaction between 4EHP (M1–F234, wild type, or the indicated mutants) and GYF2 fragments. MBP served as a negative control. Samples were analyzed as described in D. The starting material (2% for the MBP-tagged proteins and 4%–12% for the 4EHP proteins) and bound fractions (10%) were analyzed by SDS-PAGE followed by Coomassie blue staining.

Figure 2.

Overall structures of GYF1, GYF2, and 4E-BP1 bound to 4EHP. (A,B) Overview of the structures of 4EHP bound to GYF1/2 (C+L+NC+A) fragments. The 4EHP surface is shown in yellow, and surface residues within a radius of 4 Å of the bound GYF1 or GYF2 peptides are colored in orange. The GYF1 and GYF2 peptides are colored in purple and blue, respectively. Selected secondary structure elements in the GYF1/2 peptides are indicated. The invariant PLAL motif of GYF1/2 is circled with a dashed line. (C,D) Cartoon representation of the structures of 4EHP bound to GYF1/2. Selected secondary structure elements are labeled in black for 4EHP and in color for GYF1/2. (E) Superposition of the structures of 4EHP bound to GYF1 and GYF2. For clarity, the 4EHP molecule from the 4EHP–GYF1 complex was omitted. The structures of the complexes are very similar, and overall root mean square deviations do not exceed 0.32 Å over 227 Cα atoms. (F) Structure of 4EHP bound to 4E-BP1. Selected secondary structure elements are labeled in black for 4EHP and in cyan for 4E-BP1. (G) Structure of 4EHP bound to the GYF2 C+L+NC fragment. Selected secondary structure elements are labeled in black and red for 4EHP and GYF2, respectively. (H) Superposition of the structures of 4EHP bound to the 4E-BP1 and GYF2 C+L+NC peptides. For clarity, the 4EHP molecule from the 4EHP–4E-BP1 complex was omitted. (I,J) Schematic representations of 4EHP bound to GYF1/2 and 4E-BP1.

Figure 3.

The interactions between the canonical and noncanonical sequences of GYF2 and 4E-BP1 with 4EHP. (A,B) Close-up views of the interactions between the 4EHP dorsal surface and the GYF2 and 4E-BP1 canonical helices. 4E-BP1 residue R63 is colored in dark blue after its Cγ atom and is highlighted with a black dashed box. The corresponding residues in GYF2 (F52) are also highlighted by a black dashed box. (C,D) Close-up views of the interaction between the 4EHP lateral surface and the GYF2 and 4E-BP1 noncanonical linkers. (E,F) Close-up views of the interactions between the 4EHP lateral surface and the GYF2 and 4E-BP1 noncanonical loops. Selected interface residues are shown as sticks. For clarity, all residues labeled with an asterisk are shown without their side chain.

Figure 4.

Interaction between the GYF1/2 auxiliary sequences and 4EHP. (A,B) Close-up view of the arrangement of the linker a-L1 and the PLAL motif (A1) in GYF1/2 proteins at the 4EHP dorsal surface. The surface of 4EHP is shown in yellow, and the surfaces of the GYF1/2 canonical helices are shown in gray. The positions of the 4EHP unique residues R103 and E149 are highlighted in orange, and selected GYF1/2 residues are shown as either purple (GYF1) or blue (GYF2) sticks. (C–H) Close-up views of the interactions between 4EHP and the GYF1/2 auxiliary sequences (A1, A2, and A3). Selected GYF1/2 residues and 4EHP interface residues are shown as sticks. The GYF1/2 canonical helices are colored in gray.

Figure 5.

The auxiliary interactions are crucial for the formation of the 4EHP–GYF complex. (A) Western blot showing the interaction of endogenous GYF2 or GFP-4E-BP1 with V5-SBP-4EHP (wild type or the indicated mutants). The proteins were pulled down using streptavidin-coated beads. The inputs (1.5% for the V5-tagged proteins and 1% for GYF2 and GFP-4E-BP1) and bound fractions (3% for the V5-tagged proteins and 20% for GYF2 and GFP-4E-BP1) were analyzed by Western blot using the indicated antibodies. (B) Interaction of V5-SBP-4EHP with GFP-GYF2 (residues 1–180; either wild type or the indicated mutants). The proteins were immunoprecipitated using anti-GFP antibodies. GFP-MBP served as negative control. The inputs (1.5% for the GFP-tagged proteins and 0.5% for V5-SBP-4EHP) and immunoprecipitates (7.5% for the GFP-tagged proteins and 30% for V5-SBP-4EHP) were analyzed by Western blot using anti-GFP and anti-V5 antibodies. (C–E) Purified 4EHP–4E-BP1 complexes containing 4EHP-His₆ (wild type or the RE-LL mutant) were incubated in the presence of equimolar amounts of the GYF2 C+L+NC+A peptide C-terminally tagged with GB1 or MBP as a negative control. The proteins bound to 4EHP were pulled down using Ni-NTA beads at the indicated time points and analyzed by SDS-PAGE and Coomassie blue staining. C shows the quantification of the amount of 4E-BP1 still associated with 4EHP. n = 3. The half-life of the 4EHP–4E-BP1 complex (t_1/2) in the presence of the competitor protein is represented as the mean ± SD. D and E show representative SDS-PAGE gels. The positions of the GYF2 and 4E-BP1 peptides are marked by blue and black dashed boxes, respectively. The lanes labeled SM (starting material) show the purified complexes and peptides used in the assay.

Figure 6.

4EHP requires interaction with GYF1/2 proteins to repress translation. (A) A complementation assay using the R-Luc-5BoxB-A₉₅-MALAT1 reporter and λN-HA-4EHP (either wild type or the indicated mutants) was performed in control and GYF1/2-null HEK293T cells expressing GFP-MBP or GFP-GYF2 (wild type or canonical mutant). A plasmid expressing F-Luc-GFP served as the transfection control. R-Luc activity was normalized to that of the F-Luc transfection control and set to 100% in cells expressing the λN-HA peptide. Bars represent the mean values, and error bars represent standard deviations from three independent experiments. (B) Western blot analysis showing that full-length GYF1/2 levels were strongly reduced relative to control levels in the GYF1/2-null cell line. (C) Western blot analysis showing the expression of the λN-HA-4EHP and GFP-GYF2 proteins used in the assay shown in A. (D) Tethering assay using the R-Luc-5BoxB-A₉₅-MALAT1 reporter and λN-HA-4EHP (wild type or mutants) in HEK293T cells. Samples were analyzed as described in A. (+) Binding to the GYF1/2 proteins; (+/−) reduced binding to the GYF1/2 proteins; (−) no binding to the GYF1/2 proteins. (E) Western blot showing the equivalent expression of the λN-HA-4EHP proteins used in the assay shown in D. (F) Tethering assay using the R-Luc-6xMS2-A₉₅-MALAT1 reporter and MS2-HA-GYF2 (wild type or canonical mutant) in HEK293T cells. The cells were also cotransfected with GFP-MBP and F-Luc-GFP as transfection controls. R-Luc activity was normalized to that of the F-Luc transfection control and set to 100% in cells expressing MS2-HA. Samples were analyzed as described in A. (G) Western blot analysis showing the equivalent expression of the MS2-HA-GYF2 proteins. (H) Control HEK293T cells or cells depleted of GYF1/2 (KO) were transfected with the R-Luc-ARE-A₉₀-MALAT1 reporter and plasmids expressing the indicated proteins. The F-Luc-GFP reporter served as a transfection control. R-Luc activity was normalized to that of the F-Luc transfection control and set to 100% in the absence of TTP for each cell line. (I) Western blot showing the expression of the proteins in the experiment shown in H. Note that TTP is stabilized in GYF1/2-null cells expressing GYF2. However, repression did not correlate with TTP levels but with the coexpression of wild-type GYF2 and 4EHP.