Direct role for the Drosophila GIGYF protein in 4EHP-mediated mRNA repression

Abstract

The eIF4E-homologous protein (4EHP) is a translational repressor that competes with eIF4E for binding to the 5'-cap structure of specific mRNAs, to which it is recruited by protein factors such as the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins (GIGYF). Several experimental evidences suggest that GIGYF proteins are not merely facilitating 4EHP recruitment to transcripts but are actually required for the repressor activity of the complex. However, the underlying molecular mechanism is unknown. Here, we investigated the role of the uncharacterized Drosophila melanogaster (Dm) GIGYF protein in post-transcriptional mRNA regulation. We show that, when in complex with 4EHP, Dm GIGYF not only elicits translational repression but also promotes target mRNA decay via the recruitment of additional effector proteins. We identified the RNA helicase Me31B/DDX6, the decapping activator HPat and the CCR4-NOT deadenylase complex as binding partners of GIGYF proteins. Recruitment of Me31B and HPat via discrete binding motifs conserved among metazoan GIGYF proteins is required for downregulation of mRNA expression by the 4EHP-GIGYF complex. Our findings are consistent with a model in which GIGYF proteins additionally recruit decapping and deadenylation complexes to 4EHP-containing RNPs to induce translational repression and degradation of mRNA targets.

Figure 1.

Dm 4EHP requires Dm GIGYF to downregulate reporter mRNA expression. (A) Tethering assay using the firefly luciferase (F-Luc)-5BoxB reporter and the indicated λN-HA-tagged proteins in control [Ctrl knockdown (KD): neomycin dsRNA-treated cells] or GIGYF-depleted Dm S2 cells (GIGYF KD). A plasmid expressing Renilla luciferase (R-Luc)-A₉₀-HhR served as a transfection control. The F-Luc activity (green bars) and mRNA levels determined by northern blotting (blue bars) were normalized to those of the R-Luc transfection control and set to 100% in cells expressing the λN-HA peptide (black bar). Bars represent the mean values and error bars denote the standard deviation from at least three independent experiments. (B) Northern blot analysis of a representative tethering experiment shown in (A). (C) Western blot showing the expression levels of the tethered proteins. (D) Immunoprecipitation assay showing the interaction between GFP-tagged 4EHP WT or the indicated mutants (dorsal: D*; lateral: L*; and 4EHP-specific: S*) and HA-tagged GIGYF N-term. The proteins were immunoprecipitated using an anti-GFP antibody. GFP-MBP (maltose binding protein) served as a negative control. The input (2.8% of the total lysate) and bound fractions (10%) were analyzed by western blotting using anti-GFP and anti-HA antibodies.

Figure 2.

Dm GIGYF represses translation and induces mRNA decay. (A) Schematic representation of the Dm GIGYF protein. The N-term contains a 4EHP-binding region (4EHP-BR), an Arg/Gly-rich sequence adjacent to the 4EHP-BR, a Me31B binding motif (MBM) and a glycine-tyrosine-phenylalanine (GYF) domain of the Smy2-type (13). The C-term does not contain any known domain and is predicted to contain primarily α-helices. The amino acid positions at the domain/motif boundaries are indicated below the protein. (B–D) Tethering assay using the F-Luc-5BoxB reporter and the indicated λN-HA-tagged proteins in S2 cells. Samples were analyzed as described in Figure 1A. Relative luciferase activity (green bars), relative reporter mRNA levels (blue bars) and representative northern and western blot images are depicted in panels B, C and D, respectively. (E, F) A tethering assay with the F-Luc-5BoxB reporter and the indicated λN-HA-tagged proteins performed in control cells (GFP-V5-expressing cells) and in cells expressing a DCP1 GSSG mutant. GW182 was used as a positive control for deadenylation-dependent mRNA decapping. Samples were analyzed as described in Figure 1A. In panel F, the red dashed line marks the position of the deadenylated (A₀) F-Luc-5BoxB reporter mRNA after northern blot analysis of representative RNA samples. A_n indicates the position of the adenylated reporter mRNA.

Figure 3.

Dm GIGYF interacts with components of the decapping and deadenylation machineries. (A) Immunoprecipitation assay showing the interaction between GFP-GIGYF full length (WT), and the indicated fragments, and endogenous HPat and Me31B. The proteins were immunoprecipitated in the presence of RNaseA using an anti-GFP antibody. GFP-F-Luc-V5 served as a negative control. The input (3% for GFP-tagged proteins, 0.2% for HPat and 0.4% for Me31B) and bound fractions (30% for GFP-tagged proteins and HPat, and 40% for Me31B) were analyzed by western blotting using anti-GFP, anti-HPat and anti-Me31B antibodies. (B) The interaction among full length (WT) and fragments of GFP-GIGYF and HA-NOT1 was determined by anti-GFP immunoprecipitation in S2 cell lysates. GFP-F-Luc-V5 served as a negative control. The input (3% for GFP-tagged proteins and 1% for HA-tagged proteins) and bound fractions (15% for GFP-tagged proteins and 30% for HA-tagged proteins) were analyzed by western blotting using anti-GFP and anti-HA antibodies. (C) Sequence alignment of the MBM present in Drosophila melanogaster (Dm) GIGYF and human (Hs) GIGYF1 and 2 proteins with the CUP-homology region from the Hs and Dm 4E-T proteins or the Dm CUP protein. Identical residues are highlighted in red boxes and printed in white whereas residues with >70% similarity are shown with a light color background. (D) Immunoprecipitation assay showing the interaction between GFP-tagged GIGYF proteins (WT and indicated mutants) and endogenous Me31B. The proteins were immunoprecipitated using anti-GFP antibodies. GFP-F-Luc-V5 served as a negative control. The input (4% for GFP-tagged proteins and 0.18% for endogenous Me31B) and bound fractions (40% for GFP-tagged proteins and 45% for endogenous Me31B) were analyzed by western blotting using anti-GFP, anti-Me31B antibodies. (E) The interaction of HA-NOT1 and endogenous HPat with WT and mutant GIGYF proteins was analyzed by immunoprecipitation using anti-GFP antibodies. GFP-F-Luc-V5 served as a negative control. The input (4% for GFP-tagged proteins and 0.8% for HA-tagged proteins and endogenous HPat) and bound fractions (15% for GFP-tagged proteins and 35% for HA-NOT1 and endogenous HPat) were analyzed by western blotting using anti-GFP, anti-HA and anti-HPat antibodies. (F) Western blot analysis of the interaction of GFP-HPat [WT and PPGF motif mutant (PPGF*)] with HA-GIGYF N-term. The input (2% for GFP-tagged proteins and 0.8% for HA-tagged proteins) and bound fractions (10% for GFP-tagged proteins and 20% for HA-tagged proteins) were analyzed by western blotting using anti-GFP and anti-HA antibodies.

Figure 4.

Translational repression by GIGYF requires the decapping factors Me31B and HPat. (A–C) S2 cells were transfected with the F-Luc-5BoxB reporter in which the cleavage and polyadenylation signal were replaced by a polyadenosine stretch of 96 residues followed by seven cytosines and a self-cleaving Hammerhead ribozyme (F-Luc-5BoxB-A₉₆-HhR). λN-HA-GIGYF WT, Me31B-binding mutant (ΔMBM), or double ΔMBM+GYF* mutant and the transfection control R-Luc-A₉₀-HhR plasmids were cotransfected with the F-Luc reporter. Luciferase activity and mRNA levels were analyzed as described in Figure 1A. The P value (***P < 0.0005) was determined using the two-tailed Student's t test between WT GIGYF and the other GIGYF proteins. A representative northern blot is shown next to the corresponding graph (B). Panel C shows expression levels of the tethered proteins as analyzed by western blotting. (D–G) S2 cells were treated with the indicated dsRNAs and transfected with a mixture of three plasmids coding for F-Luc-5BoxB-A₉₆-HhR, R-Luc-A₉₀-HhR and the indicated λN-HA-tagged proteins. In panels D and F, F-Luc activity and mRNA levels were normalized to that of R-Luc in the presence of the different tethered proteins. Panels E and G show representative northern blots. The P value (**P < 0.005, ***P < 0.0005) was determined using the two-tailed Student's t test between WT GIGYF in Ctrl KD and GIGYF proteins in the other experimental conditions.

Figure 5.

GIGYF recruits Me31B and HPat to 4EHP-repressor complexes. (A) A complementation assay using the F-Luc-5BoxB-A₉₆-HhR reporter and λN-HA-4EHP was performed in Ctrl and GIGYF-depleted cells expressing HA-GIGYF proteins (dsRNA-resistant and either WT, mutants or the N-term). A plasmid expressing R-Luc-A₉₀-HhR served as a transfection control. Samples were analyzed as described in Figure 1A. (B) Western blot analysis showing the expression of the λN-HA-4EHP, HA-GIGYF and F-Luc-V5 proteins used in the complementation assay. (C) Interaction of GFP-4EHP with endogenous Me31B and HPat proteins assayed by anti-GFP immunoprecipitation in the presence or absence of HA-GIGYF (WT or mutants). GFP-F-Luc-V5 was used as a negative control. The inputs (4% for GFP- and HA-tagged proteins and 0.1% for Me31B and HPat) and bound fractions (5% for GFP- and HA-tagged proteins and 20% for Me31B and HPat) were analyzed by western blotting using anti-GFP, anti-HA, anti-Me31B and anti-HPat antibodies. (D, E) S2 cells were treated with dsRNA targeting neomycin (Ctrl KD) or Me31B and HPat mRNAs (Me31B+HPat KD) and transfected with a mixture of three plasmids coding for F-Luc-5BoxB-A₉₆-HhR, R-Luc-A₉₀-HhR and the indicated λN-HA-tagged proteins. In panel D, F-Luc activity and mRNA levels were normalized to that of R-Luc in the presence of the different tethered proteins. Samples were analyzed as described in Figure 1A. Panel E shows a representative northern blot. The P value (***P < 0.0005) was determined using the two-tailed Student's t test. (F) Model for the assembly of a 4EHP-repressor complex. 4EHP (orange) is the cap-binding protein that blocks eIF4F mRNA recruitment. GIGYF (red) provides binding sites for 4EHP (4EHP-BR), Me31B (MBM), HPat (GYF domain) and the CCR4–NOT deadenylase complex. Additionally, GIGYF might also bind an unknown RNA-binding protein (X) or directly interact with the target mRNA. HPat and the CCR4–NOT complex also can interact with Me31B (24,40,41). The formation of this complex ensures that multiple mechanisms are employed to robustly suppress target mRNA expression: translational repression, deadenylation and decapping, which is subsequently followed by degradation of the mRNA.