4EHP-independent repression of endogenous mRNAs by the RNA-binding protein GIGYF2

Abstract

Initially identified as a factor involved in tyrosine kinase receptor signaling, Grb10-interacting GYF protein 2 (GIGYF2) has later been shown to interact with the 5' cap-binding protein 4EHP as part of a translation repression complex, and to mediate post-transcriptional repression of tethered reporter mRNAs. A current model proposes that GIGYF2 is indirectly recruited to mRNAs by specific RNA-binding proteins (RBPs) leading to translation repression through its association with 4EHP. Accordingly, we recently observed that GIGYF2 also interacts with the miRNA-induced silencing complex and probably modulates its translation repression activity. Here we have further investigated how GIGYF2 represses mRNA function. In a tethering reporter assay, we identify three independent domains of GIGYF2 with repressive activity. In this assay, GIGYF2-mediated repression is independent of 4EHP but largely dependent on the CCR4/NOT complex that GIGYF2 recruits through multiple interfaces. Importantly, we show that GIGYF2 is an RBP and identify for the first time endogenous mRNA targets that recapitulate 4EHP-independent repression. Altogether, we propose that GIGYF2 has two distinct mechanisms of repression: one depends on 4EHP binding and mainly affects translation; the other is 4EHP-independent and involves the CCR4/NOT complex and its deadenylation activity.

Figure 1.

GIGYF2 is a repressor of mRNA function. (A) Schematic representation of the RNA tethering assay, POI: protein of interest. (B) Normalized expression levels of the FL-5boxB reporter measured with a dual luciferase assay (protein) or (C) by RT-qPCR (mRNA). In both cases FL-5boxB readings were normalized to RL values. The normalized FL values obtained in the presence of the proteins without λ-peptide (HA-tagged (non-tethered) protein) were set to 100%. Mean values are shown with s.e.m. from six independent experiments. (D) Expression of the fusion proteins analyzed by western blotting. (E) Schematic representation of human GIGYF2. Positions of binding motif for 4EHP, GYF, Q-rich1, Q-rich2 and RQCD1-binding domains are indicated. (F) Left: Normalized expression of RL-5boxB measured with a dual luciferase assay. HeLa cells were co-transfected with plasmids expressing the indicated constructs, RL-5BoxB and FL transfection control reporters. RL was normalized to FL and values of normalized RL produced in the presence of HA-GIGYF2 were set to 100%. Mean values are shown with s.e.m. from four to six independent experiments. Right: Protein expression was analyzed by western blotting. P-values were calculated from the average of the ratios with a two-tailed Student’s t-test. *P-value < 0.05, **P < 0.01, *P-value < 0.05, ***P < 0.001.

Figure 2.

GIGYF2-mediated silencing relies on the CCR4/NOT complex and its deadenylation activity. (A) Dual luciferase assay of HeLa cell lysates co-transfected with RL-5boxB, FL and λHA (dark gray bars)- or HA (black bars)-TNRC6C plasmids, or λHA (light gray bars)- or HA (white bars)-GIGYF2 plasmids. The cells were additionally co-transfected with siRNAs against CNOT1, CNOT1/7/8 or a control siRNA as indicated. (B) Same as in (A) except that in addition to the reporters and fusion proteins plasmids, the cells were co-transfected with a plasmids coding for dominant negative variants of CNOT6 and CNOT7 (CNOT6/7 mut) or with a control plasmid (mock) as indicated. In both (A) and (B), RL readings were normalized to FL and values of normalized RL produced in the presence of the HA-GIGYF2 were set to 100%. Mean values are shown with s.e.m. from four to six independent experiments. *P-value < 0.05; **P < 0.005; ***P < 0.001, n.s.: non-significant. (C). Expression levels of the RL-5BoxB mRNA reporter over time following Actinomycin D-mediated transcription arrest in the presence of λHA—or HA-GIGYF2. (D) Same as (C) assessing the p21 mRNA as a control for effectiveness of the ActD treatment. RL-5boxB or p21 levels (normalized to GAPDH) were analyzed at the indicated time points by RT-qPCR in five independent experiments and values obtained at time 0 were set to 100%. Error bars are s.e.m.

Figure 3.

Characterization of the interaction between GIGYF2 and the CCR4/NOT complex. Blots of GFP-pull-down experiments. GFP-fusions of CNOT1 (A), CNOT7 (B) and CNOT9 (C) were co-expressed with the indicated constructs in HeLa cells. The GFP fusions were affinity purified from cells lysates using GFP-trap magnetic beads. (D) Dual luciferase assay of cells transfected with RL-5boxB, FL and the indicated fusion proteins in the presence (CNOT6/7 mut) or absence (mock) of dominant negative variants of CNOT6 and CNOT7. Mean values are shown with s.e.m. for four to six independent experiments. *P-value < 0.05; **P < 0.01; n.s., not significant.

Figure 4.

GIGYF2 is an RBP. (A) Schematic representation of the RNA binding assay. (B) HeLa cells transfected with the indicated constructs were irradiated with 254 nm UV light. The GFP fusions were then affinity purified from cells lysates using GFP Trap Agarose beads. Co-immunoprecipitated mRNAs were detected by hybridization with WellRED-labeled oligo(dT)₂₅. Shown are western blot analysis of the expression of each fusion protein (left) and the corresponding red (WellRED) to green (GFP) fluorescence ratios (right), the ratio obtained for the GFP condition was set to 1. The graph was generated from three biological replicates each done in three technical replicates. (C) same as in (B) with the indicated fusion proteins. *P-value < 0.05, **P < 0.01.

Figure 5.

Identification of endogenous targets of GIGYF2. (A) Volcano plot showing mRNA expression level changes upon depletion of GIGYF2 from HeLa cells analyzed with microarrays. (B) Same as in (A) but showing changes in mRNA expression levels upon overexpression of GIGYF2. In both (A) and (B) the logarithmic ratios of mRNA levels were plotted against negative logarithmic P values of a two-sided two samples t-test (n = 3 biological replicates). Red and green points are significantly (P < 0.05) upregulated and downregulated transcripts respectively. Highlighted in purple are the transcripts that are both upregulated upon GIGYF2 depletion and downregulated upon GIGYF2 overexpression. (C) Venn graph showing the transcripts that are upregulated upon GIGYF2 depletion and downregulated upon GIGYF2 overexpression, and the corresponding overlap between the two datasets. (D) Top: enrichment of selected transcripts in GIGYF2 immunoprecipitates analyzed by RT-qPCR. RNA samples were purified from input cell lysates and immuno-precipitation samples performed with an antibody against GIGYF2, or a negative control antibody (Control IgG). Transcripts levels in the IP were normalized to the input RNA fraction and enrichment in the GIGYF2 IP over the control IP was calculated. As a negative control, enrichment obtained for GUSB mRNA was set to 1 (indicated by the dotted line). Mean values are shown with s.e.m. Bottom: efficiency and specificity of the GIGYF2 immunoprecipitation was analyzed by western blotting.

Figure 6.

4EHP is not essential to GIGYF2-mediated repression of endogenous targets. (A) Western blot analysis of the expression levels of the indicated proteins in GIGYF2 knockout cells that were rescued with a control plasmid (mock) or plasmids encoding GIGYF2 or a variant thereof that cannot bind to 4EHP (mut GIGYF2). (B) Expression levels, normalized to GUSB mRNA, of the five validated endogenous target transcripts and a control mRNA (PPIA) analyzed by RT-qPCR from the rescued cells analyzed in (A). Log₂ values obtained from mock-transfected cells were set to 0. Mean values are shown with s.e.m. from two to seven independent experiments. *P-value < 0.01; **P < 0.05; n.s., not significant. (C) A speculative model for two modes of GIGYF2-mediated repression depending on direct or indirect binding to target mRNAs.