Translational repression by 4E-T is crucial to maintain the prophase-I arrest in vertebrate oocytes

Abstract

Meiotic maturation of vertebrate oocytes occurs in the near-absence of transcription. Thus, female fertility relies on timely translational activation of maternal transcripts stockpiled in full-grown prophase-I-arrested oocytes. However, how expression of these mRNAs is suppressed to maintain the long-lasting prophase-I arrest remains mysterious. Utilizing fast-acting TRIM-Away, we demonstrate that acute loss of the translation repressor 4E-T triggers spontaneous release from prophase-I arrest in mouse and frog oocytes. This is due to untimely expression of key meiotic drivers like c-Mos and cyclin-B1. Notably, mutant 4E-T associated with premature ovarian insufficiency in women fails to maintain the prophase-I arrest in Xenopus oocytes. We further show that 4E-T association with eIF4E and PATL2 is critical for target mRNA binding and repression. Thus, 4E-T is a central factor in translational repression of mRNAs stockpiled in full-grown oocytes for later activation and, therefore, essential to sustain the oocyte pool throughout the reproductive lifespan of female vertebrates.

Fig. 1. 4E-T depletion triggers spontaneous meiotic resumption and accelerates hormone-induced meiotic maturation.

a Xenopus stage-VI oocytes were injected with the indicated 4E-T or unspecific control (Ctrl) antibodies and mRNA encoding Flag-TRIM21. Samples were taken 22 h after injection and immunoblotted as indicated. One representative experiment of three independent biological replicates is shown. b Xenopus stage-VI oocytes were injected with water or mRNA encoding Flag-4E-T^TRIM. 18 h after injection, oocytes were co-injected with mRNA encoding Flag-TRIM21 and either 4E-T^Ab1 or unspecific control (Ctrl) antibodies. 35 h after the second injection, the occurrence of GVBD was determined by the appearance of a white spot in the animal hemisphere of the oocytes (Ctrl TRIM + H₂O, n = 107 oocytes; 4E-T^Ab1 TRIM + H₂O, n = 105 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM, n = 104 oocytes). Percentage of oocytes with GVBD spots is given as mean ± s.d. from four independent biological replicates. p-values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. c Mouse oocytes were injected with the indicated 4E-T or unspecific control (Ctrl) antibodies and mRNA encoding mouse TRIM21. Samples were kept in medium containing 10 µM RO-3306 for 6 h after injection and immunoblotted as indicated. One representative experiment of two independent replicates is shown. d Representative stills from time-lapse movies of control and 4E-T-depleted mouse oocytes stained with 5-SiR-Hoechst. Oocytes were incubated with dbcAMP as indicated. Cyan, DNA. Arrowheads indicate oil droplets injected to ensure quantitative microinjections. e Percentage of control and 4E-T-depleted mouse oocytes that resume meiosis (upper panel) or extrude polar bodies (lower panel) in the presence or absence of dbcAMP. Data are from four (upper panel) or two (lower panel) independent experiments. f TRIM-Away of 4E-T and expression of Flag-4E-T^TRIM was performed as described in (b). 22 h after the second injection, oocytes were lysed for immunoblotting as indicated. One representative experiment of three independent biological replicates is shown. g Oocytes from (f) (Ctrl TRIM + H₂O, n = 76 oocytes; 4E-T^Ab1 TRIM + H₂O, n = 68 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM +, n = 75 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM ++, n = 72 oocytes) were treated with PG 22 h after the second injection. Percentage of oocytes undergoing GVBD in 760 min after PG addition and median time to GVBD are given as mean±s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. h Upper panel: Scheme of c-Mos expression, Cdk1 and MAPK activation as shown by loss of inhibitory and gain of activating phosphorylation, respectively, during prophase-I arrest and oocyte maturation. TRIM-Away of 4E-T and expression of Flag-4E-T^TRIM was performed as described in (b). 22 h after the second injection, oocytes were treated with PG, samples were taken at the indicated time points and immunoblotted as indicated. One representative experiment of three independent biological replicates is shown. i, j Experiment was performed with the indicated Flag-4E-T^TRIM constructs as described in (f, g). Oocytes (Ctrl TRIM + H₂O, n = 105 oocytes; 4E-T^Ab1 TRIM + H₂O, n = 111 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM, n = 94 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM 1-319, n = 101 oocytes) from four independent biological replicates were analyzed. Percentage of oocytes undergoing GVBD in 460 min after PG addition and median time to GVBD are given as mean±s.d. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Source data including additional loading controls are provided as a Source Data file.

Fig. 2. 4E-T controls the translation of cell cycle mRNAs in Xenopus oocytes.

a Xenopus stage-VI oocytes were injected with water or mRNA encoding Flag-4E-T^TRIM. 18 h after injection, oocytes were co-injected with mRNA encoding Flag-TRIM21 and either 4E-T^Ab1 or unspecific control (Ctrl) antibodies. 22 h after the second injection, oocytes were treated with puromycin and as indicated with cycloheximide (CHX) for 2 h. Oocytes were lysed and immunoblotted as indicated. Note, that we specifically selected oocytes that have not yet undergone spontaneous GVBD for analysis, to exclude indirect translational effects caused by pathways activated during meiotic maturation. Puromycin signals were quantified and normalized intensities from four independent biological replicates are given as mean ± s.d. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. b Xenopus stage-VI oocytes were lysed and subjected to immunoprecipitation with unspecific control (Ctrl) or 4E-T^Ab1 antibodies. Samples were immunoblotted as indicated. In parallel, RNA was isolated from the same IP samples and analyzed by qRT-PCR for the indicated mRNAs. ΔC_t values between IP Ctrl and IP 4E-T^Ab1 samples are given as mean ± s.d. from three independent biological replicates. c Xenopus stage-VI oocytes were injected with water or mRNA encoding Flag-4E-T^TRIM. 18 h after injection, oocytes were co-injected with mRNA encoding Flag-TRIM21, with 4E-T^Ab1 or unspecific control (Ctrl) antibodies, with mRNA encoding Myc-eGFP_HBG1 3´UTR and with mRNA encoding Flag-eGFP fused to the indicated 3´UTR. Oocytes were lysed after 22 h and immunoblotted as indicated. Note, that we specifically selected oocytes that have not yet undergone spontaneous GVBD for analysis, to exclude indirect translational effects caused by pathways activated during meiotic maturation. One representative experiment of three independent biological replicates is shown. d Quantification of eGFP signals in (c). Values are given as mean ± s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. e Xenopus stage-VI oocytes were injected with water or mRNA encoding Flag-4E-T^TRIM. 18 h after injection, oocytes were co-injected with mRNA encoding Flag-TRIM21 and with 4E-T^Ab1 or unspecific control (Ctrl) antibodies. Oocytes were incubated in medium containing the Cdk inhibitor Roscovitine. 48 h after the second injection, oocytes were lysed and immunoblotted as indicated. Cyclin-B1 and c-Mos signals were quantified and normalized to p150. Values were normalized to the Ctrl TRIM condition and are given as mean ± s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Asterisks indicate unspecific bands. f Xenopus stage-VI oocytes were treated with PG and lysed at the indicated time points. Lysates were subjected to immunoprecipitation with 4E-T^Ab1. Samples were immunoblotted as indicated. In parallel, RNA was isolated from the same samples and analyzed by qRT-PCR for the indicated mRNAs. Values in input and IP samples were normalized to t = 0 h conditions and are given as mean±s.d. from three independent biological replicates. p values within Input and IP conditions were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. g Summary of results obtained for the c-Mos, CCNB1 and CCNA1 mRNAs. Source data including additional loading controls are provided as a Source Data file.

Fig. 3. Characterization of meiotic RNPs containing 4E-T.

a Xenopus stage-VI oocytes were lysed and treated with RNaseA as indicated. Lysates were subjected to immunoprecipitation with unspecific control (Ctrl) or 4E-T^Ab1 antibodies. Samples were immunoblotted as indicated. One representative experiment of three independent biological replicates is shown. Quantification of this figure can be found in Fig. S4a. b Xenopus stage-VI oocytes were injected with mRNA encoding Myc-LSM14A. 18 h after injection, oocytes were treated with PG and lysed at the indicated time points. Lysates were treated with RNaseA as indicated and subjected to immunoprecipitation with 4E-T^Ab1 antibodies. Samples were immunoblotted as indicated. One representative experiment of three independent biological replicates is shown. c Signals in IP samples from (b) were quantified and normalized to 4E-T signal in IP samples. Values were normalized to t = 0 h / −RNaseA condition and are given as mean±s.d. from three independent biological replicates. p values within –RNaseA and +RNaseA conditions were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. d Xenopus stage-VI oocytes were injected with the indicated 4E-T or unspecific control (Ctrl) antibodies and mRNAs encoding Flag-TRIM21, Myc-PATL2 and Myc-LSM14A. 18 h after injection, oocytes were lysed and separated by sucrose density gradient centrifugation. Gradient fractions were analyzed by immunoblot as indicated. As reference, fractions containing 4E-T in Ctrl-depleted oocytes are shown as dashed rectangles in both conditions. Asterisk indicates IgG HC. One representative experiment of three independent biological replicates is shown. Source data including additional loading controls are provided as a Source Data file.

Fig. 4. 4E-T directly interacts with eIF4E and PATL2 in immature Xenopus oocytes.

a Schematic representation of 4E-T.S from Xenopus laevis. Regions required for interaction with the indicated proteins in somatic cells are highlighted. b Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated Flag-4E-T^TRIM variant and a mixture of mRNAs encoding Myc-eIF4E1b, Myc-PATL2 and Myc-LSM14A. 18 h after injection, oocytes were lysed and subjected to immunoprecipitation with Flag antibodies. Samples were immunoblotted as indicated. One representative experiment of three independent biological replicates is shown. Quantification of this figure can be found in Fig. S5a. c Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated Flag-4E-T^TRIM variant. 18 h after injection, oocytes were lysed and subjected to immunoprecipitation with Flag antibodies. Samples were immunoblotted as indicated. One representative experiment of four independent biological replicates is shown. Quantification of this figure can be found in Fig. S5a. d Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated Flag-4E-T^TRIM variant and a mixture of mRNAs encoding Myc-PATL2 and Myc-LSM14A. 18 h after injection, oocytes were lysed and subjected to immunoprecipitation with Flag antibodies. Samples were immunoblotted as indicated. Asterisk indicates unspecific bands. One representative experiment of three independent biological replicates is shown. e Signals in IP samples from (d) were quantified. Signals in IP samples of water-injected oocytes were subtracted and values were normalized to Flag. All conditions were normalized to WT and values are given as mean±s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Source data including additional loading controls are provided as a Source Data file.

Fig. 5. Interactions of 4E-T with eIF4E and PATL2 are required for its meiotic function.

a Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated Flag-4E-T^TRIM variant. 18 h after injection, oocytes were co-injected with mRNA encoding Flag-TRIM21 and either 4E-T^Ab1 or unspecific control (Ctrl) antibodies. 22 h after the second injection, oocytes were lysed and immunoblotted as indicated. One representative experiment of three independent biological replicates is shown. b Oocytes from (a) (Ctrl TRIM + H₂O, n = 50 oocytes; 4E-T^Ab1 TRIM + H₂O, n = 51 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM, n = 51 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM Δ4Ec, n = 53 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM Δ4Enc, n = 53 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM ΔPATL, n = 54 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM ΔLSM14A, n = 59 oocytes) were treated with PG 22 h after the second injection. Percentage of oocytes undergoing GVBD in 390 min after PG addition and median time to GVBD are given as mean ± s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. c TRIM-Away of 4E-T and expression of the indicated Flag-4E-T^TRIM variants was performed as described in (a). 22 h after the second injection, oocytes were lysed for immunoblotting as indicated. One representative experiment of three independent biological replicates is shown. d Oocytes from (c) (Ctrl TRIM + H₂O, n = 67 oocytes; 4E-T^Ab1 TRIM + H₂O, n = 64 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM, n = 63 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM Δ4Ec ΔPATL, n = 62 oocytes) were treated with PG 22 h after the second injection. Percentage of oocytes undergoing GVBD in 600 min after PG addition and median time to GVBD are given as mean ± s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. A detailed analysis of the GVBD timing in the different conditions can be found in Fig. S6a. e Xenopus stage-VI oocytes were injected with mRNA encoding Flag-4E-T^TRIM or Flag-4E-T^TRIM Δ4Ec ΔPATL. 18 h after injection, oocytes were lysed and separated by sucrose density gradient centrifugation. Gradient fractions were analyzed by immunoblot as indicated. One representative experiment of three independent biological replicates is shown. f Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated Flag-4E-T^TRIM variant. 18 h after injection, oocytes were lysed and subjected to immunoprecipitation with Flag antibodies. Samples were immunoblotted as indicated. Asterisk indicates IgG HC. In parallel, RNA was isolated from the same samples and analyzed by qRT-PCR for the indicated mRNAs. Values in input and IP samples were normalized to the Flag-4E-T^TRIM conditions and are given as mean ± s.d. from three independent biological replicates. p values within input and IP conditions were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Source data including additional loading controls are provided as a Source Data file.

Fig. 6. PATL2 is required to anchor 4E-T to its target mRNAs.

a Schematic representation of the λN/boxB tethering assay in (b). b Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated λN-Flag-4E-T^TRIM variants. 18 h after injection, oocytes were co-injected with a mixture of mRNAs encoding Myc-eGFP_5xboxB 3´UTR and Myc-dtTomato. 22 h after the second injection, oocytes were lysed and immunoblotted as indicated. Asterisk indicates unspecific bands. Myc signal of Myc-eGFP and Myc-dtTomato from three independent biological replicates was quantified. Values were normalized to water conditions and are given as mean ± s.d. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. c Working model for the function of 4E-T in suppressing mRNA translation in full-grown prophase-I-arrested oocytes by blocking access of eIF4G to eIF4E at the 5´cap. The RNA-binding proteins Zar1l and PATL2 anchor 4E-T to selected target mRNAs. PG stimulation triggers early partial destruction of Zar1l (before GVBD) and late destruction of PATL2 (after GVBD). Source data including additional loading controls are provided as a Source Data file.