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.