Fig 2: Eif4e1c is both unique to and shared by all aquatic vertebrates. Shown is a phylogeny of eIF4E1 orthologs from a sampling of the species considered (see Fig. S1 for full analysis). Eif4e1c is ancestral to the canonical eIF4E1A split from its variant, eIF4E1B. Terrestrial species have a canonical and eIF4E1B ortholog (pink and purple). All aquatic species have a canonical variant: eight species with a duplication (blue and green), two species retain only one variant of the duplication, and the Scyliorhinus canicula (small-spotted catshark) canonical clusters with terrestrial eIF4E1A. All 12 aquatic vertebrate retain an Eif4e1c family member. Shown are images of each of the aquatic vertebrate species to highlight the diversity considered. Only five of 12 aquatic vertebrates shown here retain an eIF4E1B variant. Estimates are made using maximum likelihood and the IQ-TREE. Nodes with bootstrap support <0.85 are marked with their respective values, all other nodes had support values of 0.85 or higher.

Fig 3: 4E-BP1 is involved in cysteine deprivation-induced repression of D-type cyclin translation.A, schematic diagram of hypothesis for cysteine-mediated regulation for the translation of D-type cyclins. B, the protein levels of pGCN2, ATF4, and 4E-BP1 in BNL 1ME A.7 R.1 cells 16 h after incubation in cystine-containing [Cyss (+)] or cystine-deficient [Cyss (−)] media. The protein levels were normalized to those of total GCN2 (for pGCN2) or β-actin (for ATF4 and 4E-BP1). Each value represents the mean with SD (n = 4). ∗∗p < 0.01, significant difference between the two groups (t6 = −3.958, p = 0.008 for pGCN2; t6 = −10.744, p < 0.001 for ATF4; t6 = −4.046, p = 0.007 for 4E-BP1; Student’s t test). C, the protein levels of 4E-BP1 bound to eIF4E in BNL 1ME A.7 R.1 cells 16 h after incubation in Cyss (+) or Cyss (−) media. Cell extracts were immunoprecipitated with anti-eIF4E antibodies. The protein levels were normalized to those of eIF4E. Each value represents the mean with SD (n = 3). ∗∗p < 0.01, significant difference between the two groups (t6 = −6.678, p = 0.003; Student’s t test). D, downregulation of 4E-BP1 restores cysteine deprivation–induced repression cyclin D1 and D2 protein levels in BNL 1ME A.7 R.1 cells. Cells were transfected with scrambled siRNA (control) or two types of siRNA against Eif4ebp1 gene encoding 4E-BP1 (Eif4ebp1#1 and Eif4ebp1#2) and then incubated in Cyss (+) or Cyss (−) media for 16 h. The protein levels were normalized to those of β-actin. Each value represents the mean with SD (n = 3–4). For upper panels, ∗∗p < 0.01, ∗p < 0.05, significant difference between the two groups (F3,8 = 32.627, p < 0.001 for cyclin D1, F3,8 = 14.316, p < 0.001 for cyclin D2; ANOVA with Tukey–Kramer’s post hoc test). For lower panels, ∗∗p < 0.01, significant difference between the two groups (F3,12 = 35.369, p < 0.001 for cyclin D1; F3,12 = 20.061, p < 0.001 for cyclin D2; ANOVA with Tukey–Kramer’s post hoc test). 4E-BP, 4E-binding protein; ATF4, activating transcription factor-4; eIF4, eukaryotic translation initiation factor 4; GCN2, general control nonderepressible 2.

Fig 4: The impact of eIF4E overexpression on hippocampal oxytocin levels in mice. A, ELISA experiment reveals the concentrations of hippocampal oxytocin in control and eIF4Eki/ki mice (3 animals in each group). B, qPCR experiment demonstrates the mRNA expression levels of hippocampal oxytocin in control and eIF4Eki/ki mice (3 animals in each group). C, D, Western blot assay displays the protein expression levels of oxytocin in the hippocampus of control and eIF4Eki/ki mice (3 animals in each group). Mann–Whitney U test was used for comparison between the two groups. The data are presented as mean ± standard deviation, and statistical significance is denoted as *p < 0.05 and **p < 0.01.

Fig 5: Effect of eIF4E overexpression on mouse microglia in immunofluorescence assay. A, Immunofluorescence staining of eIF4E and Iba-1 in hippocampal CA3 region of mice in each group; the arrow indicates the activation state of microglia. B, Comparison of the fluorescence intensity of eIF4E protein expression in the hippocampal CA3 region of mice in each group. C, Comparison of the fluorescence intensity of the expression of the microglial activation marker protein Iba-1 in the hippocampal CA3 region of mice in the two groups. D, Colocalization of eIF4E and microglia in the hippocampal CA3 region of the two groups of mice. E, Analysis of colocalization data of eIF4E and microglia in the hippocampal CA3 region of the two groups of mice. F, G, Western blot assay, expression of Iba-1 protein in the hippocampus of control and eIF4Eki/ki mice. H, qPCR assay. Oxytocin mRNA expression levels in the hippocampus of control and eIF4Eki/ki mice. I, eIF4E overexpression causes autism-like social cognitive impairment. Mann–Whitney U test was used for comparison between the two groups. The data are presented as mean ± standard deviation, and statistical significance is denoted as *p < 0.05 and **p < 0.01. In the study, n refers to the number of animals, with five acquisitions from each (hippocampus) slice, with a maximum of three slices obtained from each experimental animal used for each protocol (3 animals in each group).