Figure 2

eIF4E1b interacts with the mRNA cap, eIF4EBP1, and eIF4ENIF1 and localizes to cytoplasmic foci in oocytes and embryos.

(A) eIF4E interaction with the mRNA cap (top, PDB-5BXV, Sekiyama et al, 2015) is mediated by two tryptophans that are conserved in eIF4E1B (bottom, AlphaFold (AF) prediction of mouse eIF4E1B). The superimposition of both structures is shown on the right, with eIF4E in light gray, eIF4E1B in dark gray, and m⁷G in red. (B) (Left) Coomassie-stained gels from immunoprecipitation assays with E. coli lysates containing zebrafish eIF4Ea and human eIF4E1B (wild-type and tryptophan mutants, see (A)) using m⁷G-coated beads. The eIF4E-binding motif (4EBM) of human eIF4EBP1 is used as a negative control. Quantification of eIF4E binding to m⁷G (relative to eIF4Ea) is shown on the right. (C) Scheme of in vitro pulldown assays. Lysates of E. coli cells expressing His- and MBP-tagged eIF4Es were incubated with Ni²⁺ beads. Lysates containing MBP-tagged 4EBMs of human eIF4G, eIF4EBP1, or eIF4ENIF1 (see Appendix Fig. S2C–E) were added to the beads. After elution, binding of 4EBMs to eIF4E was assessed by SDS-PAGE and Coomassie staining. (D) (Left) Coomassie-stained gels of pulldowns with mouse eIF4E, eIF4Es from zebrafish (eIF4Ea, eIF4E2, and eIF4E3), and mouse eIF4E1B with 4EBMs from eIF4G, eIF4EBP1, and eIF4ENIF1. Quantifications are shown on the right. (E, F) Confocal microscopy images of fixed transgenic zebrafish oocytes and embryos expressing 3xflag-sfGFP-eIF4E1c (E) and 3xflag-sfGFP-eIF4E1b (F). Mitochondria were stained with Mitotracker (in magenta). Images at two different magnifications are shown at the top and bottom (scale bars correspond to 100 and 10 μm, respectively). The Balbiany body (Bb) is indicated by a dashed box; individual channels are shown in boxes for the Bb. Cytoplasmic foci are highlighted with arrows. Data information: (B, D) n = 3 independent experiments. Significance was determined using two-way ANOVA (B) or one-way ANOVA (D) followed by Tukey’s (B) or Dunnett’s (D) multiple comparisons test (****P value < 0.0001). Lines indicate mean with SD. (B, D) Predicted molecular weights (in kDa) are: Dr eIF4Ea, Hs eIF4E1B, Mm eIF4E1B, Dr eIF4E1c and Dr eIF4E2 = 65; Mm eIF4E = 66; Dr eIF4E3 = 64; Hs eIF4G^[4EBM] and Hs eIF4ENIF1^[4EBM] = 53; Hs eIF4EBP1^[4EBM] = 52. Source data are available online for this figure.