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eIF4E1b levels negatively correlate with total protein levels in zebrafish embryos and gonads. (A, B) Volcano plots of MS data obtained from lysates of 5 hpf embryos expressing either actb2:3xflag-sfGFP-eIF4E1c (eIF4E1c OE; (A)) or actb2:3xflag-sfGFP-eIF4E1b (eIF4E1b OE; (B)) compared to wild type (WT). n = 3 biological replicates. (C) mRNA levels (assessed by RT-qPCR) of proteins dysregulated in eIF4E1c OE (left) or eIF4E1b OE (right) embryos in eIF4E1b OE, eIF4E1c OE and WT embryos at 5 hpf (n = 3 biological replicates). Columns indicate mean and error bars indicate SD. (D–F) Translational efficiency (TE; (D)), mRNA levels (E) and ribosome-protected fragments (RPF; (F)) of genes encoding proteins dysregulated in eIF4E1c OE or eIF4E1b OE embryos (data from 4 hpf wild-type embryos from Subtelny et al, 2014). Lines indicate median and first and third quartiles. (G, H) Volcano plots representing mass spectrometry (MS; (G)) and RNA sequencing (RNA-seq; (H)) data from eif4e1b mutant and wild-type female gonads isolated from juvenile fish with high ziwi:GFP expression (n = 3 biological replicates). (I) Venn diagram showing the overlap between up- or downregulated proteins with up- or downregulated mRNAs in eif4e1b mutant gonads isolated from high ziwi:GFP-expressing juvenile fish. Data information: significance was calculated using an ordinary one-way ANOVA followed by Sidak’s (C), Dunett’s multiple comparisons tests (D–F), limma (A, B, G, Smyth, 2005), and Benjamini–Hochberg-corrected Wald test (DeSeq2; H). ****P value < 0.001. RPKM reads per kilobase of transcript per million mapped reads, Hpf hours post fertilization. Source data are available online for this figure.
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