Fig. 5

a An alignment of the sequences of the translational regulatory regions of human ATF4 and zebrafish atf4a and atf4b, showing the highly conserved upstream open reading frames (uORF1 and uORF2; red boxes) and coding ORFs (green box). These regions of zebrafish atf4a and atf4b were cloned into the reporter vectors, and their coding ORFs were fused with the ORF of EGFP. b Fluorescence microscopy images showing the enhanced expression (i.e., translation) levels of the atf4a- and atf4b-EGFP fusion ORFs (red arrows) in the truck and head of tars^−/− embryos, which were inhibited by the knockout of gcn2, but not perk. The Tol2 transposase-based transgenic system containing a ubiquitin promoter (ubi:) was used to drive the expression of the reporters. c Mutation analysis of the zebrafish atf4a and atf4b uORFs for their eIF2α phosphorylation-dependent translational regulation. The start codon (ATG) of uORF1 or uORF2 was mutated into AGG. The expression levels of the WT and uORF1- and uORF2-mutated (Mut) atf4a- and atf4b-EGFP reporters were demonstrated by representative fluorescence microscopy images. The lower panels present the working model in which uORF1 can reduce the usage of the inhibitory uORF2, so that the uORF1 Mut eliminates the AAR-dependent upregulation of atf4a/b expression, whereas the uORF2 Mut leads to an extremely high AAR-independent atf4a/b expression.