FIGURE 3.

GADD34 translation control involves bypass of an inhibitory uORF that relies on a Pro-Pro-Gly juxtaposed to the uORF2 stop codon. A, WT and mutant versions of P_TK-GADD34-Luc were transfected into WT MEFs, treated for 6 h or left untreated, and measured using a dual luciferase assay and qRT-PCR. Mutant versions of P_TK-GADD34-Luc include mutation of the GADD34 uORF2 poor start codon context to “*, strong Kozak context,” and mutation of the stop codon of uORF2 to generate and overlapping out-of-frame uORF (TGA to GGA). Relative values are represented as histograms for each with the S.D. indicated. B, polypeptide sequence encoded by GADD34 uORF2 from different vertebrates. The uORF2 polypeptide sequences were from cDNAs derived from GADD34 orthologs, including human (GenBank^TM accession number NM_014330), mouse (NM_008654), rat (NM_133546), hamster (L28147), naked mole rat (XM_004889808), pig (XM_003127275), cow (NM_001046178), horse (XM_001489532), and dog (XM_533626). Residues conserved between the uORF sequences are listed in the consensus and are highlighted. C, WT and mutant versions of P_TK-GADD34-Luc were transfected into WT MEFs, treated for 6 h or left untreated, and measured using a dual luciferase assay and qRT-PCR. Mutant versions of P_TK-GADD34-Luc include deletion of codons 5–25 or 15–25 (ΔAA 5–25 or ΔAA 15–25) and a frameshift construct in which a nucleotide was inserted just following the uORF2 ATG start codon and deleted just prior to the uORF2 stop codon. Additional constructs included mutation of the codons encoding Pro-Pro-Gly to codons for Ala-Ala-Ala (PPG to AAA), simultaneous mutation of the uORF2 start codon to Kozak consensus sequence with the Pro-Pro-Gly to Ala-Ala-Ala mutation (* Strong Kozak Context, PPG to AAA), and insertion of codons encoding Ala-Ala-Ala just prior to the uORF2 stop codon (insert AAA). Relative values are represented as histograms for each with the S.D. indicated.