FIGURE 2.

Preferential translation of GADD34 features an inhibitory uORF. A, top panel, 5′-RACE was carried out for GADD34 using WT MEFs treated with thapsigargin for 6 h or left untreated; total RNA was prepared and DNA products were separated by gel electrophoresis, with markers of the indicated base pair sizes illustrated on the left. A, bottom panel, Representation of GADD34 5′-leader in lowercase letters, with uppercase letters representing the 5′-linker added during the 5′-RACE procedure and the beginning of the CDS of the GADD34-Luc fusion. Colored boxes represent the GADD34 uORFs and the coding region of the GADD34-Luc fusion. The transcription start site is indicated with an arrow, and location of the stem loop insertion is illustrated. B, the P_TK-GADD34-Luc construct and a Renilla luciferase reporter were co-transfected into WT or A/A MEF cells and treated for 6 h with thapsigargin or left untreated. GADD34 translation control was measured via dual luciferase assay and corresponding GADD34-Luc mRNA was measured by qRT-PCR. The P_TK-GADD34-Luc construct contains the cDNA sequence corresponding to the GADD34 5-leader fused to the luciferase reporter gene with both GADD34 uORFs and the CDS of the GADD34-Luc fusion indicated with colored boxes. 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 a stem loop insertion and mutation of the initiation codons for uORFs individually or together, as represented by ΔATG. Relative values are represented as histograms for each with the S.D. indicated.