FIGURE 6.
The overlapping uORFs of the mouse 5′UTR mGADD34 mediate translational control during eIF2α phosphorylation. Top row, immunoprecipitates of [35S]methionine-labeled YFP from lysates of cells stably expressing wild-type or mutant mouse 5′UTR mGADD34-YFP reporter RNAs as indicated above under untreated or 1μm thapsigargin treatment for 45 min. 1&2KO, 1KO, and 2KO represent mutant mouse mGADD34 5′UTR reporter RNAs in which the AUG start codon of uORF1 (1KO), uORF2 (2KO), or both (1&2KO) uORFs was mutated. Mutations that inserted one (1cod) or two (2cod) codons between the AUG codon of the uORF2 and the stop codon of the upstream uORF1 were engineered into the 5′UTR mGADD34-YFP reporter RNA (see “Experimental Procedures” for sequence). Second row, immunoblots of lysates using antibodies that recognize phospho-eIF2α or total eIF2α. Third row, cells were pulse-labeled with [35S]methionine for 20 min prior to harvesting. Lysates were subjected to SDS-PAGE analysis and exposed to autoradiograph. Bottom row, in parallel, RNA from treated cells was subjected to Northern blot analysis using probes specific for YFP or GAPDH. B, quantitation of [35S]methionine-labeled YFP immunoprecipitates (top left) and of YFP RNA levels normalized to GAPDH (top right). Bottom, each bar represents the percentage of [35S]methionine-labeled YFP expressed or YFP RNA levels during the thapsigargin treatment as compared with that in untreated cells. Translational efficiency of reporter YFP RNA in unstressed cells was calculated by the amount of newly synthesized [35S]methionine precipitates normalized to YFP/GAPDH mRNA levels. Above each bar shows the average translational efficiency normalized to the wild-type mGADD34-YFP RNA. Shown are averages ± S.D. from at least three independent experiments.