FIGURE 5.

The uORFs of hGADD34 5′UTR mediate translation regulation during eIF2α phosphorylation. In A: Top row, immunoprecipitates of [³⁵S]methionine-labeled YFP from lysates of cells expressing IGR IRES-YFP, hATF4-YFP, wild-type hGADD34-YFP, YFP alone, or mutant human GADD34 5′UTR fused with YFP where the AUG codon of the upstream uORF (1KO), downstream uORF (2KO), or both uORFs were mutated (1KO, 2KO, and 1&2KO, respectively) were either left untreated (U) or treated with 1 μm thapsigargin (T), 2 mm DTT (D), or 100 μg/ml arsenite for 45 min (A). Cells were pulse-labeled with [³⁵S]methionine for 20 min prior to harvesting. Immunoprecipitates were separated by SDS-PAGE and exposed by phosphorimaging analysis. Second row, immunoblots of lysates using antibodies that recognize phospho-eIF2α or total eIF2α. Third row, lysates were subjected to SDS-PAGE analysis and exposed to autoradiography. Bottom row, in parallel, RNA from treated cells was subjected to Northern blot analysis using probes specific for YFP or GAPDH. B, quantitation of newly synthesized [³⁵S]methionine-labeled YFP immunoprecipitates (top left) and levels of YFP RNA normalized to GAPDH (top right) as described in A. Each bar represents the percent of [³⁵S]methionine-labeled YFP expressed or the YFP RNA levels during the indicated drug treatments as compared with that in untreated cells, which is set at 100%. For Northern blot analysis, YFP mRNA levels were normalized to GAPDH mRNA levels. Bottom, translational efficiency of reporter YFP RNA in unstressed cells was calculated by the amount of newly synthesized [³⁵S]methionine YFP protein normalized to YFP/GAPDH mRNA. Above each bar shows the average translational efficiency normalized to the wild-type hGADD34-YFP RNA as 1. Shown are averages ± S.D. from at least three independent experiments.