FIGURE 4.
EPRS translation control involves bypass of an uORF with a noncanonical CUG initiation codon. WT and mutant versions of PTK-EPRS-Luc constructs were transfected into WT MEF cells, treated for 6 h or left untreated, and measured using a Dual-Luciferase assay, and the relative levels of the corresponding EPRS-Luc mRNAs were measured by qRT-PCR. Mutant versions of PTK-EPRS-Luc include a stem loop insertion and mutation of the CUG initiation codons individually or together, as represented by ΔCUG1, ΔCUG2, and ΔCUG3. Losses of the initiation codons CUG1, CUG2, and CUG3 are indicated in the EPRS-Luc fusion that is indicated by the gray boxes. Optimization of the CUG2 initiation codon to an AUG in optimal Kozak consensus sequence (ACCAUGG) is represented as CUG to optimized AUG (orange box). Relative values are represented as histograms for each with the S.D. indicated. TG, thapsigargin; Luc, luciferase.