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letter
. 2015 Jun 22;35(13):1736–1742. doi: 10.1038/onc.2015.233

Figure 3.

Figure 3

Reinitiating ribosomes contribute to TK translation. (a) Schematic representation of mutations introduced into the TLS reporter construct to determine the contribution of reinitiating ribosomes to TK translation. The deletion of uORF-related upstream stop codons (uStops to CUU, ΔuStop) created extended uORFs that overlapped the mAUG start site. Second, an alternative uStop codon was re-introduced between the natural uStop and the mAUG codon (uStop re-introduction). For CEBPA, CEBPB and ERBB2, the uStop re-introduction was not performed as the distances between the uStop and the CDS were as short as 7, 4 and 5 nt, respectively. All mutations were introduced by site-directed mutagenesis of the wt uORF version of the respective TLS using the PfuPlus! DNA Polymerase (Roboklon) and customized primers (Supplementary Tables 3 and 4). (b) Bar graph representing the relative luciferase activity detected in the presence of wt uORF-, ΔuStop- and re-introduced uStop-containing TLSs. (c) Bar graph indicating the relative luciferase mRNA levels of wt uORF, ΔuStop and re-introduced uStop reporter constructs for indicated TLSs. Error bars represent the s.e.m. of at least three independent experiments. Asterisks indicate statistical significance (**P<0.01 and *P<0.05).