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. 2016 Mar;22(3):456–466. doi: 10.1261/rna.054452.115

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© 2016 Beznosková et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 5. — eIF3-promoted readthrough does not interfere with the eIF3-promoted REI on REI-permissive uORF1 from the GCN4 mRNA leader. (A) The 6-nt-long context immediately following stop codons of all four uORFs from the GCN4 mRNA leader allows eIF3-dependent readthrough with varying efficiency. The PBH140 derivatives bearing TIF35 wt and tif35-KLF mutant alleles were grown in SD and processed for stop codon readthrough measurements using standard dual luciferase readthrough reporter constructs pTH460; pTH477; PBB135; PBB136; PBB137; PBB138; and PBB139 as described in Materials and Methods. Changes in the measured readthrough values between TIF35 and tif35-KLF cells were analyzed by the Student's t-test (mean ± SD; n = 6) and shown to be statistically significant only for those cases marked with the asterisk (P < 0.01). (B) The 6-nt-long sequence preceding the stop codon of uORF1 nullifies the eIF3-mediated stimulation of stop codon readthrough on this REI-permissive uORF. The PBH140 derivative bearing the TIF35 wt allele was grown in SD and processed for stop codon readthrough measurements using standard dual luciferase readthrough reporter constructs pTH460; pTH477; PBB135; PBB136; PBB137; PBB138; PBB139; PBB140; PBB141; PBB142; PBB143; and PBB144 as described in Materials and Methods. Changes in the measured readthrough values between selected constructs were analyzed by the Student's t-test (mean ± SD; n = 6) and shown to be statistically significant only for those cases marked with the asterisk (P < 0.01).