Fig. 6

Sequence-dependent effects of novel CPuORFs on main ORF translation. a Schematic representation of the WT (35S::UTR (WT):Fluc) and frameshift (fs) mutant (35S::UTR (fs):Fluc) reporter constructs. The 5′-UTR containing each CPuORF tested was inserted between the 35S promoter (35Spro) and the Fluc coding sequence. The hatched box in the CPuORF (fs) indicates the frame-shifted region. The dotted boxes represent the first five nucleotides of the mORF. See Supplementary Figure S2 for the exact position and length of each CPuORF and the exact frame-shifted region. HSPt: the AtHSP18.2 polyadenylation signal. b Transient expression assay for luciferase activity. Each reporter plasmid containing a WT or fs CPuORF, belonging to an indicated HG, was co-transfected into MM2d protoplasts with the 35S::Rluc internal control plasmid by PEG treatment. After 24-h incubation, dual luciferase assays were performed. Fluc activity was normalized to Rluc activity, and the normalized activity relative to that of the corresponding WT reporter construct is shown. c Transient expression assay for translation efficiency. For the five CPuORFs that showed a significant sequence-dependent effect in (b), the WT and fs reporter plasmids were each co-transfected into MM2d protoplasts with the 35S::Rluc plasmid again. After 24-h incubation, the Fluc and Rluc activities and the Fluc and Rluc mRNA levels were measured. Fluc activity was normalized to Rluc activity, and the Fluc mRNA level was normalized to the Rluc mRNA level. The normalized Fluc activity was divided by the normalized Fluc mRNA level to calculate the translation efficiency of Fluc. The Fluc translation efficiency relative to that of the corresponding WT reporter construct was calculated to determine the relative translation efficiency. In (b) and (c), means ± SD of at least three biological replicates are shown. Single and double asterisks indicate significant differences between the WT and fs constructs at p < 0.05 and p < 0.01, respectively, as determined by Student’s t-test