Small and large uORFs both robustly stimulate NMD in HeLa cells. A Tet-Off system triggered with 2 µg/mL doxycycline (dox) was used to determine reporter mRNA half-lives in HeLa cells. Schematic of reporter is located above each mRNA decay curve. The small and large uORFs (same design as in Fig. 1) are shown in black and blue, respectively. nLuc ORF is in purple. Start and stop codons are green and red circles, respectively. Introns in the nLuc ORF and 3′ UTR are shown as dotted, angled lines. As controls to rescue mRNA half-life from NMD, a strong hairpin in the 5′ UTR was inserted to limit translation initiation (middle column) and stop codons were mutated from UAA to UAC to prevent termination upstream of a spliced intron (right column). n = 3 biological replicates. A nonlinear regression was used to calculate the mRNA half-lives and is shown as the line with the 95% confidence interval included as a watermark. The half-life is reported with the error for the 95% confidence interval range.
