Fig. 6: RG4 does not increase mRNA lifetime but may stabilize ribosome bound to mRNA.

a, b Hypothesis 3: RG4 increases the mRNA lifetime. a Illustration of how RG4 could prevent the mRNA degradation. b RT-PCR is performed with two primers, proximal and distal to the 5′ end. No significant difference is observed among the control, Template (T), and Non-template (NT) transcript levels with either primer, indicating that RG4 does not affect mRNA lifetime. Data are presented as mean ± SEM of independent experiments (n = 6). NS: nonsignificant (two-sided paired t test). Exact mean values are provided in Supplementary Table 3.7. Raw data points are provided as a Source Data file. c–g Hypothesis 4: RG4 stabilizes the ribosome-bound state. c A potential mechanism of RG4 stabilizing the ribosome bound to the mRNA by preventing ribosomes from dislodging off the mRNA. d RG4 specific helicase, DHX36, is used to remove RG4 from mRNA during translation. e Translation readout as real-time GFP intensity. The titration of helicase demonstrates a dose-dependence such that the more addition of helicase decreases the translation level further. The curves shown are one representative result from three independent experiments. f Normalized transcription rate of NT construct with DHX36 titration. g Normalized translation efficiencies are calculated from the early linear part of the curve in (e), and normalized to transcription level in (f). It reveals a dose-dependent decrease in translation, indicating that RG4 is essential for translation enhancement. For (b, f and g), data are presented as mean ± SEM of independent experiments (n = 3). **P < 0.005, ***P < 0.0005 and NS nonsignificant (two-sided paired t test). Exact mean values are provided in Supplementary Tables 3.8 and 3.9. Raw data points are provided as a Source Data file.