Fig. 2: PQS orientation and presence of RNA G4 increases translation efficiency.

a Schematic of RNA G4 insertion in the 5′UTR. The potential G-quadruplex sequence (PQS) in non-template (NT) strand leads to the formation of RG4 in the mRNA, while insertion in template (T) strand and control (C) does not. b, d, f Real-time intensities of transcription, translation and G4 formation assays. The constructs, non-template (NT), control (C), and template (T) are colored in blue, black, and purple, respectively. The curves shown are one representative result from multiple independent experiments. The control and tested PQS sequences are scramble sequence and cMyc. b Transcription assay is quantified by Cy3 probe intensity. d Translation is measured as sfGFP intensity. f RNA G4 formation is quantified by real-time N-methyl mesoporphyrin IX (NMM) signal. NT shows NMM signal while C and T have no signal. c Transcription rates are calculated from the early linear part of the curve in (b) and normalized to the transcription rate of the control sequence. The transcription rate of NT is 30% higher than T. e NT construct enhanced the translation fivefold higher than C and T. Translation efficiencies are calculated from the translation rates obtained from the early linear part of the curve in (d) and the normalized transcription rates in (c). The translation efficiency was normalized to the control sequence. For (c) and (e) data are presented as mean ± SEM of independent experiments (n > 6). Exact mean values are provided in Supplementary Tables 3.1 and 3.2. NS: nonsignificant, *P < 0.05, ***P < 0.0005 (two-sided unpaired t test). g Halftime to saturation of NT. The halftimes of transcription (in b), translation (in d), and NMM RG4 formation (in f) are 32 ± 2.8, 47 ± 7.5, and 71.7 ± 4.6 min, respectively. Data are presented as mean ± SEM of independent experiments (n = 3). Raw data points are provided as a Source Data file.