Fig. 4: Cryptic splicing enhances PTRE-seq reporter expression via RE-excision and enhanced production.

A Relationship between observed splicing fraction and reporter expression. GFP_B4 and GFP_P4 families of spliced transcripts are shown in dark and light blue, respectively. Regression lines of respective colors show the fits of RE-excision model to GFP_B4 and GFP_P4 reporters. Translucent bands show the 95% confidence interval for the regression estimates. The RE-excision model assumes constant production and is computed via Eq. 4 (Methods). Expression measurements are from the original PTRE-seq study²⁴ using barcode primers and normalized to the BBBB reporter. B Illustration of the mechanisms through which splicing impacts PTRE-seq reporter expression. C Estimated production rate for GFP_B4 and GFP_P4 reporters as a function of observed splicing fraction. Production rate is computed via Eq. 6 (Methods). D Effect of ARE module copy number on RNA steady state expression and translation efficiency. Shown are reporters consisting of all arrangements of blank modules with the indicated number of ARE modules. The original PTRE-seq study was unaware of cryptic splicing, making it appear that AREs have widely divergent impacts on mRNA stability and translation (grey boxplots). When spliced reporters are excluded, AREs have a uniformly destabilizing impact on reporter expression and translation efficiency (purple boxplots). Relative expression and translation efficiency were calculated as the median RNA and polysome sequencing measurements across 4 biological replicates reported in the original PTRE-seq study²⁴. Box plots span the 25^th and 75^th percentile and the centers indicate the median. Whiskers indicate the furthest datum 1.5x outside the interquartile range.