Figure 1. A standardized reporter library uncovers splicing regulatory information encoded within introns.

A) Overview of the reporter approach for studying splicing mediated gene expression regulation. Intron insertion cassettes were constructed in-vitro, each comprised of a selection marker (URA3), a constitutive promoter, the first 195 nucleotides (nt) of the YFP gene, and one of 240 native S. cerevisiae introns followed by an additional 60 nt of the YFP gene. Each insertion cassette was transformed into the genome of a master strain which contained a promoter-less YFP gene, thus creating an in-vivo intron-reporter yeast library (YiFP). Culture growth and YFP expression levels of each variant in the library were monitored using a micro-plate reader. B) All strains in the YiFP reporter library grew similarly. C) However, each intron conferred unique YFP expression levels. D) Each strain's average expression levels, YiFP(i), were compared to that of an intron-less reference strain, YFP(wt), to get an assessment of “splicing efficiency”. YiFP strains whose YFP levels did not pass the detection limit were considered as “non-spliced” (marked with circles). Error bars represent ±1 SD from four independent experiments. E) Splicing efficiency of ribosomal and non-ribosomal protein genes (RPGs) is distributed in a similar manner. F) Analysis of YFP expression at the single cell level (using automated microscopic imaging) validated splicing efficiency measurements of spliced introns (inset graph, r = 0.94), and enabled assessment of splicing efficiency noise in a population. Noise is represented by the (squared) YFP expression coefficient of variation (CV²), i.e. the variance (σ²) normalized by the squared mean YFP expression (μ²), for each intron strain as determined using microscopic imaging analysis. Gene names of introns that presented noise higher (red) or lower (blue) than normal are indicated (outliers of linear regression; p<0.1). G) Splicing efficiency in a synthetic context is robust to environmental change. Yeast were grown in several stress conditions (Amino acid starvation, Rapamycin, 1M KCl) known to affect the splicing machinery. Error bars represent ±1 SD from three independent experiments.