Accurate, Model-Based Tuning of Synthetic Gene Expression Using Introns in S. cerevisiae

Abstract

Introns are key regulators of eukaryotic gene expression and present a potentially powerful tool for the design of synthetic eukaryotic gene expression systems. However, intronic control over gene expression is governed by a multitude of complex, incompletely understood, regulatory mechanisms. Despite this lack of detailed mechanistic understanding, here we show how a relatively simple model enables accurate and predictable tuning of synthetic gene expression system in yeast using several predictive intron features such as transcript folding and sequence motifs. Using only natural Saccharomyces cerevisiae introns as regulators, we demonstrate fine and accurate control over gene expression spanning a 100 fold expression range. These results broaden the engineering toolbox of synthetic gene expression systems and provide a framework in which precise and robust tuning of gene expression is accomplished.

Nucleotide sequence composition adjacent to intronic splice sites improves splicing efficiency via its effect on pre-mRNA local folding in fungi

“…Furthermore, they can help us understand how silent mutations are related to human diseases via their possible effect on pre-mRNA folding near intronic splice sites (Sauna and Kimchi-Sarfaty 2011). Finally, the results suggest new methods for engineering gene expression of synthetic genes via the manipulation of pre-mRNA folding near intronic splice sites (Yofe et al 2014). …”

“…One way to validate these two hypotheses is by comparing the inferred pre-mRNA folding signal (and folding selection signal) with SE estimations. We did this using a previously reported synthetic library (Yofe et al 2014). Briefly, S. cerevisiae is transformed FIGURE 5.…”

“…YiFP strains whose expression levels did not pass the detection limit were considered as nonspliced. Summary of the system can be seen in Figure 6A; for more details see Materials and Methods and Yofe et al (2014). First, we extracted the intronic SE library related measurements of 215 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 nt of the YFP gene, and one of 240 native S. cerevisiae introns followed by an additional 600 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); each strain's average expression levels were compared with that of an intron-less reference strain, to get an assessment of splicing efficiency (SE); YiFP strains whose YFP levels did not pass the detection limit were considered as nonspliced (marked with circles; error bars represent STD from four independent experiments; see Materials and Methods and Yofe et al 2014). (B) LFE analysis of endogenous introns corresponding to highly/lowly expressed YiFP genes (i.e., SE) shows that both groups have a similar tendency for weaker folding surrounding their splice sites (brown and green, respectively; intronome in blue).…”

“…The synthetic YiFP reporter library was downloaded from Yofe et al (2014). To create the synthetic intron-reporter library, S. cerevisiae was transformed with a library of DNA transformation cassettes, each containing a different native yeast intron.…”