July 03, 2014
As any good handyman knows, the more tools you have in your tool chest, the better the chance that you can find what you need to solve a problem. The same goes for synthetic biologists. The more parts they can mix and match, the more likely they are to engineer the exact level of gene expression they need.
In the last few years synthetic biologists have amassed a wide variety of transcription and translation elements that can be combined in different ways to exquisitely tune the level of expression of their gene of interest. And now, in a new study out in PLOS Genetics, Yofe and coworkers have added introns to the list of parts available for our favorite yeast Saccharomyces cerevisiae.
Yeast isn’t loaded with introns, but it does have a reasonable number that can be co-opted for synthetic biology. The authors inserted 240 of these introns individually into the same position near the 5’ end of the yellow fluorescent protein (YFP) gene and monitored the level of fluorescence of each individual strain over a 24 hour period. They chose the 5’ end of the gene because yeast has a bias for introns being located there.
The authors found that these reporters spanned a 100-fold range of gene expression, that every intron caused a decrease in the level of gene expression, and that even though many of these introns respond to environmental stimuli in their natural context, their effect on gene expression here was immune to the environmental changes the authors tested. Taken together, these results suggest that introns could be used in yeast systems for dampening over-exuberant gene expression in ways that are independent of growth conditions. If all of this holds up, introns will prove to be very useful tools indeed.
Yofe and coworkers next wanted to use this library to figure out some of the rules for why some introns cause lowered activity compared to others. The simplest possibility, that longer introns cause a larger decrease in gene expression, turned out not to be true. There was no correlation between the size of the intron and its effect on the level of fluorescence.
Next they scanned the sequences of their constructs to look for elements that might increase or decrease splicing efficiency. These splicing regulatory elements (SREs) are better understood in larger eukaryotes, but there is evidence that they are important in yeast as well. The authors identified a number of intron splicing enhancers (ISEs) and intron splicing silencers (ISSs) that were highly enriched near the splice sites.
To confirm that these sequences did in fact affect splicing efficiency (and hence gene expression), they showed that mutating the enhancer motif TTTATGCT to the silencer motif TTTGTGTA in two reporters resulted in a 22% and a 13% decrease in gene expression. This proof of principle experiment suggests that future synthetic biologists may be able to further tweak the expression of their genes by manipulating these SREs.
In a final set of experiments the authors used the library to identify rules that can be used to predict how inserting various introns into different positions will affect a gene’s activity. They found that the most important features were the presence of SREs and the RNA structures at the intron-exon junction. Synthetic biologists should be able to use these rules to intelligently design their reporter systems.
These experiments are the first step towards adding introns to the ever growing set of tools available to synthetic biologists for modulating gene expression. We are getting closer to figuring out how genes are controlled and being able to use that knowledge to our advantage. Or to put it another way, we have taken another baby step towards being able to control a gene as well as a yeast cell does.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight