Determinants of splicing efficiency in yeast and their effect on cellular function

Pre-mRNA splicing is a crucial step during gene expression regulation in eukaryotic cells. However, the control and regulation of splicing itself is complex and not yet fully understood. In addition, we hypothesize that the efficiency of splicing on one transcript might affect cellular functions in-trans as it influences the cellular supply and demand economy of the splicing machinery.

We aim to map the effect of different features on splicing efficiency using a large library of synthetic intron-containing non-coding RNAs in S.Cerevisiae. We measured splicing efficiency and RNA abundance of each library variant using RNA sequencing. Moreover, we measured global cellular effects of the library by measuring cellular fitness of each strain using a competition assay, and by measuring splicing efficiency in-trans using an intron-containing fluorescent reporter.

Splicing is regulated through three functional sites, 5’ donor, branch site, and 3’ acceptor. We explored the contribution of variations in these sites to splicing efficiency and demonstrate that different sites have different tolerance to perturbations. We also demonstrate that intronic and exonic sequences have a major effect on splicing efficiency. Moreover, we successfully designed sequence elements that are alternatively spliced to up to four different isoforms, which is intriguing since there is almost no evidence for alternative splicing in S. Cerevisiae.

Finally, we show that certain design features influence other intron-containing genes, and the cellular fitness, which demonstrates how changes to one transcript might affect the wellbeing of the cell by non-optimal usage of a common resource such as the splicing machinery.