A broad analysis of splicing regulation in yeast using a large library of synthetic introns

Abstract

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5’ donor site, the branch site, and the 3’ acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3’ splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns’ 3’ ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism’s splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract

“…We cannot speculate why CTG is also depleted. A difference between our study and that of Schirman et al (2021) may be the strategy used to identify depletion of 3’ splice signal motifs. They did not use strand asymmetry but compared the sequence context around introns’ 3’ ends to that of 1000 randomly selected genomic loci and did not investigate depletion of the motifs TAG and CAG separately, but together.…”

“…Recently, Schirman et al (2021) reported that the absence of the U2AF1 protein in certain yeast species, including S. cerevisiae , leads to increased selective pressure to avoid a premature splice signal. In our study, the pattern of asymmetry scores looks grossly similar in S. cerevisiae and L. thermotolerans .…”

“…Once ESE motifs started to appear, ESEs and trans-acting splicing factors would co-evolve due to selection on splicing. A study in yeast suggested that the avoidance of cryptic splicing through depletion of the 3’ splice signal around the 3’ end drives intron sequence evolution and splicing factors can co-evolve with this (Schirman et al, 2021). In the light of our results, a positive feedback loop is conceivable: once the pyrimidine content in the 3PT increased due to avoidance of the 3’ splice signal, splicing factors co-evolved to develop higher binding specificity to pyrimidines.…”

Biochemical activity is the default DNA state in eukaryotes

“…2D ), indicating that evolution must optimize to get very high expression levels. Splicing, which is rare in the yeast genome (306/5028 transcripts spliced), was about half as common in naïve DNA (10/356 transcripts spliced; 6% vs 2.8%; two sided binomial p=0.007), with naïve intron sequences closely matching known splicing motifs (39) ( Supplementary Fig. 2 ).…”

RBP45d is required for 5’splice site selection via binding to intronic U-rich elements and interaction with PRP39a inArabidopsis thaliana

“…Spliceosome has evolved intrinsic ability to prevent from uses of cryptic splice sites in pre-mRNA splicing. Interestingly, targeted RNA-seq analysis using a large library of synthetic introns in yeast revealed a similar mechanism by which splicing machinery can prefer to utilize alternative 3' ss downstream of the original site, thus avoiding selection of cryptic splice sites nearby the 3' end of introns (Schirman, et al, 2021). Further analysis showed that the vast majority of cryptic splicing sites located downstream of 3'ss sites are activated by a loss of a specific splicing factor U2AF35 (Schirman, et al, 2021).…”