An exon three-way junction structure modulates splicing and degradation of the SUS1 yeast pre-mRNA

The SUS1 gene of Saccharomyces cerevisiae is unusual as it contains two introns and undergoes alternative splicing, retaining one or both introns depending on growth conditions. The exon located between the two introns can be skipped during splicing and has been detected in circular form. This exon (E2) has also been found to influence the splicing of the flanking introns, an unusual situation in budding yeast where splicing mainly relies on intron recognition. Using SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension), NMR spectroscopy, gel electrophoresis and UV thermal denaturation experiments combined with computational predictions, we show that E2 of SUS1 comprises a conserved double-helical stem topped by a three-way junction. One of the hairpins emerging from the junction exhibited significant thermal stability and was capped by a purine-rich loop structurally related to the substrate loop of the VS ribozyme. Cellular assays revealed that three mutants containing altered E2 structures had impaired SUS1 expression, and that a compensatory mutation restoring the conserved stem recovered expression to wild-type levels. Semi-quantitative RT-PCR measurements paralleled these results, and revealed that mutations in E2 altered splicing and transcript degradation processes. Thus, exon structure plays an important role in SUS1 RNA metabolism. •The SUS1 gene of S. cerevisiae contains two introns and undergoes alternative splicing.•The exon located between two introns is generated in circular form and can be skipped.•The RNA of this exon (E2) forms a conserved stem topped by a three-way junction.•Mutants with altered E2 RNA structures had impaired SUS1 expression.•The E2 mutations modified SUS1 pre-mRNA splicing and transcript degradation.