Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Abstract

Intron branch point (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeastSaccharomyces cerevisiaeby changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Minutes after addition of Plad-B splicing is inhibited, however different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed when we measure co-transcriptional splicing in Plad-B using single-molecule intron tracking (SMIT) to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. The results echo and extend observations from mammalian cells, where inhibitor studies are complicated by multi-intron gene architecture and alternative splicing. The more compact yeast system may hasten characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

“…Yeast is a facile model to address outstanding questions in splicing regulation, owing to its simplicity and versatility, in addition to the conservation of splicing machinery (Fabrizio et al 2009;Meyer and Vilardell 2009). As expression of human homologs can complement yeast genes of diverse pathways, yeast has been successfully used to dissect complex biological pathways relevant to human physiology and pathology (Garge et al 2020;Boonekamp et al 2022;Hunter et al 2023) as well as drug discovery (dos Santos and Sa-Correia 2009;Zimmermann et al 2018). Yeast has fewer (334; ~5% of the genome) and smaller introns with a highly conserved 5' ss, and most of them are co-transcriptionally processed (Spingola et al 1999;Aslanzadeh et al 2018;Talkish et al 2019).…”

The evolutionary dynamics of alternative splicing during primate neuronal differentiation

“…They found that individual introns had distinct sensitivities, including during co-transcriptional splicing, to different splicing inhibitors. Interestingly, they found that yeast sequences including the branch point consensus motif contribute to the differences in sensitivity (65). Osterhoudt et al (2024) explored changes in 3’SS usage upon SACY-1 perturbation in introns with pairs of 3’ splice sites ≤ 18 nucleotides away from each other.…”

Intron-lariat spliceosomes convert lariats to true circles: implications for intron transposition

“…Hits to the same target are grouped together in the output file for review. To identify yeast reads from intron circles we followed a similar process using annotated splice junctions from the track "Talkish Standard Introns" on our yeast genome browser at intron.ucsc.edu (Talkish et al 2019) and the sequencing data in GEO at GSE90105 from (Talkish et al 2019) and from (Hunter et al 2024) in the SRA at PRJNA972189. A github deposit with scripts and more technical details can be found at: https://github.com/donoyoyo/intron_circle_hunt…”