A spliceosome-independent eukaryote generated by complete intron removal

Spliceosomal introns impose a universal processing burden on eukaryotes and obstruct genome minimization because their essentiality remains unresolved. By exploiting Spo11-independent meiosis in synthetic single-chromosome Saccharomyces cerevisiae, the complete deletion of all 300 spliceosomal introns was achieved, generating an intron-free strain, SYNE27^α. Whole-genome sequencing confirmed precise excision. Unexpectedly, spliceosomal components (all five small nuclear RNAs [snRNAs], Prp8, Prp9, Prp19, Yhc1, and Luc7) were no longer required for viability, demonstrating that a eukaryotic cell can exist independently of spliceosomal function. U3 small nucleolar RNA (snoRNA) splicing bypassed the requirements for Yhc1, Luc7, Prp9, and Prp19, revealing a mechanistic divergence from pre-mRNA splicing. Cumulative intron loss caused slow growth via ribosomal dysregulation, yet SYNE27^α maintained genetic stability. Fitness costs were fully recessive in diploids, confirming intron loss as the primary driver. These findings establish an intron-free, spliceosome-independent eukaryote, resolving the essential function of the spliceosome and enabling minimal-system studies of genome evolution.