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, SYNE27α. 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 SYNE27α 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.