The RNA isoform repertoire is regulated by splicing factor (SF) expression, and alterations in SF levels are associated with disease. SFs contain ultraconserved poison exon (PE) sequences that exhibit greater identity across species than nearby coding exons, but their physiological role and molecular regulation is incompletely understood. We show that PEs in serine-arginine-rich (SR) proteins, a family of 14 essential SFs, are differentially spliced during induced pluripotent stem cell (iPSC) differentiation and in tumors versus normal tissues. We uncover an extensive cross-regulatory network of SR proteins controlling their expression via alternative splicing coupled to nonsense-mediated decay. We define sequences that regulate PE inclusion and protein expression of the oncogenic SF TRA2β using an RNA-targeting CRISPR screen. We demonstrate location dependency of RS domain activity on regulation of TRA2β-PE using CRISPR artificial SFs. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2β-PE detected in breast tumors, altering breast cancer cell viability, proliferation, and migration. Display omitted •SR proteins levels are coordinated through splicing of their poison exons•SR protein poison exon splicing is altered during cell differentiation and in tumors•ASOs promotingTRA2β poison exon inclusion have anti-cancer effects in vitro Leclair et al. demonstrate that expression of 14 SR proteins is coordinated through splicing of their poison exons during cell differentiation and tumorigenesis. Using the TRA2β poison exon as an example, they identify splicing-regulatory sequences and design splice-switching antisense oligonucleotides that decrease TRA2β protein expression and exhibit anti-cancer effects.