Specificity and exon target space of splicing modifying compounds

Abstract

Abstract Modulation of splicing is an established therapeutic strategy with clinical applications and potential to target specific exons to influence gene expression. Small-molecule splicing modifiers such as Risdiplam and Branaplam induce inclusion of exons typically skipped due to weak $${5}^{{\prime} }$$ 5 ′ splice sites. Risdiplam preferentially induces exons with an N −3 G −2 A −1 sequence at the $${3}^{{\prime} }$$ 3 ′ exon end, whereas Branaplam favors A −3 G −2 A −1 -ending exons. However, determinants of specificity remain unclear, as many motif-matching exons are not induced. Here, we investigate the molecular basis of splicing-modulator specificity. Using biochemical assays, transcriptome analyses, and genetic perturbations, we identify sequence-dependent features that determine exon responsiveness to splicing-modulator induction. We further demonstrate that specificity can be reprogrammed through manipulation of U1 snRNA. These findings refine the determinants of splicing-modulator target space and may support identification of additional target exons and compounds.