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 \({5}^{{\prime} }\) splice sites. Risdiplam preferentially induces exons with an N−3G−2A−1 sequence at the \({3}^{{\prime} }\) exon end, whereas Branaplam favors A−3G−2A−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.
