Specificity and exon target space of splicing modifying compounds.

Lenkeit, Felina; Knehr, Judith; Altorfer, Marc; Byrnes, Andrea; Li, Wenjing; Hsiao, Jack; Wu, Connie; Gaitonde, Priti et al. · Nat Commun · 2026

basic_science · Level V

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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 <math xmlns="http://www.w3.org/1998/Math/MathML"><msup><mrow><mn>5</mn></mrow><mrow><mo>'</mo></mrow></msup></math> splice sites. Risdiplam preferentially induces exons with an N<sub>-3</sub>G<sub>-2</sub>A<sub>-1</sub> sequence at the <math xmlns="http://www.w3.org/1998/Math/MathML"><msup><mrow><mn>3</mn></mrow><mrow><mo>'</mo></mrow></msup></math> exon end, whereas Branaplam favors A<sub>-3</sub>G<sub>-2</sub>A<sub>-1</sub>-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.