The U1 snRNP protein U1C and Helix H of U1 snRNA are critical for small molecule splicing modulator function

Risdiplam and branaplam represent two classes of small-molecule splicing modulators that promote U1 snRNP recognition of weak non-canonical GA/GU-containing 5' splice sites (ss). We demonstrate that branaplam enhances recognition of these 5' ss by reconstituted U1 snRNP in vitro, and that this effect depends on the ZnF domain of U1C and Helix H of U1 snRNA, but not U1A or U1-70K. We also demonstrate that branaplam enhances the weak 5' ss recognition through a dual act of strengthening the U1 snRNP-5' ss interaction and U1 snRNP-U1C interaction. In cells, depletion of U1C reduces or abolishes compound-induced exon inclusion for most cassette exons. Interestingly, a subset of cassette exons become responsive to compound only upon U1C knockdown, supporting a model in which U1C stabilizes specific conformations at the 5' ss-U1 snRNA interface in a context-dependent manner that can either facilitate or hinder compound binding. Surprisingly, risdiplam shows no effect on weak 5' ss recognition in vitro, suggesting additional cellular factors are required for its activity.