Talk abstracts
Talk on Saturday 09:45-10:00am submitted by Benjamin Fair
Distinct U1 cofactor requirements link Branaplam and Risdiplam specificity to splice-site context
Benjamin Fair (Department of Medicine, University of Chicago), Bo Liu, Zhichao Tang, Junxing Zhao, Yang Li, Jingxin Wang (Department of Medicine, University of Chicago), Zhiling Kuang, Rui Zhao (Department of Biochemistry and Moleclar Genetics, University of Colorado Anschutz Medical Campus)
Abstract:
Splice-modifying small molecules can perturb genes considered undruggable with conventional protein-targeted molecules. Risdiplam and Branaplam are distinct splice-modifying small-molecule scaffolds originally identified to promote SMN2 exon 7 inclusion as a treatment for spinal muscular atrophy (SMA). Risdiplam is approved for SMA, and close analogs of both compounds are in clinical trials for other indications by modulating splicing of other genes. Selectively redirecting these scaffolds to new splice targets requires a fuller understanding of how each compound alters splice-site recognition. While canonical 5' splice sites pair with U1 snRNA through an AGGUaagu consensus, both compounds' accepted mechanism of action involves stabilization of U1 binding to weak AGGU sites to promote exon inclusion, though genome-wide analyses demonstrate that each of the molecules have different potencies for different sets of GAGU splice sites [1]. To better understand the unique mechanisms of Risdiplam and Branaplam, we investigated splicing changes that cannot be explained by the accepted GAGU 5' splice site activation mechanism. We identify Branaplam-induced exon skipping in ten genes, all with exon-internal GAGU U1 binding motifs 10-40 basepairs downstream of 3' splice site. Perturbation of the exon-internal GAGU motif or U1C expression confirms the direct role of exonic U1 binding in promoting skipping. Interestingly, Branaplam is 10-100-fold more potent than Risdiplam at promoting skipping of these exons. Biochemical reconstitution of U1 snRNP binding to GAGU motifs offers a mechanistic explanation for this specificity: Branaplam enhances GAGU motif recognition with a minimal U1 snRNP [2], even at exon-internal sites too close to the 3'ss for productive exon definition. In contrast, Risdiplam requires addition of the U1 auxiliary factor LUC7L [3] and GAGU motif placement conducive to traditional exon-definition assembly. Thus, auxiliary splice factors and GAGU motif context may fine-tune Risdiplam/Branaplam specificity beyond 5' splice site sequence motifs. These findings also suggest that U1 auxillary factors help determine when stabilized U1 binding supports, or interferes with, neighboring splice-site recognition.
References:
[1] Ishigami Y et al. Specificity, synergy, and mechanisms of splice-modifying drugs. Nat Commun 15, 1880 (2024). doi:10.1038/s41467-024-46090-5.
[2] Kuang Z et al. The U1 snRNP protein U1C and Helix H of U1 snRNA are critical for small molecule splicing modulator function. Nat Commun 17, 8837 (2026). doi:10.1038/s41467-026-74765-8.
[3] Liu B et al. Depleting prion protein using splice-switching small molecules. bioRxiv (2026), preprint. doi:10.64898/2026.06.26.734742.
Keywords: splicing, small molecules, therapeutics