Knehr, Judith, Altorfer, Marc, Byrnes, Andrea, Li, Wenjing, Hsiao, Jack, Kolter, Christian, Skaanderup, Philip, Mullin, Steve, Krueger, Andrew, Ottl, Johannes, Gubser Keller, Caroline, Naumann, Ulrike and Ottis, Philipp (2025) Primary sequence dependencies define the specificity and exon target space of splicing-modifying compounds. biorXiv.

Abstract

Modulation of splicing has emerged as a promising therapeutic strategy for various diseases, offering the potential to target specific exons to influence gene expression. Recent advancements led to the identification of small molecule splicing modifiers such as Risdiplam and Branaplam. These compounds induce the inclusion of exons that are typically found to be skipped due to their weak 5’ splice site. While Risdiplam has a preference to induce exons with a N-3G-2A-1 sequence at the 3’ exon end, Branaplam has a proclivity towards introducing A-3G-2A-1 -ending exons. However, the variables that determine the selectivity and specificity of splicing modulators are still not completely understood, as evidenced by the hundreds of untargeted N-3G-2A-1 ending exons present in the human genome. In this study, we delve into the molecular mechanisms governing the specificity of splicing-modifying compounds, focusing on their interactions with RNA structures at splice sites. Using biochemical assays, whole transcriptome analyses, and genetic perturbation approaches, our findings reveal contributions of primary structure dependencies that contribute to different aspects to the required secondary structural conformation, thus governing responsiveness to splicing-modulator induction. Based on these learnings, we were able to reprogram the specificity of splicing modulators by genetic manipulation of the U1 snRNA component of the spliceosome. Our findings improve the understanding of splicing modulators and might help to identify novel targets and to design new splicing-modifying compounds.

Item Type:	Article
Date Deposited:	05 Sep 2025 00:46
Last Modified:	05 Sep 2025 00:46
URI:	https://oak.novartis.com/id/eprint/56398