Barraza, S. J. | Search bioRxiv

bioRxiv2025

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 demonstrated that branaplam enhanced recognition of these 5 ss by reconstituted U1 snRNP in vitro, and that this effect depended on the ZnF domain of U1C and Helix H of U1 snRNA, but not U1A or U1-70K. In cells, depletion of U1C generally reduced compound-induced exon inclusion for most cassette exons. Interestingly, a subset of cassette exons became 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.

biochemistry↗

bioRxiv2025

MAPT Splicing Modulators as a Therapeutic Strategy for Tauopathies

Tauopathies are neurodegenerative diseases characterized by the abnormal accumulation of microtubule-associated protein tau (MAPT) in the brain. These disorders, like frontotemporal dementia (FTD-Tau), currently lack effective therapies and can occur sporadically or be inherited when associated with MAPT gene mutations. The MAPT gene region encompassing exon 10 and adjacent introns is a hotspot for pathogenic variants, including splicing mutations that enhance exon 10 inclusion and increase 4R tau expression, and gain-of-function mutations that generate aggregation-prone mutant 4R tau protein. For these 4R-specific tauopathies, a targeted mRNA splicing approach that promotes exon 10 exclusion may offer therapeutic benefit. In this study, we discovered novel splicing modulator compounds (SMCs) that promote MAPT exon 10 exclusion, and demonstrated their efficacy in FTD patient-derived neuronal models carrying the tau-P301L gain-of-function mutation or the tau-S305N splicing mutation. Treatment with SMC reduced 4R tau expression and decreased the accumulation of hyperphosphorylated tau (pTau), oligomeric and insoluble tau, thereby rescuing tau-associated neuronal toxicity. Importantly, our lead SMC corrected the 3R/4R splice ratio in vivo and significantly reduced pTau in the brain of a gene- replacement (GR) mouse model expressing the human tau-N279K splicing mutation. These findings support the therapeutic potential of this class of small molecules and establish MAPT pre- mRNA splicing modulation as a promising strategy for the treatment of 4R tauopathies. One Sentence SummaryDiscovery of SMCs that correct MAPT splicing, reduce 4R tau, and rescue pathology in patient- derived neuronal and in vivo models of 4R tauopathies.

neuroscience↗