Human-specific sequence features in HTT exon 1 promote toxic misprocessing via splicing factor SRSF7

Abstract

Expansion of CAG repeats in HTT exon 1 is the acknowledged driver of Huntington’s disease. Alternative processing of HTT pre-mRNA generates the truncated HTT1a transcript, translated into a toxic peptide. While its dependence on CAG length is well documented, the role of adjacent sequences - particularly the Proline-Rich Domain (PRD) - remains unexplored. Using our HuntEx1-engineered mouse embryonic stem cell platform, we show that human PRD promotes HTT1a production, whereas its replacement with mouse PRD in an otherwise human exon 1 markedly reduces HTT1a levels. Mechanistically, we find that the PRD shapes mRNA structure, and motif analysis identifies Serine-Arginine Splicing Factor 7 (SRSF7) binding sites in mouse but not in human PRD. Their targeted mutation confirms SRSF7’s regulatory role in suppressing HTT1a production. Our findings establish the PRD as a key cis-regulator of HTT1a biogenesis, demonstrating that HTT toxicity also depends on sequence context, and highlighting splicing-based, PRD-focused therapeutic avenues. CAG repeat expansion drives Huntington’s disease, but additional sequence features influencing HTT toxicity are less defined. Here, the authors show that the proline‑rich domain regulates HTT1a transcript production via mRNA structure and SRSF7 binding, revealing sequence context-dependent control of toxic HTT generation.