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

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.