Allelic Dissection of Pkd1 3′-UTR Architecture Uncovers Regulatory Elements Affecting ADPKD Progression

Background: The 3′-untranslated region (3′-UTR) of mRNA plays a central role in regulating transcript stability and translation efficiency, yet the cis-regulatory logic underlying these functions remains poorly understood. Autosomal dominant polycystic kidney disease (ADPKD), a monogenic disorder driven primarily by heterozygous loss-of-function mutations in PKD1, provides a clinically relevant model to investigate these mechanisms. We previously identified a miR-17 binding motif within the PKD1 3′-UTR that suppresses translation. Here, we leverage PKD1 and ADPKD as model systems to interrogate the structural and sequence determinants of 3′–UTR–mediated regulation in vivo and uncover targetable elements for therapeutic PKD1 upregulation. Methods: We performed in-cell and in-vitro SHAPE (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension) analysis of the PKD1 3’-UTR to determine the secondary structures and RNA-binding protein (RBP) interaction sites. Using CRISPR-Cas9, we engineered an in vivo allelic series in a mouse ADPKD model to disrupt microRNA motifs, loop-forming regions, or predicted RBP-binding elements within the Pkd1 3’-UTR. We generated and characterized 20 independent founder mouse lines. Phenotypic evaluation included kidney-to-body weight ratio, histology, serum creatinine, blood urea nitrogen levels, and survival analysis. Results: We made four key observations. First, SHAPE analysis revealed extensive double-strand secondary structure in the PKD1 3′-UTR and identified stem-loops harboring miRNA and other motifs that are accessible to RBPs. Second, deletion or base-editing of either the miR-17 or miR-200 motifs stabilized Pkd1 mRNA, reduced disease progression, and prevented mortality in monoallelic Pkd1-mutant mice. Third, deletion of 3’-UTR regions that minimized the secondary structure also alleviated disease. Finally, combined deletion of both miRNA motifs, or larger deletions affecting miRNA sites and 3’-UTR structure, had an additive effect, completely preventing disease onset. Conclusion: Eliminating miRNA motifs or altering Pkd1 3′-UTR structure alleviates ADPKD, with effects that appear summative. We uncover broader principles of 3′-UTR biology with potential implications for endogenous PKD1 gene therapy for ADPKD. Funding: NIDDK Support, Other U.S. Government Support