Differentiation in the human urothelia is defined by distinct alternative transcript polyadenylation

Tissue epithelia comprise distinct cell states, yet the mechanisms that diversify their transcriptomes remain incompletely understood. The human ureter urothelium contains basal progenitors, intermediate cells, and terminally differentiated umbrella cells that maintain urinary tract barrier integrity. Although transcriptional differences are widely assumed to define these states, prior single-cell RNA sequencing (scRNA-seq) revealed highly similar global gene expression profiles. Here, we show that alternative cleavage and polyadenylation (APA) introduces a major layer of transcriptomic diversity during urothelial differentiation, largely independent of changes in total mRNA abundance. Analysis of 13,544 urothelial cells identified hundreds of differentiation-associated APA events. By single-cell imaging, we visualized APA events in the tissue, revealing their spatial specificity within the adult human ureter. Using a reporter assay, we demonstrated that the alternative 3′ UTRs in these differentiation-associated APA genes directly impacted protein expression levels. We discovered that key APA genes share conserved motifs in their 3′ UTRs, including transcription factor binding sites and Alu elements, suggesting a potential mechanism regulating poly(A) site selection. Our study establishes APA as a major source of urothelial transcriptome diversity.