Nudt21 KO and Nudt21 Hypomorph esophageal basal cells
Stem cells continually self-renew and differentiate to sustain tissue homeostasis, yet the role of post-transcriptional mechanisms in guiding these processes remains incompletely understood. Here, we demonstrate that the regulation of 3'UTR length via alternative mRNA polyadenylation (APA) is essential for stem cell function across diverse tissues. Modulating the APA regulator Nudt21 reveals that stem cell self-renewal and differentiation depend on distinct dosage thresholds and thus can be uncoupled. Specifically, moderate Nudt21 suppression  elicits a maturation arrest of stem cells due to 3'UTR-shortening of differentiation-associated mRNAs that escape miRNA regulation and perturb ceRNA networks. By contrast, complete Nudt21 suppression additionally shortens the 3'UTRs of mRNAs encoding essential multiprotein complexes, including the nuclear pore, leading to complex destabilization, proteotoxic stress, DNA damage, and cell cycle arrest. Critically, deletion of the alternative 3'UTRs of individual nucleoporins recapitulates defects observed with Nudt21 loss. We further demonstrate that the co-translational assembly of dozens of protein complexes is impaired in Nudt21-deficient cells, providing a mechanistic framework for compromised complex integrity. Collectively, our results show that APA plays distinct, dose-dependent roles in stem cell homeostasis by fine-tuning the expression of differentiation-associated genes and coordinating the biogenesis of multiprotein complexes essential for cell cycle progression. Overall design: RNA-seq timecourse for Nudt21 KO and Nudt21 Hypomorph esophageal basal cells: RNA-seq was performed for Nudt21 KO (d0, d2, d4, d6) and Nudt21 Hypomorph (d4, d6) in triplicate. Day 0 are cells that were treated with ETOH instead of 4OHT, thus they retain the floxed Nudt21 allel PAS-seq for Nudt21 KO and Nudt21 Hypomorph esophageal basal cells: PAS-seq was performed for Control, Nudt21 KO (d4) and Nudt21 Hypomorph (d4) in triplicate. Control samples  were treated with ETOH instead of 4OHT, thus they retain the floxed Nudt21 allel Small-RNA-seq for Control and Nudt21 KO esophageal basal cells: small-RNA-seq was performed for Control and Nudt21 KO (d4) in triplicate. Control samples  were treated with ETOH instead of 4OHT, thus they retain the floxed Nudt21 allel