Alternative cleavage and polyadenylation (APA) allows genes that contain multiple cleavage and polyadenylation signals (CPAs) to encode multiple RNA isoforms and has an important role in the regulation of gene expression. Now, Neve et al. report the differential regulation of APA isoforms in cytoplasmic and nuclear RNA fractions of human cell lines. APA isoforms with shorter 3′ untranslated regions (UTRs), owing to cleavage at promoter-proximal versus promoter-distal CPAs, were over-represented in the cytoplasm in all non-neuronal cell lines analysed, but not in neuroblastoma-derived cells. Further experiments indicated that the nuclear retention of distal CPA isoforms (with longer 3′ UTRs) can be partly attributed to incomplete splicing, and demonstrated that the nuclear endoribonuclease DICER1 controls subcellular APA profiles by influencing CPA site selection and through microRNA-mediated stabilization.