Hyperosmotic stress-induced redistribution of pre-mRNA cleavage factor I subunits is associated with shifts in alternative polyadenylation.

Alternative polyadenylation (APA) is an important mechanism of cellular stress response mediated in part by the cleavage factor Im (CFIm) complex. However, the spatiotemporal dynamics and regulatory activity of the mammalian CFIm complex during stress remain poorly understood. In this study, we determined the effect of moderate hyperosmotic stress on CFIm localization and APA profiles in HEK293 cells. Using a dual-normalization strategy that included 18S rRNA- and a CDS-based ratiometric qPCR, we identified a significant shift toward proximal polyadenylation sites (PAS) in the established CFIm targets, NUDT21 (encoding CFIm25) and DICER1. Notably, these APA dynamics displayed distinct kinetic profiles influenced by the metabolic environment: While the NUDT21 L-3'UTR/CDS ratio recovered to baseline by Day 4, DICER1 exhibited a serum-dependent response, showing a progressive decline under low-serum conditions but recovering under high-serum conditions. Crucially, these alterations were absent in non-target multi-PAS genes such as GOLGA2 and preceded any substantial reduction in total mRNA abundance, suggesting these effects represent a targeted regulatory event rather than a nonspecific byproduct of transcriptional decline. Mechanistically, hyperosmotic stress triggers a transient, coordinated redistribution of CFIm25 and CFIm68 from the nucleus to the cytoplasm, while total cellular protein concentrations remain stable. We propose that this spatial shift creates a 'stoichiometric bottleneck' within the nuclear CFIm pool, effectively limiting the processing of distal PAS. This 'stoichiometric stress response' offers a robust mechanistic framework linking subnuclear protein reorganization to the rapid reprogramming of the 3'UTR landscape, providing new insights into how cells modulate gene expression potential during osmotic adaptation.