Investigating the Role of FMRP in PKR-induced Integrated Stress Response via Formation of Stress induced Aggregates that Sequester PP2A

Abstract

The Integrated Stress Response (ISR) is a conserved cellular pathway that enables adaptation to diverse stress conditions by phosphorylating eukaryotic initiation factor 2α (eIF2α) via stress response kinases, including HRI, PERK, GCN2, and PKR. This leads to translational reprogramming and induction of stress response genes. Previous findings from our laboratory indicate that Fragile X Mental Retardation Protein (FMRP) is required for effective ISR activation under PKR-specific stress conditions; however, the underlying mechanism remains unclear. In this study, we investigated the role of FMRP in regulating the PKR arm of the ISR using human lung epithelial cell lines. FMRP-deficient cells exhibited reduced viability upon exposure to stressors that activate PKR. Pharmacological inhibition of serine/threonine phosphatases restored ATF4 levels in FMRP-deficient cells, with inhibition of PP2A recapitulating this rescue across different cell lines, suggesting FMRP plays a role in regulating phosphatases. PKR formed cytoplasmic aggregates that increased in a time-dependent manner following Poly I:C transfection, and they co-localised with FMRP and were enriched for PP2A. In contrast, dsRNA aggregates formed following Poly I:C transfection did not co-localize with FMRP but G3BP1 did. This could indicate that the type of PKR aggregate formed in such cases might not be a dRIFs-like, but rather resemble other stress induced assemblies like stress granules. However, in A549 cells, PKR and FMRP did not co-localise, despite robust FMRP-G3BP1 co-localization, indicating cell-type specific heterogeneity in stress-induced aggregate composition. Collectively, these findings suggest that FMRP may facilitate ISR activation through modulation of PP2A activity, potentially aided by stress-induced compartmentalization. However, the observed cell-type variation indicates that aggregate formation may represent a supplementary rather than universal mechanism of FMRP-mediated ISR regulation.