Global identification of ER stress-regulated RNA binding Proteins in clonal pancreatic beta-cells, reveals an important role for DEAD-box helicase 3 X-linked (DDX3X) in the execution of the Unfolded Protein Response and the determination of cell fate.

Endoplasmic reticulum (ER) stress plays an important role in pancreatic β-cell dysfunction and death, contributing to both type 1 and type 2 diabetes. The Unfolded Protein Response (UPR) is a key adaptive mechanism that helps cells cope with ER stress by altering gene expression. RNA-binding proteins (RBPs), which regulate gene expression at multiple levels, are likely to influence the UPR, yet their role in the UPR remain largely unexplored. To investigate this, we examined global changes in RBP-RNA interactions in the mouse β-cell line MIN6 under ER stress. This analysis identified 339 RBPs, with 13 showing significant changes in RNA binding. Among these, DEAD-box helicase 3 X-linked (DDX3X) emerged as a candidate for further study. Functional experiments revealed that inhibiting or knocking down DDX3X in MIN6 cells suppressed the ER stress-induced expression of the pro-apoptotic protein CHOP and its upstream regulator ATF4. Transcriptomic profiling of DDX3X-deficient MIN6 cells showed that DDX3X is essential not only for the upregulation of CHOP but also 24 other ER stress-responsive genes, including apoptosis-related genes such as Bcl2 and Bid. Notably, DDX3X knockdown conferred partial protection against ER stress-induced cytotoxicity and improved cell viability. In summary, these findings reveal widespread changes in the RBP landscape during ER stress in β-cells and highlight DDX3X as a key regulator of the UPR. DDX3X promotes the expression of stress-responsive genes and contributes to β-cell death under chronic ER stress, suggesting it may be a potential therapeutic target in diabetes.