Cleavage site heterogeneity at the pre-mRNA 3′-untranslated region regulates gene expression in oxidative stress response

The endonucleolytic cleavage step of the eukaryotic mRNA 3'-end processing is considered imprecise, which leads to heterogeneity of cleavage site (CS) with hitherto unknown function. Contrary to popular belief, we show that this imprecision in the cleavage is tightly regulated, resulting in the CS heterogeneity (CSH) that controls gene expression in antioxidant response. CSH centres around a primary CS, followed by several subsidiary cleavages determined by CS's positions. Globally and using reporter antioxidant mRNA, we discovered an inverse relationship between the number of CS and the gene expression, with the primary CS exhibiting the highest cleavage efficiency. Strikingly, reducing CSH and increasing primary CS usage induces gene expression. Under oxidative stress (we employ three conditions that induce antioxidant response, tBHQ, H₂O₂, and NaAsO₂) conditions, there is a decrease in the CSH and an increase in the primary CS usage to induce antioxidant gene expression. Key oxidative stress response genes (NQO1, HMOX1, PRDX1, and CAT) also show higher CSH compared to the non-stress response genes and that the number of CSs are reduced to impart cellular response to oxidative stresses. Concomitantly, ectopic expression of one of the key antioxidant response gene (NQO1) driven by the primary CS but not from other subsidiary CSs, or reduction in CSH imparts tolerance to cellular oxidative stresses (H₂O₂, and NaAsO₂). Genome-wide CS analysis of stress response genes also shows a similar result. Compromised CSH or CSH-mediated gene control hampers cellular response to oxidative stress. We establish that oxidative stress induces affinity/strength of cleavage complex assembly, increasing the fidelity of cleavage at the primary CS, thereby reducing CSH inducing antioxidant response. Together, our study reports a novel cleavage imprecision- or CSH-mediated anti-oxidant response mechanism that is distinct and operates downstream but in concert with the transcriptional pathway of oxidative stress induction.