Catalytic activity or structural function: Investigating the role of DXO in hyperinflammation

DXO is a decapping exoribonuclease enzyme capable of recognizing, targeting, and degrading aberrantly capped mRNA, thereby contributing to RNA quality control and preventing the accumulation of aberrant transcripts. A conventional knock-out (KO) model was generated using CRISPR-Cas9 in A549 cells, in which the gene encoding for DXO was deleted. The KO exhibits a hyperinflammation phenotype compared with the wild-type (WT) A549 cells. The hyperinflammatory phenotype is characterized by increased IFIT1 and IFIT2 gene expression. This project aims to investigate the potential role of DXO in the regulation of hyperinflammation. More specifically, it focuses on determining whether the catalytic activity of the enzyme is required to rescue the hyperinflammatory phenotype or whether its structural function is sufficient to suppress it. To address the question, two rescue cell lines were created from the KO background. One cell line was rescued with the WT DXO gene, while the second was rescued with a catalytically inactive DXO mutant and the different expression levels of specific inflammatory markers, such as IFIT1 and IFIT2, were observed and compared in order to determine whether DXO plays a functional role, implicating that its catalytic activity is a necessary component in the regulation of inflammation, or a structural role. Demonstrating that the structural function alone can restore the normal cellular phenotype may suggest that the enzyme is part of a larger protein complex. On the other hand, failure of the catalytically inactive mutant to suppress the hyperinflammatory phenotype underlines the importance of the enzymatic activity of DXO in the process of RNA quality control and regulation of the inflammatory response.