Mapping the RNA interactome of DDX5 and its rewiring by the K144N mutation in the RNA helicase domain

DDX5 is a multifunctional RNA helicase that regulates RNA metabolism and gene expression programs relevant to cancer and antiviral responses. Despite its importance, the RNA-binding landscape of DDX5 remains poorly defined. Here, we use CLIP-seq to systematically map the endogenous RNA interactome of DDX5 and to determine how it is altered by the K144N point mutation within the conserved RNA helicase domain. Overall design: We employed the enhanced CrossLinking and ImmunoPrecipitation (eCLIP) protocol (1) to identify RNAs associated with DDX5. Experiments were conducted using HepaRG cell lines engineered to express doxycycline-inducible FLAG-tagged DDX5, including both the wild-type (WT) protein and the catalytically inactive K144N mutant (2). The DDX5-K144N mutant, which is deficient in ATP hydrolysis and RNA binding, served as a negative control in downstream RNA and bioinformatic analyses. The eCLIP assays were performed by Eclipse BioInnovations, a specialized RNA genomics service provider.