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. 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.