Monitoring Acetylation of the RNA Helicase DDX3X, a Protein Critical for Formation of Stress Granules

Stress granules are dynamic structures that assemble in response to various forms of stress, such as heat shock or oxidative stress, among others. We had previously shown that the lysine deacetylase HDAC6 is required for the formation of stress granules, but the substrate important for this function was not known. We recently found that the RNA helicase DDX3X is a novel HDAC6 substrate, which is critical for the formation of stress granules. Through a series of detailed experiments, we showed that, upon stress, DDX3X becomes acetylated in an intrinsically disordered region; this alters its propensity to undergo phase separation and inhibits growth of the stress granules. HDAC6, by deacetylating DDX3X, allows maturation of the stress granules. This work identified acetylation of an RNA helicase as a critical regulator of the stress response. Here, we present methods to analyze the acetylation of DDX3X; these methods can be easily adapted to study the acetylation of other helicases, or other proteins.