Assays to Study SUMOylation of RNA Helicase DHX9

RNA helicase DHX9 is a multi-functional protein governing transcriptional modulation, RNA processing, R-loop balance, and genome stability. SUMOylation is a post-translational modification that adds a critical regulatory layer to protein function by diversifying subcellular localization, stability, enzymatic activity, and protein-protein interactions. This protocol describes a basic but detailed approach for the analysis of DHX9 SUMOylation. We demonstrate that accurate site identification, specifically at Lysine 120 (K120), is the prerequisite for defining the molecular mechanism of this modification. Identifying the K120 acceptor site enables the generation of specific mutants to dissect how DHX9 SUMOylation orchestrates the protein interactions necessary to preserve genomic integrity. Furthermore, this protocol provides a versatile framework for characterizing the SUMOylation of other RNA helicases involved in RNA regulation.