Assay for In-Vitro Arginine Methylation of RNA Binding Proteins Followed by RNA Binding

Arginine methylation is a prevalent post-translational modification that plays a critical role in regulating the function of RNA-binding proteins [RBPs]. RBPs are the largest class of arginine methylated proteins. Catalyzed by protein arginine methyltransferases [PRMTs], this modification influences RNA recognition, binding affinity, and the formation of RNA-protein complexes. This impacts diverse cellular processes such as RNA splicing, transport, stability, and translation. Arginine methylation of RGG motifs modulates RBP behavior, with implications for normal physiology and disease states, including neurodegeneration, cancer, and immune disorders. In vitro methylation assays combined with RNA binding studies offer a powerful approach to elucidate how arginine methylation affects RNA-protein interactions. These techniques allow precise manipulation of methylation states and controlled assessment of binding dynamics, providing mechanistic insights into regulatory pathways. The protocols described in this chapter outline methods for recombinant protein expression, enzymatic methylation, and RNA binding assays, offering adaptable tools for researchers studying the functional consequences of arginine methylation in RNA biology. Together, these methodologies provide a framework to explore the interplay between post-translational modifications and RNA regulation, deepening our understanding of cellular processes.