Non-Enzymatic MGO-Glycation of SRSF2 Drives RNA Mis-Splicing

Methylglyoxal (MGO) is a reactive metabolic byproduct of glycolysis shown to accumulate in highly glycolytic cells such as cancer cells. MGO reacts with proteins to form covalent adducts in a process termed glycation, and MGO-glycation has been linked to oxidative stress, diabetes, cancer, neurodegenerative diseases, and inflammation. Although several protein targets of MGO-glycation and their link to disease have been reported, we lack a complete understanding of MGO-glycation targets that may underlie disease progression. Here, we take a quantitative chemoproteomic profiling approach with an alkyne-functionalized MGO probe (AlkMG) to map the proteome-wide targets of MGO. A total of 494 proteins were found to be glycated under these conditions, many of which are involved in RNA processing pathways. Focusing on the serine/arginine-rich splicing factor 2 (SRSF2), we determined the sites and characterized the role that MGO-derived modifications have on its function. Biophysical modeling of site-specific glycation of SRSF2 identified residues that destabilize the native protein-RNA complex upon glycation, which we corroborated experimentally by RNA pulldown. Importantly, we found that these glycation events attenuate SRSF2's RNA binding and alter RNA splicing, phenocopying a recurrent leukemia oncogenic SRSF2 mutation, P95H. Collectively, our study resolves glycation as a bona fide, site-specific regulatory PTM for a splicing factor and provides the first evidence for MGO-mediated mis-splicing in living cells, suggesting a new mechanistic link between MGO-glycation and disease.