RNA Chemical Proteomics Reveals the N⁶-Methyladenosine (m⁶A)-Regulated Protein-RNA Interactome

Epitranscriptomic RNA modifications can regulate mRNA function; however, there is a major gap in our understanding of the biochemical mechanisms mediating their effects. Here, we develop a chemical proteomics approach relying upon photo-cross-linking with synthetic diazirine-containing RNA probes and quantitative proteomics to profile RNA-protein interactions regulated by N⁶-methyladenosine (m⁶A), the most abundant internal modification in eukaryotic RNA. In addition to identifying YTH domain-containing proteins and ALKBH5, known interactors of this modification, we find that FMR1 and LRPPRC, two proteins associated with human disease, "read" this modification. Surprisingly, we also find that m⁶A disrupts RNA binding by the stress granule proteins G3BP1/2, USP10, CAPRIN1, and RBM42. Our work provides a general strategy for interrogating the interactome of RNA modifications and reveals the biochemical mechanisms underlying m⁶A function in the cell.