Proteome-wide Interrogation of Small GTPases Regulated by N6-Methyladenosine Modulators

N-6-Methyladenosine (m(6)A) in messenger RNA (mRNA) regulates its stability, splicing, and translation efficiency. Here, we explored how the expression levels of small GTPase proteins are regulated by m(6)A modulators. We employed a high-throughput scheduled multiple-reaction monitoring (MRM)-based targeted proteomic approach to quantify systemically the changes in expression of small GTPase proteins in cells upon genetic ablation of METTL3 (the catalytic subunit of the major m(6)A methyltransferase complex), m(6)A demethylases (ALKBHS and FTO), or m(6)A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBHS resulted in substantially diminished and augmented expression, respectively, of a subset of small GTPase proteins, including RHOB and RHOC. Our results also revealed that the stability of RHOB mRNA is significantly increased in cells depleted of METTL3, suggesting an m(6)A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBHS also displayed higher discrepancies between protein and mRNA expression in paired primary/metastatic melanoma or colorectal cancer cells than those that are not. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome regulated by epitranscriptomic modulators of m(6)A, and our study suggests the potential of an alternative therapeutic approach to target the currently "undruggable" small GTPases.