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

Abstract

N 6-Methyladenosine (m6A) 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 m6A 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 m6A methyltransferase complex), m6A demethylases (ALKBH5 and FTO), or m6A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBH5 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 m6A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBH5 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 m6A, and our study suggests the potential of an alternative therapeutic approach to target the currently “undruggable” small GTPases.

The m6A reader YTHDC2 inhibits lung adenocarcinoma tumorigenesis by suppressing SLC7A11-dependent antioxidant function

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“…The m6A regulators inhibited melanoma development by increasing the methylation of Hint2 ( Jia et al, 2019 ). Yang et al revealed the positive/negative regulatory role for METTL3/ALKBH5 in RHOB and RHOC (a subset of small GTPase proteins) in melanoma ( Yang et al, 2020 ). YTHDF1 interferes with the presentation of dendritic cells by promoting the expression of lysosomal protease in dendritic cells and gives rise to disorder in the immune response.…”

Emerging Roles of N6-Methyladenosine Demethylases and Its Interaction with Environmental Toxicants in Digestive System Cancers

“… 9 With the advance of m6A, the studies demonstrate that m6A and m6A associated proteins are involved in various biological and pathological processes, such as splicing, transport, translation and degradation. 10 , 11 In addition, the abnormal expression of m6A associated proteins are involved in regulating genes that impact cell processes and physiological function in various cancers. As Figure 1 shows, m6A modification is regulated by three types of proteins, including the m6A methyltransferase (“writer”), demethylase (“eraser”) and reading proteins (“reader”).…”