N⁶-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.

中文翻译：

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N6-甲基腺苷调节剂调节的小 GTP 酶的全蛋白质组询问。

信使 RNA (mRNA) 中的N ^{6 -}甲基腺苷 (m ⁶ A) 调节其稳定性、剪接和翻译效率。在这里，我们探讨了 m ⁶ A 调节剂如何调节小 GTPase 蛋白的表达水平。我们采用基于高通量计划多反应监测 (MRM) 的靶向蛋白质组学方法来系统量化 METTL3（主要 m ⁶ A 甲基转移酶复合物的催化亚基）基因消融后细胞中小 GTPase 蛋白表达的变化, m ⁶ A 脱甲基酶（ALKBH5 和 FTO），或 m ⁶阅读器蛋白（YTHDF1、YTHDF2 和 YTHDF3）。METTL3 和 ALKBH5 的消耗分别导致小 GTPase 蛋白子集（包括 RHOB 和 RHOC）的表达显着减少和增强。我们的结果还表明，在耗尽 METTL3 的细胞中，RHOB mRNA 的稳定性显着增加，表明该 mRNA由 m ⁶ A 引起的不稳定。METTL3 和/或 ALKBH5 靶向的那些小 GTP 酶在配对的原发性/转移性黑色素瘤或结直肠癌细胞中的蛋白质和 mRNA 表达之间的差异也比那些不是。总之，这是对 m ⁶表观转录组调节剂调控的小 GTPase 蛋白质组变化的首次综合分析A，我们的研究表明了另一种治疗方法的潜力，可以针对目前“不可成药”的小 GTP 酶。