Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m⁶A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m⁶A-modified mRNAs are enriched in SGs, and that m⁶A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m⁶A-binding YTH domain of YTHDF proteins are important for SG formation. Super-resolution imaging further reveals that YTHDF proteins appear to be in a super-saturated state, forming clusters that often reside in the periphery of or at the junctions between SG core clusters, and potentially promote SG formation by reducing the activation energy barrier and critical size for SG condensate formation. Our results suggest a new function of the m⁶A-binding YTHDF proteins in regulating SG formation.

不同的 RNA 和 RNA 结合蛋白在压力条件下在细胞中形成相分离的无膜颗粒。然而，流行的 mRNA 甲基化、m ⁶ A 及其结合蛋白在应力颗粒 (SG) 组装中的作用仍不清楚。在这里，我们展示了 m ⁶ A 修饰的 mRNA 富含 SG，并且 m ⁶ A 结合 YTHDF 蛋白对 SG 的形成至关重要。YTHDF1/3 的消耗抑制了 SG 的形成和 mRNA 向 SG 的募集。N端固有无序区和C端m ⁶YTHDF 蛋白的 A 结合 YTH 结构域对于 SG 的形成很重要。超分辨率成像进一步显示 YTHDF 蛋白似乎处于超饱和状态，形成簇，这些簇通常位于 SG 核心簇的外围或连接处，并可能通过降低活化能垒和临界值来促进 SG 的形成。 SG 冷凝液形成的尺寸。我们的结果表明 m ⁶ A 结合 YTHDF 蛋白在调节 SG 形成中的新功能。