N⁶-methyladenosine (m⁶A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m⁶A is thought to mediate its effects through a complex network of interactions between different m⁶A sites and three functionally distinct cytoplasmic YTHDF m⁶A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m⁶A-modified mRNAs rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are depleted simultaneously. Our study reveals a unified model of m⁶A function in which all m⁶A-modified mRNAs are subjected to the combined action of YTHDF proteins in proportion to the number of m⁶A sites.

N ⁶ -甲基腺苷 (m ⁶ A) 是最丰富的 mRNA 核苷酸修饰，调节细胞生理学和分化的关键方面。 m ⁶ A 被认为通过不同 m ⁶ A 位点和三个功能不同的细胞质 YTHDF m ⁶ A 结合蛋白（DF1、DF2 和 DF3）之间相互作用的复杂网络来介导其作用。与流行的模型相反，我们发现 DF 蛋白结合相同的 m ⁶ A 修饰的 mRNA，而不是不同的 mRNA。此外，我们发现 DF 蛋白不会诱导 HeLa 细胞中的翻译。相反，DF 旁系同源物冗余地发挥作用来介导 mRNA 降解和细胞分化。仅当所有三种 DF 旁系同源物同时耗尽时，DF 蛋白调节稳定性和分化的能力才变得明显。我们的研究揭示了 m ⁶ A 功能的统一模型，其中所有 m ⁶ A 修饰的 mRNA 均受到 YTHDF 蛋白的联合作用，与 m ⁶ A 位点的数量成比例。