RNA 5-methylcytosine regulates YBX2-dependent liquid-liquid phase separation

5-Methylcytosine (m⁵C) is one of the most prevalent internal modifications of messenger RNA (mRNA) in higher eukaryotes. Here we report that Y box protein 2 (YBX2) serves as a novel mammalian m⁵C binding protein to undergo liquid-liquid phase separation (LLPS) both in vivo and in vitro, and this YBX2-dependent LLPS is enhanced by m⁵C marked RNA. Furthermore, the crystal structure assay revealed that W100, as a distinct m⁵C binding site of YBX2, is critical in mediating YBX2 phase separation. Our study resolved the relationship between RNA m⁵C and phase separation, providing a clue for a new regulatory layer of epigenetics.