m(6)A enhances the phase separation potential of mRNA

N(6)-methyladenosine (m(6)A) is the most prevalent modified nucleotide in mRNA(1,2), with ~25% of mRNAs containing at least one m(6)A. Methylation of mRNA to form m(6)A is required for diverse cellular and physiological processes(3). Although the presence of m(6)A in an mRNA can affect its fate in different ways, it is unclear how m(6)A directs this process and why the effects of m(6)A can vary in different cellular contexts. Here we show that the cytosolic m(6)A-binding proteins, YTHDF1–3, undergo liquid-liquid phase separation (LLPS) in vitro and in cells. This LLPS is markedly enhanced by mRNAs that contain multiple, but not single, m(6)A residues. Polymethylated mRNAs act as a multivalent scaffold for binding YTHDF proteins, juxtaposing their low-complexity domains, leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules, or neuronal RNA granules. m(6)A-mRNA is subject to compartment-specific regulation, including reduced mRNA stability and translation. These studies reveal that the number and distribution of m(6)A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome. Additionally, these findings indicate that the cellular properties of m(6)A-modified mRNAs are governed by liquid-liquid phase separation principles.