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 around 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, YTHDF2 and YTHDF3—undergo liquid–liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m 6 A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby 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 a reduction in the stability and translation of mRNA. 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, and suggest that the cellular properties of m 6 A-modified mRNAs are governed by liquid–liquid phase separation principles.