mA enhances the phase separation potential of mRNA.

N-methyladenosine (mA) is the most prevalent modified nucleotide in mRNA, with around 25% of mRNAs containing at least one mA. Methylation of mRNA to form mA is required for diverse cellular and physiological processes. Although the presence of mA in an mRNA can affect its fate in different ways, it is unclear how mA directs this process and why the effects of mA can vary in different cellular contexts. Here we show that the cytosolic mA-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, mA 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. mA-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 mA sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of mA-modified mRNAs are governed by liquid-liquid phase separation principles.