YTH domain-containing family protein 3
N6-methyladenosine-containing RNA reader activity
m<sup>6</sup>A enhances the phase separation potential of mRNA.
N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is the most prevalent modified nucleotide in mRNA<sup>1,2</sup>, with around 25% of mRNAs containing at least one m<sup>6</sup>A. Methylation of mRNA to form m<sup>6</sup>A is required for diverse cellular and physiological processes<sup>3</sup>. Although the presence of m<sup>6</sup>A in an mRNA can affect its fate in different ways, it is unclear how m<sup>6</sup>A directs this process and why the effects of m<sup>6</sup>A can vary in different cellular contexts. Here we show that the cytosolic m<sup>6</sup>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<sup>6</sup>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<sup>6</sup>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<sup>6</sup>A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m<sup>6</sup>A-modified mRNAs are governed by liquid-liquid phase separation principles.