m⁶A enhances the phase separation potential of mRNA.

Abstract

N⁶-methyladenosine (m⁶A) is the most prevalent modified nucleotide in mRNA^1,2, with around 25% of mRNAs containing at least one m⁶A. Methylation of mRNA to form m⁶A is required for diverse cellular and physiological processes³. Although the presence of m⁶A in an mRNA can affect its fate in different ways, it is unclear how m⁶A directs this process and why the effects of m⁶A can vary in different cellular contexts. Here we show that the cytosolic m⁶A-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, m⁶A 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. m⁶A-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 m⁶A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m⁶A-modified mRNAs are governed by liquid-liquid phase separation principles.