m⁶A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m⁶A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m⁶A level of polysomal and cytoplasmic mRNAs with m⁶A-LAIC-seq and m⁶A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m⁶A-methylated, whereas those enriched in P-body are hyper-m⁶A-methylated. Downregulation of the m⁶A writer METTL14 enhances translation by switching originally hyper-m⁶A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m⁶A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m⁶A-dependent manner.