The YTHDF1-3 proteins are bidirectionally influenced by the codon content of their mRNA targets

N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNAs and plays critical roles in a broad variety of biological processes. Recognition of m6A by the YTHDF1-3 proteins results in the alteration of the translation efficiency and stability of methylated mRNAs, although their mode of action is still matter of debates. To decode the molecular basis of YTHDF1-3 action in human cells, we performed an unbiased proteomic screen of their full spectrum of interacting proteins using BioID (proximity-dependent biotin identification). Our systematic BioID mapping revealed that each YTHDF protein is a dynamic hub that associates with both mRNA silencing machineries and the translation apparatus. Based on this, we identified a direct interaction between YTHDF2 and the ribosomal protein RACK1, and found that the silencing activity of YTHDF2 is bidirectionally modulated by the codon content of its targeted mRNAs. Using a tethering reporter system that recapitulates this phenomenon, we confirmed that the three YTHDF proteins selectively repress mRNAs enriched in optimal codons, while they activate those enriched in non-optimal codons. Altogether, these results have important implications for understanding the underlying multiplicity of YTHDF1-3 and could reconcile seemingly contradictory data.