Dynamic O-GlcNAcylation of YTHDF proteins controls translation of m6A-modified mRNA

N6-methyladenosine (m6A) is the most abundant internal mRNA nucleotide modification in mammals, regulating critical aspects of cell physiology and differentiation. The YTHDF proteins are the primary readers of m6A modifications and exert physiological functions of m6A in the cytosol. Elucidating the regulatory mechanisms of YTHDF proteins is critical to understanding m6A biology. Here, we report a mechanism that protein post-translational modifications control the biological functions of the YTHDF proteins. We find that YTHDF1 and YTHDF3, but not YTHDF2, carry high levels of nutrient-sensing O-GlcNAc modifications. O-GlcNAc modification attenuates the translation promoting function of YTHDF1 and YTHDF3 by blocking their interactions with proteins associated with mRNA translation. We further demonstrate that O-GlcNAc modifications on YTHDF1 and YTHDF2 regulate the assembly, stability, and disassembly of stress granule, facilitating rapid exchange of m6A-modified mRNAs in stress granules for recovery from stress. Therefore, our results discover an important regulatory pathway of YTHDF functions, adding an additional layer of complexity to the post-transcriptional regulation function of mRNA m6A. [Dataset 1] 8 samples. Duplicates for CLIP-seq samples in HEK293T cells with YTHDF1/3 knockdown and rescue with Flag-tagged YTHDF1 and YTHDF3 (WT or GlcNAcylation-null mutant (Mut)). [Dataset 2] 24 samples. Duplicates for RNA-seq and ribosome-protected fragments samples in HEK293T or HeLa cells with YTHDF1/3 knockdown and rescue with Flag-tagged YTHDF1 and YTHDF3 (na: no rescue, mut: rescue with GlcNAcylation-null mutant, wt: rescue with wild-type YTHDFs)