A unified model for the function of YTHDF proteins in the regulation of m6A-modified mRNA

N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs, rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are simultaneously depleted. Our studies reveal a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of the YTHDF proteins in proportion to the number of m6A sites. We measured transcript stability, translation rate, and transcript expression analysis upon the silencing of each of the YTHDF protein in HeLa cells. Since all three YTHDF proteins show similar sequence, binding proteins, binding sites and intracellular localization, their redundancy was tested by performing transcript stability, translation rate and transcript levels measurement upon the silencing of all three DF proteins as well. At least three replicates were performed for each measurement.