Ythdf m6A readers compensate each other in a context dependent manner [eCLIP]

YTH domain family proteins Ythdf1, Ythdf2, Ythdf3 are three readers with highly similar structure, which were shown to have different roles in the cell. However, their similarity and the fact that they tend to bind the same targets suggest that in some cases they may have an overlapping role. In this paper, we systematically knocked-out (KO) each of the three readers and the three together (triple-KO), and estimated the effect in-vitro in mouse embryonic stem cells (mESCs), and in-vivo, in viability and gametogenesis. We show that in mESCs there is a compensation between the three readers, with a dramatic hyper-pluripotent phenotype only in the triple-KO. However, in-vivo, Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, both in viability of the mouse pups and in gametogenesis. We suggest that compensation is dosage-dependent, and in-vivo we cannot detect it due to difference in the readers' expression, both in level and in spatial location. In addition, we found that Ythdf1 and Ythdf3 specifically down-regulate 2-cell genes, that Ythdf2-KO in gametogenesis affects microtubuline related genes, and that Mettl3-KO severity is increased as the deletion occurs earlier in gametogenesis. Overall design: Targets of Ythdf1, Ythdf2 and Ythdf3 were measured in mouse embryonic stem cells using eCLIP method