Systematic Analysis of the Functional Overlap among Plant YTHDF proteins

YT521-B homology (YTH) domain proteins act as readers of N6-methyladenosine (m6A), the most common internal covalent modification in eukaryotic mRNA. Members of the YTHDF subclade can determine properties of m6A-containing mRNAs in the cytoplasm of animal and plant cells. Vertebrates encode three YTHDF proteins, and whether they perform specialized or redundant molecular functions is currently debated. In land plants, the YTHDF clade has expanded from just one member in basal lineages to eleven so-called EVOLUTIONARILY CONSERVED C-TERMINAL REGION1-11 (ECT1-11) proteins in Arabidopsis thaliana, named after the conserved YTH domain found at the C-terminus following a long intrinsically disordered region (IDR) at the N-terminus. The origin and implications of YTHDF expansion in higher plants are not known, as it is unclear whether it involves acquisition of fundamentally different properties, in particular of their divergent IDRs. Here, we used the leaf formation defects in ect2/ect3/ect4 mutants to test whether the many Arabidopsis YTHDF proteins can perform the same function if expressed at similar levels in leaf primordia. We show that the ancestral molecular function of the m6A-YTHDF axis in land plants is conserved over YTHDF diversification, and currently present in all major clades of YTHDF proteins in flowering plants. Nevertheless, lineage-specific neo-functionalization of a few members also happened after late duplication events. ECT1, the closest homolog of ECT2/3/4, is one such divergent YTHDF protein. Accordingly, mutation of ECT1 does not aggravate the defective organogenesis of ect2/ect3/ect4 mutants, even though the four proteins are naturally expressed in the same population of primordial cells. Significance statement Regulation of gene expression is essential to life. It ensures correct balancing of cellular activities and the controlled proliferation and differentiation necessary for the development of multicellular organisms. Among other mechanisms, gene expression can be controlled by methylation of adenosines in mRNA (m6A). Absence of m6A impairs embryo development in plants and vertebrates, and its mis-regulation is associated with human cancers. m6A-dependent regulation can be exerted by a group of cytoplasmic proteins called YTHDFs. Plants have many more YTHDFs than animals, but the reason for this is unknown. This study addresses the origin of plant YTHDF expansion, and reveals that most, but not all, have the same molecular functions that facilitate rapid division of differentiating stem cells.