Insights into the conservation and diversification of the molecular functions of YTHDF proteins

Abstract

YT521-B homology (YTH) domain proteins act as readers of N6-methyladenosine (m6A) in mRNA. Members of the YTHDF clade determine properties of m6A-containing mRNAs in the cytoplasm. Vertebrates encode three YTHDF proteins whose possible functional specialization is debated. In land plants, the YTHDF clade has expanded from 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 placed behind a long N-terminal intrinsically disordered region (IDR). ECT2, ECT3 and ECT4 show genetic redundancy in stimulation of primed stem cell division, but the origin and implications of YTHDF expansion in higher plants are unknown, as it is unclear whether it involves acquisition of fundamentally different molecular properties, in particular of their divergent IDRs. Here, we use functional complementation of ect2/ect3/ect4 mutants to test whether different YTHDF proteins can perform the same function when similarly expressed in leaf primordia. We show that stimulation of primordial cell division relies on an ancestral molecular function of the m6A-YTHDF axis in land plants that is present in bryophytes and is conserved over YTHDF diversification, as it appears in all major clades of YTHDF proteins in flowering plants. Importantly, although our results indicate that the YTH domains of all arabidopsis ECT proteins have m6A-binding capacity, lineage-specific neo-functionalization of ECT1, ECT9 and ECT11 happened after late duplication events, and involves altered properties of both the YTH domains, and, especially, of the IDRs. We also identify two biophysical properties recurrent in IDRs of YTHDF proteins able to complement ect2 ect3 ect4 mutants, a clear phase separation propensity and a charge distribution that creates electric dipoles. Human and fly YTHDFs do not have IDRs with this combination of properties and cannot replace ECT2/3/4 function in arabidopsis, perhaps suggesting different molecular activities of YTHDF proteins between major taxa.

ALBA proteins facilitate cytoplasmic YTHDF-mediated reading of m6A in plants

“…First, deletion of N8 causes reduced RNA binding of ECT2 in vivo . Second, ECT interactors of ALBAs include ECT1 and ECT11 which have m 6 A-binding capacity but not the function of ECT2 required for leaf formation 20 . Hence, our results suggest that the ALBA-ECT interaction mediates a molecular property common to all ECT proteins, perhaps m 6 A-binding.…”

“…ALBA1 levels were not detectable in immunopurified fractions of these two mutants ( Figure 1H ), perhaps suggesting that additional determinants of ALBA interaction are located in the IDR outside of the N8 region. Second, inspection of IP-MS data with HA-ECT2 and with tagged versions of the two YTHDF paralogs ECT3 (ECT3-Venus) and ECT1 (ECT1-TFP) 21 , both of which have m 6 A-binding capacity 7,20,24,28,44 , revealed enrichment of ALBA proteins over the negative controls ( Figure S2C ). Third, comparative IP-MS analysis carried out with ALBA4-GFP and free GFP revealed a clear enrichment of several ECT proteins, including ECT1-8 and ECT11, in the ALBA4-GFP purified fractions ( Figure 1I, Figure S2D, Table S1 ).…”

“…This interaction is mediated by a conserved tyrosine-rich motif in the IDR of ECT2 and is required for developmental functions of ECT2 21 . Third, most Arabidopsis ECT paralogues across phylogenetic subclades retain the ability to complement ect2 ect3 ect4 mutants upon ectopic expression in primordial cells 20 . For the three Arabidopsis ECT proteins unable to perform this basal function, the divergence can at least in part be ascribed to differences in their N-terminal IDRs 20 , including the loss of the PAB2/4/8-interacting motif 21 .…”

“…Third, most Arabidopsis ECT paralogues across phylogenetic subclades retain the ability to complement ect2 ect3 ect4 mutants upon ectopic expression in primordial cells 20 . For the three Arabidopsis ECT proteins unable to perform this basal function, the divergence can at least in part be ascribed to differences in their N-terminal IDRs 20 , including the loss of the PAB2/4/8-interacting motif 21 . Thus, the molecular properties of the IDRs of ECT proteins are central to understand their biological functions.…”

“…Three features of the molecular functions of ECT proteins that promote growth during organogenesis have been defined. First, they are deeply conserved, because the sole YTHDF protein encoded by the liverwort Marchantia polymorpha that diverged from higher plants ∼450 million years ago 18,19 can functionally replace Arabidopsis ECT2 when expressed in primordial cells in ect2 ect3 ect4 mutants 20 . Second, ECTs interact with the major cytoplasmic poly(A)-binding proteins PAB2/4/8 21,22 .…”