Recognition Loop Dynamics Differentiates m6A-mRNA Reader Domains of YTHDF Proteins

The three human YTH-domain family proteins (YTHDF1, YTHDF2, and YTHDF3) are highly homologous and recognize the N6-methyladenosine (m6A) modification of mRNA with essentially identical binding modes and affinities. This homogeneity challenges the well-established view that different YTHDF proteins play distinct cellular functions (the specificity view), with an emerging argument that these proteins may instead play redundant roles. To study this apparent discrepancy, we investigated the mechanism of binding of m6A-containing RNA using mutagenesis, X-ray crystallography, isothermal titration calorimetry (ITC), and molecular dynamics simulations. Specifically, we compared different properties of YTHDF proteins, including their binding capacities to m6A-RNA, conformational changes, and homodimerization propensity. Our results show that the YTHDF proteins share an identical binding mode of m6A-RNA, which requires rare protonation of m6A for binding. Additionally, the three proteins share a similar open-closed switch for their recognition loop regulating the m6A binding. On the other hand, ITC data and crystal structures provide consistent evidence that the YTHDF proteins differ significantly in the flexibility of the recognition loop and its conformational preferences. Only YTHDF1 can homodimerize, which prevents binding to m6A-RNA. Furthermore, we integrate the available biophysical and biochemical evidence to define both the shared and distinct features of YTHDF1–3 in m6A-RNA recognition and propose a hybrid model that reconciles the redundancy-specificity debate. Finally, we discuss the limitations of the work and propose experiments for validating the presented model.