The affinity of at-YTH or tg-YTH for RNA or RNAm6A were determined using a MicroCal iTC200 system (Malvern Panalytical, Malvern, UK). Experiments were performed at 25°C in 20 mM Tris pH 8, 50 mM NaCl. The YTH proteins solutions (at concentration ranging from 25 µM to 60 µM) were loaded in the calorimetric cell. RNA (at concentrations ranging between 0.6 mM to 0.9 mM) were titrated in the protein sample typically by performing 16 injections of 2.5 µL aliquots. Control titrations were performed by titrating RNA into buffer. The dissociation constants (Kd), enthalpy of binding (ΔH), and stoichiometries (N) were obtained after fitting the integrated and normalized data to a single-site binding model. Data were processed using Origin 7.0 (Malvern Panalytical, Malvern, UK). All experiments were performed at least in duplicate to check for reproducibility of the data.

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