Fig. 4

YTH recognizes m⁶A in B5ʹ but not D. a–c Binding curves for YTH domain with a SS, b D, and c B5ʹ. d One-site and independent two-site binding models used for curve fitting. Data is shown for unmodified and methylated RNA with and without 3 mM Mg²⁺. Uncertainty reflects the standard deviation from three measurements. (c, left) Fits to the data assuming single (blue) versus two independent (red) binding sites (see Methods). e In vitro RNA pulldown assay using 5ʹbiotinylated RNA constructs with m⁶A. Showing YTHDF2 binds preferentially to m⁶A in unpaired context or at the junction, and Mg²⁺ decreases the enrichment of YTHDF2 by methylated B5ʹ. Blots shown are representative of results from three experiments. Uncropped blots are shown in Supplementary Fig. 6. The semi-quantitative YTHDF2 enrichment analysis was performed using ImageJ⁵⁶. The uncertainty represents the standard deviation from three experiments