Supplementary Figure 1: Synthesis of APOBEC1-YTH and APOBEC1-YTH^mut for in vitro deaminase assays.

(a) Sanger sequencing chromatograms from in vitro deamination assays demonstrate that the cytidine adjacent to m⁶A is converted to uridine when m⁶A-containing RNA is used in the assay. APOBEC1-YTH^mut, which lacks the m⁶A-binding portion of the YTH domain, fails to convert C to U in m⁶A-containing RNA. Chromatograms are representative images from 3 individual clones picked for sequencing after subjecting PCR products to TOPO cloning. (b) Western blotting for anti-HA was performed to indicate the levels of APOBEC1-YTH or APOBEC1-YTH^mut in in vitro DART-Seq assays. APOBEC1-YTH or APOBEC1-YTH^mut proteins were synthesized in vitro and an aliquot equivalent to the amount used for DART-Seq assays was removed and subjected to western blot analysis. General protein levels are also shown using ponceau S staining of the membrane. Images are representative of n = 2 independent experiments. (c) APOBEC1-YTH^mut exhibits substantially reduced binding to m⁶A in RNA pulldown assays. Anti-HA western blot results are shown after mixing HA-tagged APOBEC1-YTH or APOBEC1-YTH^mut with A- or m⁶A-containing biotinylated RNA and subsequently purifying RNA-bound protein with streptavidin pulldown. 2% of input is shown as a reference. Blots are representative of n = 2 independent experiments.