Supplementary Figure 3: Identification of a short ycf3-intron RNA sequence for THA8 interaction.

a, A schematic diagram for the principle of the AlphaScreen binding assay. b, The binding between 10 nM His6-THA8 and 10 nM of different biotin tagged RNAs was measured by AlphaScreen binding assay (n=3, error bars=SD). RNAzm1a is derived from the maize ycf3 intron 2, whereas RNACTL1 and RNACTL2 are two control RNAs that bind CRP1 and HCF152 (two other PPR proteins), respectively. The binding assay was repeated once with similar results. c, The minimal region of RNAzm1a that binds to THA8 protein. A competition assay was performed using 4 μM of different truncations of Zm-1a RNA to compete the interaction between biotin-RNAzm1a and His6-THA8 protein by AlphaScreen (n=3, error bars=SD). The competition was repeated once with similar results. d, Sequence alignment of 13-nucleotide RNAs from different species. The arrow indicates the decreasing order of affinities of BdTHA8 for different RNAs. e, Bindings between 10 nM His6-BdTHA8 and 10 nM biotin-RNAzm-1a were competed with 13-nucleotide homologous RNAs from different species using AlphaScreen assay (n=3, error bars=SD). THA8 binds to RNA sequences from different species with a preference of Zm-4 ≈ Os > Zm-1a ≈ Zm2 > Cs ≈ At. f, Bindings between 10 nM His6-BdTHA8 and 10 nM biotin-RNAZm-1a were competed with an increasing concentration of untagged Zm1a-5, Os or Zm4 RNA (n=3, error bars=SD). The IC50 values were calculated by curve fitting using Graphpad Prism. g, The minimal region of Zm-4 RNA that binds to THA8 protein. A competition assay was performed using 1 μM of different truncations of Zm-4 RNA to compete the interaction between 10 nM biotin-RNAzm1a and 10 nM His6-THA8 protein by AlphaScreen (n=3, error bars=SD). The competition was repeated once with similar results.