Supplementary Figure 4: THA8 protein prefers single-stranded RNA (ssRNA) and charge-complementary interactions are important for THA8 and RNA interactions.

a, THA8 protein binds to Zm4 ssRNA preferentially. Bindings between 10 nM His6-BdTHA8 and 10 nM biotin-Zm1a RNA were competed with untagged Zm4 ssRNA, ssDNA, dsRNA, RNA/DNA hybrid, or dsDNA (n=3, error bars=SD). THA8 protein binds Zm4 with a preference order of ssRNA > dsRNA ≈ DNA-RNA hybrid > dsDNA > ssDNA. The IC50 values were calculated by curve fitting using Graphpad Prism. b, The Zm4 RNA 2-OH group of ribose interacts with THA8 dimer protein through H-bonds. c, Mutational effects of the THA8 positively charged residues on THA8 and Zm1a RNA interaction. The binding between 10 nM biotin-Zm1a RNA and 50 nM His6-THA8 wild type or mutant proteins were measured by AlphaScreen assay (n=3, error bars=SD). The mutants with strongly reduced binding affinity are indicated by asterisks. The binding assay was repeated once with similar results. d, Mapping the positively charged residues onto the THA8 dimeric structure (shown in stick models). The positively charged residues that significantly reduced protein-RNA interaction are colored in magenta whereas those did not significantly affect protein-RNA interaction are colored in yellow. The RNA molecule is shown as a cartoon diagram.