Methylation in the β-actin zipcode changes ZBP1 binding. (A,B) Positions and labeling of the assayed m6A sites in the chicken β-actin core zipcode sequence. (C) Binding assay of the ZBP1KH34 domain to the zipcode oligonucleotide containing m6A at the different positions. A–E refers to the m6A positions presented in A and B subfigures. The bar chart shows the Kd values for the different oligonucleotides. These values were calculated using three different concentrations for each oligonucleotide. The error bars represent the standard error from three replicates. B and E oligomers are significantly different from A oligomer (t-test, one-tailed, unequal variance for A–B, P = 0.05; A–E, P = 0.02; B–E, P = 0.015; A–C, P = 0.2; A–D, P = 0.4). (D) Competition assay using cold B oligonucleotide. The bar chart shows the percentage of outcompeted fractions where the error bars are representing standard deviation from three replicates for A and B oligomers and two replicates for C and D. (*) We were not able to quantitatively assess E oligonucleotide due to close to background level intensity of its shifted band in the absence of a competitor, and complete disappearance in the presence of cold B.
