Functional and structural specificity of Spd binding to the spe2 UTR RNA. (A) Real-time PCR analysis of mRNA abundance of the spe2 UTR RNA relative to the internal control tubulin, showing the level of mRNA abundance of lacZ (representing spe2) remains unchanged on Spd titration. Error bars represent the standard deviation of three independent experiments. Statistical significance was shown compared with 0 µM, respectively, using Student's t-test, (**) P < 0.01. (B) The positions (boxed dark blue) and identity of the mutations on the secondary structure of the spe2 UTR RNA. The position boxed in purple indicates the mutation of the initiation codon of the uORF. (C) Reporter gene expression of seven inactive RNA mutations, responding to 0, 10, and 1000 µM of Spd. The error bars are the standard deviation of at least three independent experiments. Statistical significance of each mutant was calculated compared to that of WT at 0, 10, and 1000 µM, respectively, using Student's t-test, (**) P < 0.01. (D) Reporter gene expression of seven inactive RNA mutations at 0 µM of Spd. The error bars are the standard deviation of at least three independent experiments. Statistical significance of each mutant was calculated compared to that of WT at 0 µM, respectively, using Student's t-test, (**) P < 0.01. (E) Reporter gene expression of inactive RNA mutations M25–26, M28, M30, M32, M34, and M36–37, responding to 0, 10, and 1000 µM of Spd. The error bars are the standard deviation of at least three independent experiments. Statistical significance of each mutant was calculated compared to that of WT at 0, 10, and 1000 µM, respectively, using Student's t-test, (**) P < 0.01. (F) Histogram analysis of OsO4 modification for nucleotides U59 of the wild-type and three inactive mutation RNAs on Spd titrations.
