The uORFs in the spe2 5′UTR have no dominant translational function. (A) The locations of the micro-ORF in the spe2 UTR; gray indicates peptide sequence, and black rectangle filled with yellow indicates start codon mutation M6 and M22 in the peptide. (B) Sequences of the wild-type peptide and the corresponding RNAs, M6 start codon mutant and their degenerate mutants M42–45 (mutations marked pink). The peptide RNA sequences of M42–45 were altered, whereas the peptide amino acid sequences remained unchanged. The sequences of the M22 start codon mutant are also shown. (C) β-gal activity measured in the presence of 0, 10, and 1000 µM of Spd without thiamine in Δspe2 cells that were transformed with the reporter plasmid containing mutants M6, M22, and M42–45. Statistical significance was calculated compared to WT at 0, 10, and 1000 µM, respectively, using Student's t-test, P < 0.01.
