Figure 1. Sequence element near the C-terminus of the AZIN1 uCC is required for polyamine stimulation of translation initiation at the upstream AUU start codon.

(A) HEK293T cells treated with DFMO (polyamine-depleted) or DFMO+SPD (high polyamines) were transfected with mouse azin1 uCC-luciferase reporters. OF, last 10 codons of the uCC out-of-frame. Percent AUU initiation was calculated relative to corresponding AUG-initiated constructs. Error bars denote standard deviation; *p<0.05 (Student’s two-tailed t-test; n=4, assayed in duplicate). Results from tests of additional SPD concentrations are presented in Figure S1; relative reporter mRNA levels are presented in Table S1. (B) Schematic of the AZIN1 mRNA from mammals: conventional AUG initiated uORFs in cyan, uCC in red, and mORF in green. WebLogo representations of the most conserved C-terminal uCC residues from (top) 101 vertebrate AZIN1 orthologs or (middle) 320 metazoan ODC homologs, including the 101 vertebrate AZIN1 sequences; (bottom) WebLogo of the entire uCC from 34 Zygomycota ODC mRNAs. The highly conserved PPW motif is boxed. (C), azin1 uCC-Luc fusions with the indicated frameshift or point mutations (red) were tested as in panel (A). Mutations that alter the PPW motif are boxed. Firefly reporter values were normalized to a co-transfected Renilla luciferase initiated by AUG in perfect context. Error bars denote standard deviation; **p<0.01 (Student’s two-tailed t-test; n=5, assayed in duplicate). Results in panels (A) and (C) are from independent experiments; relative reporter mRNA levels are presented in Table S1.