Fig 10.

SSAT expression is elevated in mutants lacking uORFs. Red dots show locations of uORF starting codons. HEK293T cells were transiently transfected with plasmids having SSAT RNA 5′uORF plus the full coding region (5′UTR-ORF), mutations eliminating uORF starting codons plus the full coding region (ΔuORF-ORF), mutations eliminating the uORFs plus coding region deletions preventing 5′ORF stem-loop formation (ΔuORF-Δ4-3 ORF), or mutations eliminating the uORFs plus coding region deletions preventing 5′ ORF stem-loop formation and nucleolin binding (ΔuORF-Δ49-114ORF). (A) uORFs affect translation. Lanes 1 and 2 show that elimination of uORFs does not allow SSAT expression without spermine, but lanes 5 and 6 show that uORF elimination enhances spermine-induced expression. Lanes 3 and 4 show that elimination of both the uORFs and stem-loop allows greater expression, and further elimination of the nucleolin binding site enhances expression further. (B) uORF translational control is not affected by spermine. SSAT expression was compared among three constructs that require spermine for translation: 5′UTR-ORF, ΔuORF-ORF, and ORF. Since spermine-induced SSAT translation of ORF was markedly less than that for 5′UTR ORF but the difference was eliminated when the start codons of the uORFs were eliminated, that indicates that the translation repression by uORFs is independent of the presence of spermine. The slight translation leakage of ΔuORF-ORF without spermine supports this interpretation.