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. Author manuscript; available in PMC: 2022 Oct 7.
Published in final edited form as: Mol Cell. 2021 Aug 9;81(19):3904–3918.e6. doi: 10.1016/j.molcel.2021.07.020

Figure 3. Conserved uORF Confers Polyamine Control of HOL1 mRNA Translation.

Figure 3.

(A) Schematic representation of HOL1-Fluc reporter showing location of conserved uORF encoding MLLLPS peptide.

(B–E) Relative HOL1-Fluc expression (B,E), HOL1-Fluc mRNA levels (C), or endogenous HOL1 mRNA levels (D) in spe1 spe2 strain Y362 grown in polyamine-depleted SD medium in the presence of 10 nM (white), 200 nM (gray), 10 μM (orange), or 1 mM (red) additional SPD. In (E), reporters contained the indicated uORF mutations (red). mRNA levels were determined by RT-PCR and first normalized to actin mRNA; then, in each panel, measurements were normalized to samples containing 10 or 200 nM SPD (white or gray bars). Error bars denote SD; **p < 0.01, ***p < 0.001 (Student’s two-tailed t test; n = 3, assayed in duplicate).

(F) Rate of [14C]SPD uptake at the indicated SPD concentrations in spe1 spe2 hol1 strain J1570 expressing HOL1 with an intact (black, pC6464) or mutated (ALLLAA, blue, pC6465) uORF, as indicated, were fit to the Michaelis-Menten equation to calculate the Vmax (nmol/min/mg protein) for SPD uptake (n = 3).