Fig 2: AMD1 and ODC1, rate-limiting enzymes of the polyamine metabolism, are required for 60S but not 40S subunit maturation. (A) Schematic representation of polyamine pathway including eIF5A hypusination. Hits in the 60S screen are highlighted in orange. (B) Ranks of polyamine synthesis pathway genes in the 60S screen and the previous 40S screen (30). Hits in the 60S screen are marked in bold. (C) Ribosomal reporter HeLa cell lines inducibly expressing RPL29-GFP or RPS2-YFP were depleted of the indicated proteins by RNAi (48 h). Expression of RPL29-GFP was induced for 8 h with tetracycline and chased in tetracycline-free medium for 20 h prior to fixation. Expression of RPS2-YFP was induced for 16 h, followed by a 4 h chase period. Scale bar: 20 µm. (D) Quantification of the fraction of cells with ribosome biogenesis defects (hit rate) in (C). Mean + SEM, N = 3, n = 100, unpaired t-test against si-control of the respective cell line, ***P = 0.001. (E) Levels of metabolites of the polyamine pathway upon RNAi-mediated depletion of AMD1 and ODC1 in HeLa K cells expressed as fold change relative to si-control. Mean + SEM. N = 3. (F) For polysome profiling, HeLa K cells were treated with the indicated siRNAs, followed by sucrose gradient centrifugation (15–45% sucrose) of cell extracts and recording the absorption at 254 nm along the gradient. (G) Quantification of four biological replicates of polysome profiles as shown in (F) determining the relative areas beneath the A254 peaks of 40S, 60S, 80S, and the first three polysome peaks. Mean ± SEM, N = 4, unpaired t-test against si-control, * P = 0.05, **P = 0.01, ***P = 0.001, ns = non-significant.

Fig 3: Inhibition of ODC1 with DFMO leads to defects in 60S subunit maturation. (A) Levels of metabolites of the polyamine pathway in HeLa K cells upon ODC1 inhibition with DFMO (2.5 mM, 48 h) expressed as fold change relative to solvent control. Mean + SEM, N = 3. Measurements of cells treated with si-ODC1 (Figure 3E) are shown for comparison. (B) Ribosomal reporter HeLa cell lines inducibly expressing RPL29-GFP or RPS2-YFP were treated with 2.5 mM DFMO or solvent control for 48 h. Expression of RPL29-GFP was induced for 8 h with tetracycline and chased in tetracycline-free medium for 20 h prior to fixation. Expression of RPS2-YFP was induced for 16 h, followed by a 4 h chase period. Scale bar: 20 µm. (C) Quantification of cells with ribosome biogenesis defects (hit rate) in (B). Mean + SEM, N = 4, n = 160, unpaired t-test against solvent control of the same cell line, **P = 0.01. (D) Northern blot analysis of total RNA extracted from HeLa K cells treated with DFMO (2.5 mM, 48 h) or solvent control using radioactively labelled probes targeting the ITS2 or ITS1 region. Levels of mature 18S rRNA were visualized with GelRed as a loading control. (E) Quantification of rRNA precursors detected with ITS2 and ITS1 probes as shown in (D) normalized to the amount of 47S rRNA precursor and expressed as fold change relative to solvent control. Mean + SEM, N = 3, unpaired t-test, *P = 0.05, ns = non-significant.