Fig 2: Upregulation of 5′-TOP mRNAs in p53−/− MEFs. p53fl/fl MEFs were transduced with control AAV-LacZ or AAV-Cre to knockout p53. (a) Polysome profiling showing increased polysome peaks following knockout of p53 (Cre). (b) Immunoblot analysis showing protein abundance of 5′-TOP encoded proteins (TPT1, RPL22, RPL23A, EEF2 and PABPC1) and 5′-TOP regulators (eIF4G1, LARP1, eIF4E and mTORC1) following knockout of p53 (AAV-Cre). (c) Luciferase activity of a 5′-TOP reporter is increased in p53−/− MEFs. Luciferase activity was normalized to firefly luciferase transfection control and set relative to LacZ control. Mean ± SD, n = 3. (d) qPCR shows no increase in mRNA expression of the 5′-TOP reporter. Normalized to firefly luciferase transfection control. Mean ± SD, n = 3. (e) Quantitation of blots in panel (b) for TPT1, RPL22, RPL23A, EEF2 and PABPC1. Total protein was used for normalization. Mean ± SD, n = 3–7. (f) qPCR shows no change in mRNA expression of 5′-TOP mRNAs following knockout of p53. Mean ± SD, n = 6. (g) Quantitation of blot in (b) and qPCR for LARP1. Mean ± SD, n = 7(protein), 6(RNA). (h) Quantitation of blot in (b) and qPCR for eIF4G1. Mean ± SD, n = 6(protein), 6(RNA). (i) Quantitation of blot in (b) and for mTORC1 pathway. Mean ± SD, n = 4. *, **, *** p-value < 0.05, < 0.01, < 0.001; two-tailed t-test with Bonferoni correction.

Fig 3: Oxygen-sensitive RBPs collaborate with the hypoxic protein synthesis machinery.a Representative immunoblots of HuR, PCBP1 (hypoxia-adaptive, blue), and LARP1 (non-hypoxia-activated, black) co-immunoprecipitations in U87MG. Three independent experiments (n = 3) were performed with similar results. Global RNA sequencing analysis of (b, c) HIF-2a- and (d, e) HIF-1a-dependent changes in (b, d) translation efficiency (TE) and (c, e) steady-state mRNA levels. f Representative immunoblots of hypoxia-adaptive RBPs in hypoxic U87MG ribosome density fractions with and without HIF-2a siRNA-mediated knockdown (for 48 h prior to following experimentation). Color scheme: red, normoxia; blue, hypoxia. Three independent experiments (n = 3) were performed with similar results. g Representative immunoblots of HuR co-immunoprecipitations with and without HIF-2a siRNA-mediated knockdown in U87MG. Three independent experiments (n = 3) were performed with similar results. h Representative immunoblots of U87MG treated with indicated siRNAs. Puromycin incorporation was used as a measure of global translational intensity. NS: non-silencing. Quantitation represents mean of three independent experiments (n = 3). i Empirically derived model of functional integration between hypoxia-adaptive RBPs and elements of the hypoxic protein synthesis machinery. Source data are provided as a Source data file.

Fig 4: Loss of LARP1 partially alleviates 5′TOP translational repression during Torin1 treatment.(A) Quantification of translation site intensities in LARP1 KO cells ± Torin1 (250 nM, 1 hour). SunTag intensities are plotted for all mRNAs overlaid with the mean ± SD (≥652 mRNAs per condition, n = 3). (B) Fraction of mRNAs undergoing translation quantified per cell in LARP1 KO cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥91 cells per condition, n = 3). (C) Quantification of translation site intensities in LARP1/1B KO cells ± Torin1 (250 nM, 1 hour). SunTag intensities are plotted for all mRNAs (colored circles) overlaid with the mean ± SD (≥218 mRNAs per condition, n = 3). (D) Fraction of mRNAs undergoing translation quantified per cell in LARP1/1B KO cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥30 cells per condition, n = 3). For statistics, unpaired t tests were performed, with statistical significance claimed when P < 0.05 (*P < 0.05, **P < 0.01, ****P < 0.0001). (E) Time course of fraction of translating mRNAs per cell for canonical and 5′TOP reporter cell lines in the presence (WT) or absence of LARP1/1B (KO). Cells were treated with 0-, 30-, 60-, 120-, and 240-min Torin1. Values are plotted as the mean ± SEM (≥30 cells per condition, n = 3).

Fig 5: Loss of 4EBP1/2 is sufficient to alleviate 5′TOP translational repression during Torin1 treatment.(A) Quantification of translation site intensities of reporter mRNAs in 4EBP1/2 KD cells ± Torin1 (250 nM, 1 hour). SunTag intensites are plotted for all mRNAs (colored circles) overlaid with the mean ± SD (≥1344 mRNAs per condition, n = 5). (B) Fraction of mRNAs undergoing translation quantified per cell for reporter mRNAs in 4EBP1/2 KD cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥204 cells per condition, n = 5). (C) Quantification of translation site intensities of reporter mRNAs in 4EBP1/2 KD_LARP1/1B KO cells ±1 hour Torin1 (250 nM). SunTag intensities are plotted for all mRNAs overlaid with the mean ± SD (≥783 mRNAs per condition, n = 3). (D) Fraction of mRNAs undergoing translation quantified per cell of reporter mRNAs in 4EBP1/2 KD_LARP1/1B KO cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥97 cells per condition, n = 3). For statistics, unpaired t tests were performed, with statistical significance claimed when P < 0.05. (E) Time course of fraction of translating mRNAs per cell for canonical and 5′TOP reporters in 4EBP1/2 KD cells with WT LARP1 or with LARP1/1B KO. Cells were treated with 0-, 30-, 60-, 120-, and 240-min Torin1. Values are plotted as the mean ± SEM (≥97 cells per condition, n = 3 to 5).