Fig 1: Construction of a system for monitoring ribophagy flux in human cells(a) Schematic of ribophagy detection in mammalian cells using a Ribo-Keima reporter system. (b) View of the C terminus of RPL28 and RPS3 in a structure of 80S complex showing the solvent exposed site for Keima protein tagging. (PDB: 5AJ0) (c) Generation of homozygous RPL28-Keima and heterozygous RPS3-Keima reporter cell lines via CRISPR-Cas9 based gene editing. (d) Confirmation of the RPL28-Keima and RPS3-Keima expression in HEK293 and HCT116 cells by immunoblotting. Asterisk indicates Ribo-Keima protein fragments formed after the hydrolysis of the N-acyl imine in Keima chromophore during heat denaturation (see Supplemental Fig. 1d–f). Data shown represent more than three independent experiments with similar results. (e-f) Sucrose density gradient centrifugation shows proper assembly of RPL28-Keima and RPS3-Keima with 60S, 40S, 80S, and polysomes with or without 4h BTZ treatment (0.1 μM) to stabilize any unassembled subunits. Two independent experiments showed similar results. Unprocessed original scans of blots are shown in Supplemental Fig. 6.
Fig 2: Ribophagy in response to mTOR inhibition in HEK293 cells is ATG5-independent but BECN1-dependent(a) Frequency distributions of 561/488 nm excitation ratios measured on HEK293 RPS3-Keima cells lacking ATG5 or BECN1 were compared after Torin1 or Torin1/BafA treatment using flow cytometry. (n = 10,000 cells per condition) (b) Average 561/488 nm excitation ratios calculated from the biological triplicate experiments from panel a. Mean ± S.E.M. (****p < 0.0001, **p < 0.01, Two-way ANOVA) (c) Confocal images of live HEK293 RPS3-Keima cells lacking ATG5 after Torin1 (150 nM, 24h) or Torin1 (150 nM, 24h)/SAR405 (1 μM, 24h) co-treatment. (Scale bar = 20 μm) (d) Number of red Keima puncta/cell was measured from the live-cell images of HEK293 RPS3-Keima WT, ATG5−/−, or BECN1−/− cells taken after Torin1 (150 nM, 24h) or Torin1 (150 nM, 24h)/SAR405 (1 μM, 24h) co-treatment. Mean ± S.E.M. (Total number of cells from three biologically independent samples are indicated in the graph) (e,f) HEK293 RPS3-Keima cells (with or without ATG5) were incubated with HBSS in the presence of lysosomal hydrolase inhibitors (L.H.I., E64d and Pepstatin, 30 μM each) for the indicated times prior to live cell imaging. (Scale bar = 20 μm) (g) Unbiased quantification of the live-cell images obtained as shown in panels e and f. (h) Unbiased quantification of red Keima puncta obtained from live HCT116 RPL28-Keima cells (with and without ATG5) as shown in Supplemental Fig. 2g,h. In panels g and h, total number of cells from three biologically independent samples are indicated in the graph, and Mean ± S.E.M. is shown. (i) Electron microscopy images of HEK293 RPS3-Keima WT, ATG5−/− and BECN1−/− cells 4.5 h after HBSS treatment in the presence of BafA (50 nM). Red arrow: ribosomes in autophagosomes or autophagolysosomes, yellow arrow: ribosomes bound to ER in cytosol (Scale bar = 500 μm). The data shown represents two independent experiments. Statistical source data for b can be found in Supplementary Table 2. All experiments were repeated at least three times unless otherwise indicated.
Fig 3: A screen of ribosome stress agents identifies sodium arsenite and reversine as ribophagy inducers(a) HEK293 RPS3-Keima cells were exposed to (Tor1, 150nM, 24h; Reversine, 0.5μM, 48h; Sodium Arsenite (AS), 10μM, 24h; H2O2, 250μM, 24h; Cycloheximide, 10μM, 5h; Diazaborine, 200μM, 24h; Bortezomib, 250nM, 5h; Puromycin, 1μM, 5h; p97 inhibitor CB5083, 100nM, 24h; Tunicamycin, 0.6μM, 24h), and 561/488 ratio measured. (n=10,000 cells/condition) (b) HEK293 RPS3-Keima:WT, ATG5−/−, and BECN1−/− cells treated as in a.. Mean 561/488 ratios are plotted. (n=3 independent experiments, Supplemetary Table 2). (c) Frequency distributions of 561/488 ratios measured in HEK293 RPS3-Keima:ATG5+/+, ATG5−/− cells treated with AS. (n=4200 cells/condition) (d) The average 561/488 ratios from triplicate experiments as in panel c. (e) HEK293 RPS3-Keima:ATG5−/− cells transduced with a lentivirus expressing either ATG5 or conjugation defective ATG5K130R mutant were treated with AS (10μM, 24h). The average 561/488 ratios of biological triplicate experiments are shown. Mean of two independent experiments is shown for ATG5K130R cells. (f) Immunoblotting of indicated cells treated with AS (20μM, 20h). (g) The average 561/488 ratios of the indicated cell lines treated with Reversine (0.5μM, 48h) ± BafA (50nM, 1h). (n=3 independent experiments) (h) Frequency distributions of 561/488 ratios for HCT116 RPL28-Keima cells with or without Reversine treatment. (n=10,000 cells/condition) (i) Immunoblot of HCT116 RPL28-Keima cells treated with Reversine ± SAR405. (j) Imaging of HCT116 RPL28-Keima:ATG5+/+, ATG5−/− cells ± Reversine (0.5μM, 48h). (Scale bar=20μm) (k) Quantification of the images in panel j. Mean ± S.E.M. (Total number of cells are indicated as n) (l) The average 561/488 ratios of cells treated with Reversine (0.5μM, 48h). (n=3 independent experiments) (m) Immunoblots of indicated cell lines treated as in panel l. Mean ± S.E.M. is shown in panel b,d,e,g,l. (****p<0.0001, ***p<0.001, **p<0.01, panel b:One-way ANOVA, panel d,e,g,l:Two-way ANOVA) All experiments were repeated three times unless otherwise indicated. Statistical source data are in Supplementary Table 2. Unprocessed original scans are shown in Supplementary Fig. 6.
Fig 4: IFN stimulation induces changes in ribosome composition to regulate ISG synthesis. a Schematic overview of the sucrose gradient experiments to determine changes in composition of free (40S, 60S, and 80S) and actively translating ribosomes (polysomes). b Representative traces of sucrose density gradients from cells stimulated with IFN for 24 h or unstimulated cells. c Median protein abundance across three replicates from pooled free ribosome or polysome fractions by label-free quantification (LFQ) in IFN-stimulated or unstimulated cells. PCC, Pearson’s correlation coefficient. d Western blot of ribosomes isolated from IFN-stimulated or unstimulated cells by sucrose cushion. e Experimental workflow for shotgun proteomics analysis on RPL28-depleted cells after IFN stimulation. f Volcano plot showing differential protein abundance in cells stimulated with IFN for 8 h and treated with siRPL28, relative to controls. gp values from gene set enrichment analysis (GSEA) of 5453 GO terms in a comparison of siRPL28-treated and IFN-stimulated cells compared to untreated controls. Dotted line shows the statistical significance of ISGs by GSEA (Fig. S9E). Points in red represent GO terms more significantly enriched than ISGs. h Heatmap showing abundance of select well-studied ISGs in siRPL28-treated and IFN-stimulated cells compared to untreated controls. i Western blots of select ISGs in cells treated with siRPL28 or control siRNA after 4 h, 8 h, and 24 h of IFN stimulation. j Crystal structure of the human 80S ribosome (PDB: 4UG0), with RPL28 highlighted in blue
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