Fig 2: Distinct sets of RRub events are hierarchically organized.(A,B) HCT116 mutant RRub knock-in (KI) cell lines eS10-KI (K138RK139R) or uS10-KI (K4RK8R) (A,B) were treated with ANS (5 μg/ml) and MG132 (10 μM) continuously for the indicated times. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (C,D) HCT116 mutant uS5-KI (K54RK58R) or uS3-KI (K214R) were either untreated or pre-treated with DTT (5 mM) alone or with CHX (100 μg/ml) for 4 hr followed by DTT washout in media with or without CHX (100 μg/ml) and MG132 (10 μM). Cells were collected at the indicated times post DTT washout. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The ubiquitin-modified and unmodified ribosomal protein is indicated by the arrow and asterisk, respectively. S and L denote short and long exposures (n = 1).

Fig 3: Stress-induced RRub events are reversible.(A) HCT116 cells were treated with anisomycin (ANS, 5 μg/ml) alone or with MG132 (10 μM) for the indicated times. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The ubiquitin-modified and unmodified ribosomal protein is indicated by the arrow and asterisk, respectively. S and L denote short and long exposures (n = 1). (B) The amount of ubiquitylated eS10 (red line) and uS10 (blue dashed line) and unmodified eS10 (black column) and uS10 (gray column) after the indicated treatments compared to untreated cells quantified from panel A. (C) HCT116 cells were treated with DTT (5 mM) alone or with cycloheximide (CHX, 100 μg/ml) for 4 hr followed by DTT washout in media with or without CHX alone or with MG132 (10 μM) for the indicated times. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The ubiquitin-modified and unmodified ribosomal protein is indicated by the arrow and asterisk, respectively. S and L denote short and long exposures (n = 1). (D) The amount of ubiquitylated uS3 (red line) and uS5 (blue dashed line) and unmodified uS3 (black column) and uS5 (gray column) after the indicated treatments compared to untreated cells quantified from panel C. (E) 293 T cells were exposed to UV and allowed to recover for the indicated times. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The ubiquitin-modified ribosomal protein is indicated by the arrow. S and L denote short and long exposures (n = 1). (F) The amount of ubiquitylated eS10 (blue line), uS10 (red line), uS5 (black line) and uS3 (gray line) after UV exposure compared to untreated cells quantified from panel E.

Fig 4: RIOK3 is required for RNF10-dependent 40S decay(A) Relative mean intensity of RIOK3 protein as quantified by mass spectrometry in 293T cells transfected with non-targeting (Con) or 60S-biogenesis-factor-targeting siRNA as indicated.(B) Relative median intensity of 60S and 40S proteins, normalized to non-targeting siRNA, as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 or RIOK3.(C) Ubiquitylated uS5-K58 and uS3-K214 peptide intensity quantified by mass spectrometry from 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 or RIOK3 as indicated.(D) Relative mean intensity of indicated proteins as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 or RIOK3 as indicated.For (A)–(D), n = 3, error bars indicate SEM; NS, not significant; *p < 0.05 and **p < 0.01 by Student’s t test. Red lines, text, or asterisks indicate a comparison to siGTPBP4 alone.

Fig 5: Impairing 60S biogenesis stimulates ribosome ubiquitylation(A) 293T cells transfected with non-targeting siRNA (siCon) or three independent siRNA oligonucleotides targeting the indicated translation factor were either untreated or treated with harringtonine (HTN) for 2 h. Whole-cell extracts were analyzed by SDS-PAGE followed by immunoblotting with antibodies against uS3 and uS5. The ubiquitin-modified uS3 and uS5 are indicated by the arrows. S, short exposure; L, long exposure.(B) 293T cells transfected with control siRNA or siRNAs targeting EIF4A1 or EIF6 were either untreated or treated with the indicated inhibitors for 2 h. Whole-cell extracts were analyzed by SDS-PAGE followed by immunoblotting with antibodies against uS3. The ubiquitin-modified uS3 is indicated by the arrows. S, short exposure; L, long exposure.(C) Schematic representation of ribosome biogenesis pathways with targeted 60S biogenesis factors indicated.(D) Whole-cell extracts from 293 Flp-In cells transfected with non-targeting or 60S-biogenesis-factor-targeting siRNAs were fractionated on sucrose gradients.40S, 60S, and 80S peaks are as indicated. (Top) Absorbance at 260 nm indicating ribosomal abundance across the gradient. (Bottom) The 60S:40S and 80S:40S ratio upon knockdown of the indicated 60S biogenesis factor as calculated from the sucrose gradient absorbance profile.(E) Whole-cell extracts from 293T cells transfected with siRNAs targeting the indicated 60S biogenesis factor were immunoblotted with antibodies against uS3 and uS5. The ubiquitin-modified uS3 and uS5 are indicated by the arrows. S, short exposure; L, long exposure.(F) Ubiquitylated uS5-K58 peptide intensity quantified by mass spectrometry from 293T cells transfected with non-targeting siRNA or siRNAs targeting indicated biogenesis factors. n = 3, error bars indicate SEM; **p < 0.01 by Student’s t test.