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. 2021 Aug 31;36(9):109642.
doi: 10.1016/j.celrep.2021.109642.

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

Danielle M Garshott  1 , Heeseon An  2 , Elayanambi Sundaramoorthy  1 , Marilyn Leonard  1 , Alison Vicary  1 , J Wade Harper  2 , Eric J Bennett  3
Affiliations

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

Danielle M Garshott et al. Cell Rep. .

Abstract

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.

Keywords: RNF10; protein homeostasis; ribosome-associated quality control; translation initiation; ubiquitin.

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Conflict of interest statement

Declaration of interests E.J.B. serves on the scientific advisory board of Plexium. J.W.H. is a consultant and founder of Caraway Therapeutics and a founding board member of Interline Therapeutics.

Figures

Figure 1.
Figure 1.. RNF10 catalyzes uS3 and uS5 ubiquitylation, See also Figure S1.
(A) Cell lysates from 293T cells transfected with either control siRNA oligos or three separate siRNA oligos targeting RNF10, followed by treatment with dithiothreitol (DTT) for 2 hours were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. * indicates non-specific background signal. Arrow indicates RNF10-specific immunoreactivity. For all blots the ubiquitin-modified ribosomal protein is indicated by the arrow. S and L denote short and long exposures, respectively. (B) Cell extracts from parental 293T or RNF10 knockout (KO) cells were either untreated or treated with DTT or anisomycin (ANS) then analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (C) HEK293-FlpIn cells expressing tet-inducible Flag-HA tagged RNF10 were treated with doxycycline (Dox) and cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (D) In vitro ubiquitylation assay utilizing purified 40S ribosomal subunits and RNF10. Samples were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (E) (top) RNF10 knockout (KO) cells were transfected with Myc-tagged wild type (WT) or inactive mutant (CS) RNF10 and parental 293T or RNF10-KO cells were either untreated or treated with DTT. Cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (bottom) Quantitative representation of percent ubiquitylated uS3 and uS5, and percent relative total abundance from immunoblots (bottom). (F) (top) 293T cells with and without Myc-tagged wild type RNF10 expression were drug treated as indicated. UV indicates that cells were exposed to UV and were allowed to recover for 1 or 4 hours. Cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (bottom) Quantitative representation of percent ubiquitylated uS3 and uS5, and percent relative total abundance from immunoblots.
Figure 2.
Figure 2.. Persistent uS3 and uS5 ubiquitylation targets 40S ribosomal proteins for degradation, See also Figure S2.
(A) 293T USP10-knockout (KO) cells constitutively expressing wild type (WT) or inactive mutant (CS) USP10 were treated with HTN and cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. For all blots the ubiquitin-modified ribosomal protein is indicated by the arrow. S and L denote short and long exposures, respectively. (B) (top) USP10-KO cells were treated as indicated and cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (bottom) Percent ubiquitylated uS3 and uS5, and percent total relative abundance quantified from immunoblots (C) (top) Parental 293T or USP10-KO cells expressing Myc-tagged wild type RNF10 were either untreated, treated as indicated and cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (bottom) Quantitative representation of uS3 and uS5 percent ubiquitylation, and percent relative total abundance for uS3, uS5 and uL30. (D) The median normalized log2 SILAC ratio (H:L) for all quantified 40S and 60S ribosomal proteins comparing parental cells (light label) to cells of the indicated genotype (heavy label) with or without RNF10 overexpression (O/E). Each point represents a biological replicate, Bars denote mean value for replicate experiments with error bars displaying SEM. *=pvalue<0.05 by student’s t test compared to parental controls. (E) The median normalized log2 SILAC ratio (H:L) for individual 40S and 60S ribosomal proteins comparing parental cells (light label) to cells of the indicated genotype with or without RNF10 overexpression (O/E). Bars denote mean and error bars denote SD.
Figure 3.
Figure 3.. Enhanced ubiquitylation results in turnover of 40S ribosomal proteins in an autophagy-independent manner.
(A) (top) Cell extracts from parental 293T or RB1CC1-KO cells transfected with either a control siRNA oligo or siRNA oligo targeting USP10, followed by transfection with Myc-tagged wild type RNF10 treated as indicated were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (bottom) Quantitative representation of percent relative total abundance and uS3 and uS5 percent ubiquitylation. (B) HEK293 uS3 or eL28 (RPL28) Keima-tagged cells were treated as indicated and frequency distributions of the red (561nm) to green (488nm) ratio are plotted. (C) HEK293 uS3 or eL28 Keima-tagged cells were transfected with either a control siRNA oligo (black line), siRNA targeting USP10 (yellow line) or in combination with Bafilomycin A (50nM, 1h) treatment (green line). Frequency distributions of the red (561nm) to green (488nm) ratio are plotted. (D) HEK293 uS3 or eL28 Keima-tagged cells expressing either a control plasmid (grey line), RNF10 wild type (blue line) or the catalytic mutant (red line) 48 hours post transfection were collected and analyzed via FACS. Frequency distributions of the red (561nm) to green (488nm) ratio are plotted. (E) HEK293 uS3 or eL28 Keima-tagged cells transfected with either a control siRNA oligo (grey line), or siRNA targeting USP10 and expressing either a control plasmid (green line), RNF10 wild type (blue line) or the catalytic mutant (red line) 48 hours post transfection were collected and analyzed via FACS (bottom). All bar graphs denote median red:green ratio from triplicate experiments. N=3, error bars denote SD of triplicate experiments. *=pvalue<0.05, ns = non-significant by unpaired student’s t test.
Figure 4.
Figure 4.. RNF10-dependent uS5 ubiquitylation accelerates 40S protein turnover, See also Figure S3.
(A) Schematic of Ribo-Halo fluorescent pulse-chase assay (top). Microscopy images of HCT116 uS3 or uL24-Halo tagged cells expressing GFP-tagged wild type (WT) or inactive mutant (CS) RNF10. Ribosomes were labeled with TMR ligand for 2 hours prior to imaging. Arrows indicate the same cells across panels (bottom). (B) The normalized (to control at 0h washout) TMR-fluorescence intensity for uS3 or eL29-Halo tagged cells expressing a control plasmid (grey bars), Myc-RNF10-WT (blue bars), or CS mutant (red bars) expression plasmid is depicted at the indicated time points post TMR washout (left). N=3, error bars denote SD of triplicate experiments. *=pvalue<0.05, ns = non-significant by multiple unpaired t tests compared to control protein. Frequency distribution of the normalized TMR signal at 24h is plotted (right). (C) Normalized (to control at 0h washout) TMR-fluorescence intensities for uS3-Halo tagged cells expressing a control plasmid, Myc-RNF10-WT, or RNF10 mutant (CS) expression plasmid at time 0h (grey bars), 8h post TMR washout (blue bars) or with MG132 included during the 8h TMR washout (red bars) is depicted (left). TMR fluorescence intensities for cells expressing a control plasmid (grey bars), Myc-RNF10-WT (blue bars), or CS mutant (red bars) expression plasmid at 0 or 8h post TMR washout with or without BafA or SAR405 included in the TMR washout (right). N=3, error bars denote SD of triplicate experiments. *=pvalue<0.05 by unpaired student’s t test. (D) TMR fluorescence intensities 12h post washout from parental 293T uS3-Halo tagged cells alone or with stable expression of wild type (WT) or K54R/K58R mutant (Mut) uS5 and transfected with a control plasmid (grey bars), GFP-RNF10-WT (blue bars), or GFP-RNF10-CS mutant (red bars) expression plasmids are depicted. The normalized (to control at 0h washout) TMR intensities are depicted. N=3, error bars denote SD of triplicate experiments. *=pvalue<0.05, ns = non-significant by unpaired student’s t tests compared to control protein.
Figure 5.
Figure 5.. Translational initiation inhibition induces ribosomal ubiquitylation, See also Figure S4.
(A) (top) Cell extracts from 293T cells treated with increasing doses of HTN were analyzed by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. For all blots, the ubiquitin-modified ribosomal protein is indicated by the arrow. S and L denote short and long exposures, respectively. (bottom) Percent ubiquitylated uS3 and uS5 quantified from immunoblots. (B) Cell extracts from 293T cells treated with HTN or lactimidomycin (LTM) for the indicated times were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (C) Quantification of uS3 or uS5 percent ubiquitylation from 293T cells treated with increasing doses of either rocaglates (RocA) or patamineA (PatA) from blots in S4A,B. (D) (top) Cell extracts from 293T cells treated with increasing concentration of cycloheximide (CHX) were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (bottom) Quantitative representation of uS3, uS5, and eS10 percent ubiquitylation from immunoblots.
Figure 6.
Figure 6.. Moderate integrated stress response activation induces uS3 and uS5 ubiquitylation, See also Figure S4.
(A) Cell extracts from 293T cells treated as indicated were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. For all blots, the ubiquitin-modified ribosomal protein is indicated by the arrow. S and L denote short and long exposures, respectively. (B) Cell extracts from 293T cells treated with increasing concentrations of sodium arsenite (NaAsO2) were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (C) Quantification of uS3 percent ubiquitylation and eIF2α percent phosphorylation (from Phos-tag gels) following NaAsO2 treatment from B.
Figure 7.
Figure 7.. HTN induces 40S ubiquitylation in density gradient fractions with excess 40S relative to 60S ribosomal proteins, See also Figure S5.
(A) RNaseA treated cell extracts from untreated (black line) or HTN treated (red line) 293T cells were fractionated on 10–30% sucrose gradients. The 254nm absorbance trace is depicted. (B) Fractions (designated in A) 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 respectively. (C) RNaseA treated cell lysates 293T cells treated as indicated were fractionated on 10–30% sucrose gradients. The relative 254nm absorbance trace is depicted. (D,E) The ratio of the summed molecular weight (MW) normalized LFQ intensities 40S proteins:60S proteins from untreated, light labeled (black bars) or HTN treated, heavy labeled (purple bars) 293T cells. Cell extracts with either untreated (E) or treated (D) with RNaseA prior to density gradient centrifugation. Bars denote mean value for replicate experiments (n=3) with error bars displaying SEM. *=pvalue<0.05 by unpaired two-tailed Student’s t test.
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