Translational Activity Controls Ribophagic Flux and Turnover of Distinct Ribosome Pools
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Abstract
Ribosomes are among the most abundant and complex machineries in the cell, however, the turnover of their subunits remains poorly understood. Here, we apply proteomic flux and cryo-electron microscopy analyses to interrogate the ribosome life cycle in human cells. We show that subpopulations of ribosomal subunits coexist, which vary in turnover kinetics and structure. Specifically, 80S ribosomes have a much longer half-life than free 40S and 60S ribosomal subunits, indicating that they represent distinct subunit pools that poorly intermix. Translation inhibition starkly increases the pool-size of 80S ribosomes in a translationally idle state and induces ribophagy of old ribosomes, ultimately rejuvenating the ribosome fleet. Our findings provide a comprehensive model for ribosome turnover and its regulation via translational activity.
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Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj, Bobby Hollingsworth, Gary McDowell and Michael Robicheaux. Review synthesized by Michael Robicheaux.
The preprint manuscript by Trendel et al., “Translational Activity Controls Ribophagic Flux and Turnover of Distinct Ribosome Pools”, presents a dataset that examines the lifecycle of human ribosomes, and their constituent subunit proteins, in response to translational inhibition using proteomics and cryo-EM approaches. The study focuses on the fate of 80S monosomes, which are shown to be inactive and to form a dynamic pool separate from active polysomes and nascent ribosomal subunits.
General comments
The manuscript is well-written and organized, and the methodology is thorough and detailed.
The effort to validate mass …
Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj, Bobby Hollingsworth, Gary McDowell and Michael Robicheaux. Review synthesized by Michael Robicheaux.
The preprint manuscript by Trendel et al., “Translational Activity Controls Ribophagic Flux and Turnover of Distinct Ribosome Pools”, presents a dataset that examines the lifecycle of human ribosomes, and their constituent subunit proteins, in response to translational inhibition using proteomics and cryo-EM approaches. The study focuses on the fate of 80S monosomes, which are shown to be inactive and to form a dynamic pool separate from active polysomes and nascent ribosomal subunits.
General comments
The manuscript is well-written and organized, and the methodology is thorough and detailed.
The effort to validate mass spectrometry quantitative measurements, particularly the peptide sum normalization (PSN), is commendable. The description of total sum normalization and its weaknesses in this methodology is well articulated. This work will be useful for others working on similar problems in quantitative mass spectrometry.
The described pulse-SILAC methods are quite successful at monitoring protein stability in response to different perturbations; however, the statements in favor of ribosome subunit decay through ribophagy/selective autophagy require further support. Since ribosome component decay can be due to a variety of additional pathways (see cited reference #17, An et al., 2020), it may be necessary to soften the conclusions regarding ribophagy. Additional pulse-SILAC experiments in cell lines that lack key autophagy components (e.g., ATG12/FIP200 KO cells) could be considered to directly test the ribophagy model.
There are questions as to whether the cryo-EM processing supports the conclusions stated in the manuscript. Specific comments regarding this are provided below. In addition, additional processing detail in the flowcharts presented within the supplemental data would be helpful to better understand processing choices (e.g., D classes that move forward for additional analysis/classification/refinement).
It would be relevant to discuss how the proteomic half-life measurements compare to those published by Li et al. 2021 (Mol Cell), which use a different method (cyclohexamide chase).
The manuscript reports significant differences in the half-lives of the 40S/60S ribosomal subunits vs 80S/polysome fractions (Fig 1E), and states that these make up separate ribosomal pools without free exchange. However, it should be considered as an alternative that the decay rate of assembled ribosomes could be much less than the unassembled group so that the pool of free components becomes gradually depleted. In this case, exchange could still occur with a decreasing rate as the pool of free ribosome proteins are degraded faster than assembled ones. It would also be relevant to discuss the possibility that nascent 40S and 60S subunits form 80S monosomes in an alternative “life cycle” pathway.
Specific comments and suggestions
In paragraph 1 of the Introduction, please specify the context of “serum withdrawal” as the stimulus for idle 80S ribosome accumulation. Is this from cell culture or some other system?
In paragraph 1 of the Introduction, the sentence, “Degradation of ribosomal complexes, especially under nutrient-poor conditions, is mediated by ribophagy, a selective form of autophagy [14–17]” could be more nuanced as it does not describe other non-autophagic ribosomal degradation pathways, such as those described in cited reference #17 (An et al., 2020).
In the “A Highly Robust Normalization Procedure...” Results section, the manuscript states that the intensive ribosomal purification methods lead to high variability in the mass spectrometry measurements. Based on this, have alternative methodologies been considered for ribosome purification?
In panel E of Figure 1, the color scheme makes the data difficult to differentiate, could also consider separate figures for the large and small subunit datasets.
In the “Protein Half-Lives in Polysome Profiling Fractions...'' Results section, “On average ribosomal proteins of the small subunit had 3-fold longer half-lives within the 80S fraction compared to the 40S fraction (p=5.2E-8, Wilcoxon ranksum test), whereas large subunit proteins had 4.6-fold longer half-lives within the 60S fraction compared to the 80S fraction (p=1.0E-10).” Are the “60S” and “80S” fractions mixed up at the end of the sentence?
In the “The Monosome Fraction Predominantly Contains Inactive 80S Ribosomes...” Results section, the manuscript reports, based on their cryo-EM data (Fig. 2), that 80S monosomal complexes are idle and distinct from polysomal 80S complexes. This conclusion of a single ribosome state would need supportive evidence. From the initial particle stack (>1 million) that yielded <60k high-resolution particles after classification: were there other low-resolution class averages or heterogeneous particles that may represent actively translating ribosomes? Conclusions about ribosome activity from less than 5% of the total pool of ribosomes could be due to the conformational plasticity of translating ribosomes. In a different paper (Brown et al., eLife. 2018), several structures/states of the ribosome come out of a smaller dataset. Furthermore, a structure of comparable resolution from the polysome fraction appears necessary to support the conclusion that the 80s monosome complex is functionally distinct. The same comparative data is recommended for conclusions drawn from the cryo-EM structural analysis of arsenite treated 80S particles (Fig .S6).
In the “The Monosome Fraction Predominantly Contains Inactive 80S Ribosomes..” Results section, this section introduces ribosomal P-stalk proteins, their plasticity and role in active ribosomes, which are concepts that could be included in the Introduction section of the manuscript.
In Figure 2, it is unclear from the figure legend if the 80s monosome density in panel B is from the low-salt treated preparation in panel A or from a different prep.
In the “Inhibition of Translation Produces Inactive 80S Ribosomes...” Results section, recommend revising the text to reframe the conclusion as "supports our model".
In the “An Increased Pool of Inactive 80S Ribosomes..” Results section, recommend toning down the claims about decay rates which may require control experiments in cells lacking key autophagy proteins, such as ATG12.
In the “An Increased Pool of Inactive 80S Ribosomes...” Results section, consider reframing the conclusions from the previous study (Trendel et al. 2019) to indicate that ribophagy is the predominant mechanism of ribosomal protein turnover in response to arsenite treatment. The prior study did not examine ribosomes treated with arsenite when autophagy was blocked. Additional quantitative tests for flux into lysosomes (Lyso-IP, Ribo-Keima shift assay) should be considered to support that ribophagic flux, specifically, eliminates proteins from ribosomal pools. Based on this comment, the inclusion of ribophagy in Fig. 5 and the statements in the final paragraph of the Discussion may require revision.
In the “An Increased Pool of Inactive 80S Ribosomes...” Results section, the manuscript describes proteomic data in response to increasing concentrations of arsenite. The effects of these treatments on polysome profiles could be useful future experiments.
In the “Constrained Conformational Plasticity...” Results section, there are questions about this analysis due to the small size of the final particle stack for both proteins. An alternative analysis pipeline is to mix the particles from both datasets for the simultaneous analysis of all pooled particles, from which the number of particles in a given state can be quantified.
In the “Distinct Pools of Ribosomal Subunits...” Discussion section, the discussion of inactive 80S complexes potentially re-entering the polysome “assembly line” is quite interesting to consider in terms of its dynamics and follow-up experiments that would test this theory (including subcellular localization).
In the “Distinct Pools of Ribosomal Subunits...” Discussion section, the manuscript posits that the degradation of newly synthesized ribosomal subunits is not energetically favorable; however, it should be considered that intrinsically disordered proteins, such as transcription factors, can be produced and quickly degraded in oscillatory patterns (e.g. see https://pubmed.ncbi.nlm.nih.... A quality control pathway that would eliminate immature or nascent ribosomal subunits is conceivable.
Consider depositing all EM data in EMPIAR and relevant structures in EMDB/PDB, and depositing the mass spectrometry raw data in ProteomeXchange or similar database. A data availability statement could be added with relevant accession links and IDs.
It would be helpful to build a tool to browse protein-level half lives and re-analyze raw data (e.g., tidy script depositing in Github or similar).
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