Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition

Abstract

Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies1,2,3. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response4 or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity—which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy5—reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases.

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Fig. 1: Apoptosis and depressed protein synthesis in RP-deficient tissue.
Fig. 2: Hallmarks of proteotoxic stress in RP-deficient cells.
Fig. 3: Proteomic analysis of RP-deficient imaginal discs.
Fig. 4: The ISR and autophagy limit tissue damage caused by RP deficiency.
Fig. 5: Inhibition of TOR signalling reduces the accumulation of aggregates and apoptosis in RP-deficient cells.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. The mass spectrometry dataset is available at ProteomeXchange under the identifier PXD023021. Accession numbers and names for the proteins identified by mass spectrometry are available at UniProt (https://www.uniprot.org/) and FlyBase (http://flybase.org/). Source data are provided with this paper.

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Acknowledgements

We thank T. E. Rusten (University of Oslo) for the gift of anti-p62 antibodies and F. Zhang (Broad Institute of MIT and Harvard) for the PX459 vector. We also acknowledge the staff at the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa for the provision of antibodies. Drosophila stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. We also thank M. Cockman, I. McGough and P. Ratcliffe (all at the Crick Institute), as well as M. Pieterse (Mill, The Netherlands) for discussions. This work was supported by a Wellcome Trust Investigator award (no. 206341/Z/17/Z to J.P.V.) and the Francis Crick Institute, which receives its core funding from Cancer Research UK (no. FC001204), the UK Medical Research Council (no. FC001204) and the Wellcome Trust (no. FC001204).

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Contributions

This project was conceived by C.R.-A., H.N. and J.-P.V.; H.N. created the RPS23R67K strain, as well as the RPS26attP-KO strain, which was used as the starting point for generating RPS26cKO by C.A. and C.R.-A.; H.N. also performed the developmental timing measurements. D.J.H. generated the RPS23R67K HEK293 cells. C.A. and C.R.-A. generated the translation fidelity reporter. J.K. and A.P.S. generated and analysed the mass spectrometry data. The manuscript was written by C.R.-A. and J.-P.V.

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Correspondence to Carles Recasens-Alvarez or Jean-Paul Vincent.

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The authors declare no competing interests.

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Peer review information: Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Phenotypes of Minute heterozygotes.

(a, b) Scutellar region of control and RPS23R67K/+ flies showing the short-bristle phenotype that characterises Minute heterozygotes. c, Cumulative distribution of pupariation time for control (n = 143 larvae) and RPS23R67K/+ (n = 134 larvae). Error bars represent standard deviation (d) Control mosaic imaginal disc harbouring wildtype clones (2X GFP) and their wild type twin clones (absence of GFP), induced by heat shock-mediated expression of FLP (hs-FLP). Note the low number of Dcp1-positive cells (red and grey). e, Mosaic imaginal discs harbouring wild type clones (2X GFP) in a RPS23R67K/+ background (1X GFP), also induced with hs-FLP. Here the twin clones (RPS23R67K/R67K) are rapidly eliminated and the wild type cells outcompete the RPS23R67K/+ cells, which undergo a high rate of apoptosis (Dcp1, red and grey). (f, g) JNK signalling (indicated by expression of the TRE-GFP reporter and apoptosis (Dcp1) in RPS23R67K/+ are fully supressed by a wild type copy of RPS23 from a genomic duplication (g). (h–j) JNK signalling and apoptosis in a panel of heterozygous Minute mutants (RPL5, RPL14, and RPS13). Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Source data is available for this figure. Source data

Extended Data Fig. 2 Xrp1 is required for activation of apoptosis in RPS23R67K/+.

a, Imaginal disc of a RPS23R67K/+ larva expressing an RNAi transgene against Xrp1 in the anterior compartment (marked with anti-Ci). The number of Dcp1-positive cells is lower in the anterior than in the posterior compartment where Xrp1 activity is unaffected. Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Experiments were repeated independently three times with similar results.

Extended Data Fig. 3 Manipulation of Rheb activity affects OPP incorporation in wild type and RPS23R67K/ + imaginal discs.

(a–d) Wing imaginal discs (wild type and RPS23R67K/+, as indicated) overexpressing Rheb or RhebRNAi in the anterior compartment (left hand side of the disc). The discs were explanted and incubated for a 15 min in 1 µM OPP before staining for puromycilated peptides (grey scale). Rheb overexpression stimulated OPP incorporation in both genotypes, while RhebRNAi had the opposite effect. Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Experiments were repeated independently three times with similar results.

Extended Data Fig. 4 RPS23R67K/ + imaginal discs develop tumours upon inhibition of apoptosis.

(a,b) Wing disc from a RPS23R67K/+ larva expressing P35, a baculovirus-derived inhibitor of effector caspases in the pouch (under the control of rotund-GAL4). Note the overgrowth characterised by epithelial folds and ectopic Wingless expression (green), shown in grey scale at higher magnification in b. Formation of these tumours shows that RP-deficient cells are not inherently incapable of growth. Tumour formation may be relevant to the increase cancer risk associated with human ribosomopathies as well as to the observation that ribosomal protein genes are frequently deleted in human cancers, often in concert with the loss of TP533. Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Experiments were repeated independently three times with similar results.

Extended Data Fig. 5 A toxic form of Huntingtin (HTT96Q) triggers apoptosis.

(a,b) Expression of HTT96Q throughout the pouch (with rotund-gal4) triggers an increased rate of apoptosis relative to that seen with GFP expression, which is expected to be innocuous. c, Quantification of Dcp1 coverage in the two genotypes shown in panels a and b (n = 5 discs per genotype). Error bars denote standard deviation. For statistical analysis, a two-tailed unpaired t-test was carried out. **P = 4.11E-03. Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Source data is available for this figure. Source data

Extended Data Fig. 6 Accumulation of p62 and P-eIF2α in RPS26KO/+.

(a, b) RPS26cKO was inactivated (and tubulin-mCherry deleted) by crossing to hedgehog-GAL4, UAS-FLP. In the resulting RPS26KO/+ posterior compartment, immunoreactivity against p62 and P-eIF2α was higher than in the control anterior compartment. Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Experiments were repeated independently three times with similar results.

Extended Data Fig. 7 RP deficiency alters the activity of an autophagy reporte.

a, Cartoon showing progression through autophagy as monitored by the GFP-mCherry-Atg8a reporter. Yellow indicates the simultaneous presence of GFP and mCherry in the phagophore and autophagosome. Autolysosomes only retain the red colour because of the drop in pH, which quenches GFP fluorescence. (b, c) Fluorescence from GFP-mCherry-Atg8a, expressed with tubulin-GAL4 in wild type or RPS23R67K/+. Single fluorescence channels are also shown in grey. Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Experiments were repeated independently three times with similar results.

Extended Data Fig. 8 Translation fidelity is unaffected in RPS23R67K/+.

a, The stop codon readthrough reporter comprises 10X Upstream Activator Sequences (UAS), which confers GAL4 responsiveness, the 5’ UTR from Syn21, the coding region of Firefly luciferase (Fluc), a STOP codon (UGAC), a flexible linker, the coding region of Nanoluciferase (Nluc), and the 3’UTR from p10. b, Quantification of the Nluc/Fluc ratio, measured from whole larvae lysates and normalised to that in control larvae. Statistical analysis: 4 replicates for each condition. Error bars denote standard deviation. A two-tailed unpaired t-test was carried out. P>0.05, no significant increase was seen in RPS23R67K/+ larvae. Genotypes for each figure panel are available in Supplementary Table 1. Source data is available for this figure. Source data

Extended Data Fig. 9 Validating the effect of GADD34 overexpression.

a, P-eIF2α immunoreactivity in a wild type imaginal disc. b, This is reduced by GADD34 overexpression (GADD34OE) driven by nubbin-GAL4. (c) P-eIF2α immunoreactivity is similarly decreased in RPS23R67K/+ larvae overexpressing GADD34. d, Schematic representation of the domain where GADD34 was overexpressed. (e,f) GADD34 overexpression causes a mild but significant increase in wing size in otherwise wild type flies but not in RPS23R67K heterozygotes. Note that the wing of RPS23R67K heterozygotes is smaller than that of wildtype. (g-i) GADD34 overexpression exacerbates the formation of HTT25Q punctae in RPS23R67K heterozygotes. Statistics: error bars denote standard deviation. In f, n = 12 adult wings for each genotype. In i, from left to right, n = 10 and 8 discs. A two-tailed unpaired t-test was carried out. P-values in f, from top to bottom: 2.48E-01, 1.31E-07 and 7.09E-06. P-value in i, 9.65E-04. Scale bars represent 50 µm. Genotypes for each figure panel are available in Supplementary Table 1. Source data is available for this. Source data

Extended Data Fig. 10 Effect of proteasome inhibition on the rate of apoptosis in RP-deficient tissues.

a, Extent of apoptosis (coverage of Dcp1 immuno-reactivity) in the pouch of discs of genotypes indicated. Rpt6RNAi denotes rotund-gal4-driven expression of a Rpt6RNAi transgene.. This particular Rpt6RNAi transgene had only a minor effect on apoptosis in wildtype tissue. Expression of this RNAi transgene did not enhance the rate of apoptosis in RPS23R67K heterozygotes. b, The effect of stronger proteasome knockdown (with Rpn2RNAi) on the rate of apoptosis in RP-deficient tissue could not be assessed because it triggered extensive apoptosis in otherwise wild type imaginal discs. Statistics: error bars denote standard deviation. n = 9 discs for each genotype. A two-tailed unpaired t-test was carried out. P > 0.05, no significant difference was seen. Genotypes for each figure panel are available in Supplementary Table 1. Source data is available for this figure. Source data

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Supplementary Tables 1 and 2

Supplementary Table 1: genotypes analysed. Supplementary Table 2: proteins highlighted in Fig. 3.

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Recasens-Alvarez, C., Alexandre, C., Kirkpatrick, J. et al. Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition. Nat Cell Biol 23, 127–135 (2021). https://doi.org/10.1038/s41556-020-00626-1

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