INTRODUCTION

      In stark contrast to the well-established effect of DNA mutations on multi-cellular organismal aging and disease (Garinis et al., 2008), the role of translation errors is far less studied and understood. This is despite mistranslation being the most erroneous step in gene expression. The frequency of protein errors is estimated at 10
3 to 10
6 , depending on the organism and codon (Ke et al., 2017; Kramer et al., 2010; Salas-Marco and Bedwell, 2005; Stansfield et al., 1998). This is several orders of magnitude higher compared to DNA mutations, which are estimated at 1.4 3 10
8 per nucleotide site per generation for base substitutions in humans (Lynch et al., 2016). Proteostasis disruption is a critical factor underlying aging and age-related diseases, with translation being one of its key determinants (Hipp et al., 2019; Labbadia and Morimoto, 2015; Lo´pez-Otı´n et al., 2013; Steffen and Dillin, 2016). Therefore, an improved understanding of the biological impact of translation errors in the context of organismal aging is very much needed. The role of protein errors in aging was heavily debated in the past (Gallant et al., 1997), mostly due to the lack of causal evidence linking this mechanism to organismal aging. To date, evidence linking translation fidelity and aging is correlative in mammals, and evidence that translation errors are detrimental for aging is exclusively based on single-cell organisms (Anisimova et al., 2018). Recently, the connection between translation fidelity and aging was shown in Saccharomyces cerevisiae, where error-prone or ribosomal ambiguity mutants (ram) with a point mutation in Rps2 (Rps2 Y143C and L148S) have a shorter chronological lifespan (vonder Haar et al., 2017). Similarly, a hypoaccurate mutant in mitochondrial ribosomes of yeast S12 (MRPS12 P50R) has a shorter lifespan, while a hyperaccuracy mutant (MRPS12 K71T) shows extended lifespan and improved cytosolic proteostasis (Suhm et al., 2018). Additionally, slowing down translation elongation by eEF2K-mediated inhibition of eEF2 resulted in improved translation fidelity in mammalian cells in vitro (Xie et al., 2019). There is tantalizing evidence from rodent cells, where a correlation exists between translation accuracy and maximum lifespan of different species (Ke et al., 2017). However, translation errors are rarely investigated in the context of multi-cellular organismal physiology, and their effect on aging of metazoan organisms remains unexplored (Rosset and Gorini, 1969). In addition, how to modulate fidelity of protein synthesis to increase lifespan in multi-cellular organisms has not been investigated.

      Decoding by the ribosomal accuracy center dictates translation fidelity and is separated into two steps. During the initial tRNA selection, cognate aminoacyl-tRNAs induce domain closure in the small ribosomal subunit, leading to the activation of EF-Tu/EF1A for GTP hydrolysis. In a subsequent proofreading step, the correct aminoacyl-tRNA is inserted into the peptidyl transferase center (Ogle and Ramakrishnan, 2005; Zaher and Green, 2009). Major error contributing factors are misacylation of tRNAs and peptidyl transfer to the mismatched tRNA at the ribosomal A-site (Ogle et al., 2003; Reynolds et al., 2010; Zaher and Green, 2009). We hypothesized that improving fidelity of protein synthesis could be an anti-aging intervention in multicellular organisms. Here, we investigated the physiological consequences of directly mutating a single evolutionarily conserved residue in the decoding center of the ribosome and examined for the first time in metazoan species the effect of increased protein synthesis fidelity on aging.

RESULTS AND DISCUSSION

A single substitution in the ribosomal decoding center, RPS23 K60R, reduces stop-codon readthrough translation errors and is evolutionarily conserved in certain archaea

      Structural studies of the ribosomal decoding center in evolutionarily distant organisms point to the importance of the RPS23 protein for translation accuracy due to its role in domain closure and insertion of the aminoacyl-tRNA into the peptidyl transferase center (Figures 1A, 1B, and S1A–S1C) (Loveland et al., 2017; Rodnina et al., 2017; Schmeing and Ramakrishnan, 2009). Indeed, the most well-described hyperaccuracy mutants found in E. coli contain mutations in E. coli’s RPS23 homolog S12 (Agarwal et al., 2011; Funatsu and Wittmann, 1972; Ogle et al., 2003; Sharma et al., 2007). Therefore, we performed an extensive unbiased phylogenetic analysis of RPS23 in organisms ranging from archaea to eukaryotes, using different databases (see STAR Methods for details), and we have consistently found a lysine residue to be remarkably conserved in the KQPNSA region of ribosomal RPS23, nearly invariant throughout evolution. The only exceptions to this rule are in the thermophilic and hyperthermophilic archea, where the amino acid lysine is replaced by arginine, an event that likely occurred three times independently during evolution (Figures 1C, S1D, and S2A). Analyses of key archaeal characteristics showed that this rare arginine is predominant in archaea that live in extreme conditions such as higher temperatures and acidic environments and that metabolize sulfur. Instead, aerobic and anaerobic metabolism did not discriminate between organisms possessing arginine or lysine in the decoding center (Figures S2B and S2C; Table S1). Moreover, we found that the lysine (K)-to-arginine (R) substitution is an isolated change in RPS23 in this group of organisms, since other regions of the protein are similarly conserved throughout the protein sequence. Therefore, to evaluate the effect of this mutation in higher organisms we focused on this K-R substitution of RPS23 because of its evolutionary presence.

      To investigate the link between this single site alteration and translation accuracy, we used CRISPR/Cas9 to introduce a K60R mutation in the KQPNSA region of Drosophila rps23 (Figures S3A and S3B). To measure translation errors in vivo, we created a dual luciferase reporter construct in flies, based on detailed translational studies and accuracy reporters in yeast (Kramer et al., 2010; Salas-Marco and Bedwell, 2005) (Figure 1D). Measurements of stop codon readthrough, which is a common type of translation error (Dunn et al., 2013), showed that in the old RPS23 K60R flies translation accuracy was improved compared to controls (Figure 1E). We also observed that this type of error significantly increased during aging in controls flies, but not in RPS23 K60R mutant (Figure 1E). For the less prevalent misincorporation errors, we did not observe a significant difference between control and RPS23 K60R mutant young flies or old control flies (Figures S3C and S3D), and only a minor significant increase in aged RPS23 K60R flies (Figures S3C and S3D). Thus, unlike stop codon readthrough, misincorporation errors were less frequent and did not increase with age, suggesting that the K60R mutation specifically mitigates age-related translation errors (Figures 1E and S3D).

     To investigate the role of the hyperaccuracy mutation in translation rates in evolutionarily distant organisms in addition to Drosophila, we introduced the RPS23 K60R mutation in both Schizosaccharomyces pombe and Caenorhabditis elegans using standard genetic techniques and CRISPR/Cas9, respectively. Next, we measured protein synthesis rates using puromycin, an aminoacyl-tRNA analog that terminates translation and enables detection of nascent polypeptides (Deliu et al., 2017). In yeast, the RPS23 K60R mutation reduced protein translation in a growth phase-dependent manner, with less pronounced effects observed during stationary growth (Figure 1F) compared to exponential growth (Figure S3E). In contrast, puromycin incorporation tests in young adult C. elegans showed that the RPS23 K60R mutation did not alter translation (Figure 1G). Similarly, in vivo measurements in adult flies showed that global protein synthesis was not affected in RPS23 K60R mutants (Figure 1H). To test if the hyperaccuracy mutation affects translation in rapidly growing and dividing tissues with high protein synthesis demand, we measured translation in the fly larval tissue. To this end, we generated mosaic larval wing imaginal discs. Side-by-side comparison of puromycilated peptides in control and RPS23 K60R heterozygote and homozygote mutant clones in the same tissue clearly showed no alteration in O-propargylpuromycin incorporation, further suggesting no difference in translation in flies (Figures 1I, S3F, and S3G). Also, the generated mutant clones were of similar size compared to wild-type clones (Figure S3F), showing this ribosomal mutation does not change competitive growth of the cell. These data suggest that the effect of this mutation on decreasing protein synthesis is observable only in single-cell organisms and is not present in multi-cellular metazoans. To exclude non-specific effects on protein translation as a result of the introduction of this genetic modification, we verified that rps23 gene and protein expression levels remained unaltered in the K60R mutant flies compared to control (Figures S3H and S3I). Finally, we examined additional readouts of altered protein synthesis in flies. We observed no changes between RPS23 K60R mutant and control flies for markers such as phosphorylation of eIF2a (Figure S3J). Similarly, no changes were detected for pS6K or p4E-BP, the downstream effectors of the major regulator of translation mTOR (Figures S3K and S3L).

      Overall, we observed a specific reduction of errors in stop codon readthrough in the mutant without an alteration in translation levels between wild-type and RPS23 K60R mutants (Figures 1E and 1H). These findings suggest the translation machinery can accommodate improvements in accuracy without global translation being affected. Given the previously suggested trade-off between translation speed and accuracy (Wohlgemuth et al., 2011), it is interesting that the only hyperaccurate mutation naturally selected by evolution does not impair global translation in metazoans.

RPS23 K60R mutants in yeast, worms, and flies are heat stress resistant and developmentally delayed

      Next, we sought to investigate the physiological consequences of this mutation. Elevated temperatures and errors in translation are major risk factors for protein misfolding (Balchin et al., 2016; Drummond and Wilke, 2008). Interestingly, propensity for misfolding of erroneous proteins is known to be a major selective pressure driving more accurate protein synthesis (Drummond and Wilke, 2008). Incorporation of erroneous amino acids, particularly in the catalytic site of a protein, could lead to detrimental consequences, and errors in proteins can impose additional energy requirements for folding or protein degradation (Pechmann et al., 2013). Erroneous and misfolded proteins are more prone to damage and aggregation, leading to diminished cellular proteostasis and sensitivity to further insults such as heat stress (Pechmann et al., 2013). This suggests that hyperaccuracy mutants could be more resilient to heat shock. Consistent with this hypothesis, archaea that possess R grow significantly better at higher temperatures than archaea with K (Figure 2A). To probe this hypothesis further, we measured heat stress resistance in all three organisms possessing the RPS23 K60R mutation. Indeed, we observed that the RPS23 K60R mutation resulted in significantly improved survival under heat stress in yeast, worms, and flies, reflecting their improved proteostatic capacity (Figures 2B–2D). Consistent with this interpretation, paromomycin treatment, which increases the error rate in ribosomal translation (Tuite and McLaughlin, 1984), made worms more sensitive to heat shock insult (Figure 2E). To understand the link between translation errors and heat shock response, we used the transcriptional reporters Phsp-16.2::GFP and Phsp-4::GFP for heat shock (Rea et al., 2005) and endoplasmic reticulum (ER) stress (Ron and Walter, 2007), respectively (Figures 2F, 2G, S4A, and S4B). Induction of Phsp-16.2::GFP, which is shown to correlate with longevity (Rea et al., 2005), was more pronounced in RPS23 K60R mutants than in controls upon heat shock, likely contributing to their heat shock resilience (Figure 2C). Further, consistent with the role of paromomycin in specifically producing translation errors, we observed a dosedependent activation of the ER stress reporter Phsp-4::GFP (Figure S4A) to greater levels than induced by heat shock treatment (Figure S4B). Importantly, the K60R mutation significantly protected against ER stress induced by both paromomycin treatment (Figure 2G) and heat shock stress (Figure S4B), suggesting that this ribosomal mutant is protected from insults inducing high levels of proteotoxic stress.

      Given these results, we asked why this mutation had not evolved more frequently in nature, given its potential benefit to maintaining a more accurate proteome and making organisms heat stress resilient. A possible explanation could be the existence of negative trade-offs. In agreement with our hypothesis, the RPS23 K60R mutant in S. pombe forms smaller colonies (Figure 2H) and shows growth retardation in liquid media (Figure 2I). Similarly, C. elegans RPS23 K60R mutants develop slower compared to wild-type controls (Figures 2J and S4C–S4E), have the same size at the last larval L4 stage, are smaller during the reproductive period than day 1 adults (Figures S4E–S4H), and are bigger at the end of the reproductive phase (Figure S4E). In addition, an exhaustive set of measurements of worm behavior, consisting of 2,090 behavioral features, showed that the RPS23 K60R mutation decreases worm size-related features in young day 1 adults, but not other behavioral traits (Figures S4F–S4L; Table S2). Consistent with data from both yeast and worms, Drosophila RPS23 K60R mutants were approximately 1 day delayed in eclosing (Figure 2K) and showed delay in pupariation, but the number of flies eclosing was unaffected (Figures S5A and S5B). Additionally, RPS23 K60R flies possess shorter  bristles (Figures S5C and S5D) and smaller wings (Figure S5E) and present a very subtle Minute phenotype (Marygold et al., 2007). Overall, these developmental data may explain the presence of the R residue in organisms that live only in extreme conditions for which increased translation fidelity is a strong selective pressure.

RPS23 K60R is the first metazoan hyperaccuracy mutation that increases lifespan and promotes health

     Collapse of proteostasis is often linked to aging and represents one of its hallmarks (Labbadia and Morimoto, 2015; Lo´pezOtı´n et al., 2013). Therefore, we asked if increased translation fidelity could promote longer life in both single and multi-cellular organisms. Notably, we observed a lifespan extension in RPS23 K60R mutants in yeast, worms, and flies (Figures 3A–3C). The lifespan extension mediated by this single point mutation was 9%–23% in all repeated assays, including Drosophila mutants bearing luciferase reporter constructs (Figures 3A–3C and S5F–S5H). In C. elegans, the lifespan extension of the RPS23 K60R mutant was equally robust regardless of the bacterial diet. Similar effects on lifespan were observed when worms were grown on standard bacterial food OP50 (Figure 3B) or a relative E. coli K-12 BW25113 strain (Figure S5H). Downregulation of translation has a well-established lifespan extension effect (Hansen et al., 2007). Here, by minimally altering the decoding center, we uncoupled increased accuracy from translation downregulation, thereby providing a novel anti-aging intervention.

      Long-lived mutant organisms are often healthier with age compared to controls (Kenyon, 2010). To test this premise, we measured fly health during aging using a negative geotaxis or climbing assay. Our data show an overall decline in climbing capacity over time. Importantly, we observed improved climbing of RPS23 K60R flies compared to controls, suggesting healthier aging (Figure 3D). Reduced fecundity is a negative side effect of many long-lived IIS and mTOR mutants (Lo´pez-Otı´n et al., 2013), some of which become sterile (Clancy et al., 2001). Also, reproductive senescence is an additional characteristic of aging (Wang et al., 2014). Interestingly, in RPS23 K60R mutant worms we observed fewer progeny being produced at day 1 and 2; however, there was a delayed reproductive decline at day 3 and 4 when worms produced more progeny compared to controls (Figure 3E). Similarly, RPS23 K60R mutant flies also showed slower reproductive decline and produced more eggs at day 42 and day 49, further supporting the idea that adult RPS23 K60R mutants are healthier organisms with age compared to controls (Figure 3F). RPS23 K60R mutation did not affect total number of progeny produced by worms (Figure S5I) or cumulative eggs per fly (Figure S5J). In summary, the ribosomal RPS23 K60R mutation led to improved heat stress resistance and lifespan extension in an evolutionarily diverse range of organisms. This critically highlights the impact of translation fidelity on aging. It reveals for the first time that a direct improvement in translation accuracy by a single amino acid substitution borne out from evolution in the ribosome decoding center extends metazoan lifespan.

Pharmacological anti-aging interventions, rapamycin, Torin1, and trametinib, reduce translation errors

      Great interest in the biology of aging stems from a possibility to improve health in the elderly by mimicking the effect of longevity mutations on organismal physiology through pharmacological approaches (Campisi et al., 2019; Partridge et al., 2018). Interestingly, it was shown that one of the most well-studied anti-aging drugs, the mTOR inhibitor rapamycin (Johnson et al., 2013), reduces errors in translation in mammalian cells in vitro (Conn and Qian, 2013; Xie et al., 2019). We explored whether other anti-aging drugs have similar effects on improving translation fidelity. To this end, we adapted our in vivo reporters for common translation errors, stop codon readthrough, and amino acid misincorporation for Drosophila S2R+ cells (Figures 4A–4J). We validated our reporter systems using the drug paromomycin, which induces translation errors, and observed a dose-dependent increase in errors (Figures 4B and 4G). We showed that, similar to mammalian studies (Conn and Qian, 2013; Xie et al., 2019), rapamycin improved translation fidelity in Drosophila S2R+ cells and lowered both stop codon readthrough (Figure 4C) and misincorporation errors (Figure 4H). While the effect of the selective mTORC1 inhibitor rapamycin on aging is extensively studied, effects of dual mTORC1 and mTORC2 catalytic inhibitors are not well explored. We therefore tested the effect of Torin1 on aging. We found that it extends lifespan in Drosophila (Figure S6A) (Mason et al., 2018) and, like rapamycin, improves translation fidelity for both types of translation errors (Figures 4D and 4I). Subsequently, we tested trametinib, an MEK/ERK pathway inhibitor, which regulates translation via p90 ribosomal S6 kinase (RSK)-mediated phosphorylation of RPS6 (Roux et al., 2007) and extends lifespan in flies (Slack et al., 2015). Trametinib also improved translation fidelity (Figures 4E and 4J). These findings suggest a novel unifying component in the mechanism underlying anti-aging therapies based on improving translation fidelity. To explore this idea, we tested if the lifespan of RPS23 K60R mutant could be further extended by these pharmacological anti-aging interventions and treated the RPS23 K60R mutant yeast, worms, and flies with rapamycin (Figures 4K–4M). Rapamycin extended lifespan of the wild-type yeast and flies, and to a lesser extent of the long-lived RPS23 K60R mutants, leading to their similar longevity in presence of rapamycin (Figures 4K and 4M). Our results agree with the mechanism of lifespan extension by rapamycin, which is multifactorial and dependent on increased autophagy and lower pS6K (Bjedov et al., 2010) and polIII (Filer et al., 2017). In C. elegans, rapamycin extended wild-type lifespan but did not increase the longevity of RPS23 K60R mutant worms further (Figures 4L and S6B). These data suggest a potential higher dependence of worm lifespan on protein fidelity. Overall, the epistasis analysis obtained from the three organisms indicates that when translation accuracy is increased, the capacity of rapamycin to extend lifespan is likely limited to its remaining organism-specific anti-aging components.

Conclusions

      Aging in isolated cultured cells has been studied in the past, but no correlation between errors and aging was found in this context (Anisimova et al., 2018), perhaps owing to insufficient sensitivity of error measurement methods available at the time. In addition, it was shown in yeast that increasing translational errors accelerates loss of viability in yeast (von der Haar et al., 2017). In the context of organismal aging, our data imply that reducing translation errors is an effective strategy for increasing health span. Significantly, these findings add another dimension to our current understanding of the mechanisms of aging, where DNA lesions are often considered a major culprit (Garinis et al., 2008; Tian et al., 2019).

      Translation errors, similar to DNA mutations (Mao et al., 1997), may have an adaptive role under stressful conditions (Ribas de Pouplana et al., 2014). Translation accuracy has possibly been selected by evolution to be optimal for adequate cell functioning while enabling rapid, competitive growth and maximal fitness when the environment is favorable (Figure 4N). A strong selection pressure for improved translation accuracy is driven by protein errors that cause protein misfolding (Drummond and Wilke, 2008). Consistent with this view, we show that the naturally occurring RPS23 K60R hyperaccuracy mutation only appears in certain thermophilic and hyperthermophilic archaea, where protein folding needs to occur in physiologically demanding conditions. In contrast, for all other organisms, rapid growth and reproduction are more dominant selective pressures. Therefore, we propose that accurate but developmentally delayed RPS23 K60R mutant organisms would be rapidly outcompeted in the wild.

      Historically, there was a general interest in mutations of ribosomal decoding center in single-cell organisms. This particular mutation was previously described in the laboratory settings as hyperaccurate in E. coli (Funatsu and Wittmann, 1972) and hypoaccurate in S. cerevisiae (Alksne et al., 1993). Here, we show that this single highly conserved amino acid replacement in the decoding center is sufficient to decrease stop codon readthrough errors. Decoding is a complex process, and biochemical and structural studies demonstrate that translation fidelity is affected by interactions and conformational changes of all its interacting partners, the tRNA, rRNA, mRNA, and ribosomes (Zaher and Green, 2009). In an E. coli hyperaccuracy mutant rpsL141 strain, where an equivalent lysine residue is replaced by asparagine, translation fidelity is mediated through a proofreading step of the tRNA selection process (Zaher and Green, 2010). In our mutant, lysine is replaced with a larger arginine, which provides more stable ionic interactions due to its asymmetrical nitrogen atoms in the guanidium group (Sokalingam et al., 2012). Although this arginine does not directly interact with tRNA or mRNA, rearrangements in the 18s rRNA could propagate to the decoding center. Arginine-induced changes in the rRNA structure could affect the position of mRNA and its interaction with tRNA, leading to increased translation accuracy in the RPS23 K60R mutant. With regard to the mRNA, both its ribose backbone, which is critical for tRNA interactions (Ogle et al., 2001), and phosphodiester bond influencing the mRNA kink structure at the interface of the P and A sites have been shown to affect fidelity of translation (Keedy et al., 2018). Like in bacterial hyperaccuracy mutants, the K60R substitution may disrupt interactions necessary for the closed ribosome conformation (Ogle et al., 2002). Such improvements in translation accuracy lead to advantageous phenotypes including a robust lifespan extension. These effects are observed across taxa, which include multicellular organisms such as worms and flies as well as single-cell organisms such as S. pombe fission yeast. Altogether, this suggests the importance of diverse factors such as genetic architecture and environmental conditions in shaping optimal translation accuracy levels. Further exploration of the role played by ribosomal accuracy mutations during healthy aging in diverse biological contexts will be vital to understand its function.

      Reduced protein synthesis, either by downregulation of initiation factors or ribosomal proteins (Hansen et al., 2007; Steffen et al., 2008), is a well-established anti-aging intervention. The proposed underlying longevity mechanisms include differential translation (Rogers et al., 2011; Zid et al., 2009), increased Gcn4/Atf4 (Steffen et al., 2008), as well as reduced energy burden to the folding and degradation machinery (Anisimova et al., 2018). Translation can be downregulated and altered during stress in order to allow for production of selected set of proteins (Pizzinga et al., 2020), which could potentially have some shared mechanisms with longevity processes. Despite the reported trade-off between translation efficiency and accuracy (Wohlgemuth et al., 2011), the RPS23 K60R mutation in metazoans improved age-related readthrough accuracy without reducing translation, making our findings distinct from previously reported translation-related longevity mechanisms that are all based on translation downregulation (Anisimova et al., 2018).

      A single constitutively expressed misfolding-prone protein is sufficient to compromise the entire cellular proteostasis (Gidalevitz et al., 2006), and reduced translation fidelity through defective editing domain of the tRNA synthetase can cause protein misfolding and neurodegeneration (Lee et al., 2006). Our work demonstrates that increased translation accuracy can be achieved pharmacologically and argues for screening of compounds with the potential to reduce protein errors during aging. Collectively, these findings advocate for the investigation of therapies aiming at increasing translation fidelity in the context of aging and age-related diseases, particularly neurodegenerative diseases that are primarily affected by deterioration of proteostasis (Lee et al., 2006).

Limitations of study

      Our work draws attention to translation accuracy and demonstrates that having fewer protein errors is beneficial for an organism’s resilience to heat stress and longevity. Yet despite careful characterization of hyperaccurate ribosomal mutants in different organisms, it remains to be determined whether these mechanisms are conserved in mammals.

      We measured both stop codon and misincorporation errors using the most widely used dual luciferase reporters (Kramer et al., 2010; Salas-Marco and Bedwell, 2005). However, these methods only capture two of the most frequent ribosomal errors. Additional reporters covering a variety of different codons could provide for an in-depth characterization of error prevention conferred by RPS23 K60R ribosomal mutation in diverse physiological contexts. In addition, despite some predictions of the molecular mechanism, based on available ribosomal structures (Keedy et al., 2018; Loveland et al., 2017; Ogle and Ramakrishnan, 2005; Rodnina et al., 2017; Schmeing and Ramakrishnan, 2009; Zaher and Green, 2009), the exact molecular changes in RPS23 K60R mutant ribosomes leading to translation alterations are still elusive and await further investigation.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

● KEY RESOURCES TABLE

● RESOURCE AVAILABILITY

 Lead contact

 Materials availability

 Data and code availability

● EXPERIMENTAL MODEL AND SUBJECT DETAILS

 Yeast strains

 Worm strains

 Fly strains

 Yeast growth condition

 Worm growth condition

 Fly growth condition

 S2R+ cells growth condition

● METHOD DETAILS

 Structural modeling

 Phylogenetic analysis

 Generation of RPS23 K60R mutant in S. pombe

 S. pombe growth assay

 S. pombe chronological lifespan assay

 S. pombe heat shock assay

 Translation measurement using puromycin incorporation assay and western blot in S. pombe

 Worm RPS23 K60R mutant strain generation

 Worm development assays

 Worm transgenic reporter assays

 Worm reproductive assays

 Worm heat shock survival assays

 Worm behavioral assays

 Worm lifespan assays

 Translation measurements in worms using surface sensing of translation (SUnSET) assay

 CRISPR/Cas9 in Drosophila

 Backcrossing of the Drosophila RPS23 K60R mutant

 Drosophila stocks

 Drosophila longevity assays

 Western blot measurements in Drosophila

 Development time in Drosophila

 Negative geotaxis or climbing assay in Drosophila

 Heat shock stress assays in Drosophila

 Fecundity assays in Drosophila

 RNA extraction, cDNA, RT-qPCR in Drosophila

 Relative translation rates in adult Drosophila

 Generation of clones by FLP-FRT-mediated mitotic recombination in Drosophila larvae

 Translation measurements by puromycin incorporation assay in Drosophila larval wing imaginal discs

 Translation fidelity dual luciferase assays for in vivo in Drosophila

 Translation fidelity dual luciferase assays for in vitro

Drosophila S2R+ cells

● QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j. cmet.2021.08.017.

ACKNOWLEDGMENTS

      We would like to thank the Bedwell laboratory, the Baum laboratory, the Kimata laboratory, and Addgene for plasmids. We are grateful to Marc Amoyel, Diego Sainz de la Maza Redondo, Jean Paul Vincent, and Carles Recasens Alvarez for fly stocks, reagents, and protocols. We are grateful to Helena Cocheme for help with figure preparation and to Ismail Moghul for advice with data analysis. We thank all laboratory members for helpful discussions. I.B. acknowledges funding from ERC StG 311331, ERC PoC 842174, CRUK-UCL Centre Award (C416/A25145), Radiation Research Unit at the Cancer Research UK City of London Centre Award (C7893/A28990), Royal Society Research Grant (RSGyR1y180431), and the Bill Lyons Foundation. F.C. is supported by the Wellcome Trust/Royal Society (Sir Henry Dale Fellowship 102532/Z/12/Z and 102531/Z/13/A) and MRC (MC-A654-5QC80). T.v.d.H.’s work is supported by the Wellcome Trust collaborative award 201487. S.G. is supported by a UEL PhD studentship and QR funding awarded to C.R. C.R. also acknowledges funding from the Royal Society (Research Award grant number RGSyR1y201348) and BBSRC (grant number BB/V006916/1). M.H.’s work is funded by St. Jude Children’s Research Hospital, the American Lebanese Syrian Associated Charities, and NIH award 1R01GM135599-01.

AUTHOR CONTRIBUTIONS

      V.E.M.-M. performed experiments, analyzed data, helped write the manuscript, and provided intellectual input. C.L., S.G., E.R.G., and C.B. performed experiments and analyzed data. T.E.–C. and D.M.-M. performed phylogenetic and literature analysis. S.M. and A.E.X.B. performed behavioral data analysis. M.H. performed structural modeling and helped write the manuscript. K.T. performed experiments and helped with experimental design. C.R. performed experiments, analyzed data, and helped write the manuscript. T.v.d.H. provided intellectual input, helped design experiments, and helped write the manuscript. F.C. contributed to experiments, provided intellectual input, secured funding, and wrote the manuscript. I.B. conceived the project, performed experiments, secured funding, and wrote the manuscript. All authors discussed the results and commented on the manuscript.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: August 4, 2020

Revised: May 6, 2021

Accepted: August 30, 2021

Published: September 14, 2021

This article is excerpted from the Cell Metabolism 33, 2288–2300, November 2, 2021  by Wound World.