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• W3199431618 abstract "•Evolutionarily selected arginine in RPS23 is present only in hyperthermophilic archaea•RPS23 K60R mutation in flies leads to improved accuracy of protein synthesis with age•Yeast, worm, and fly RPS23 K60R mutants are longer-lived, healthier, and heat resistant•Anti-aging drugs, rapamycin, torin1, and trametinib, increase translation accuracy Loss of proteostasis is a fundamental process driving aging. Proteostasis is affected by the accuracy of translation, yet the physiological consequence of having fewer protein synthesis errors during multi-cellular organismal aging is poorly understood. Our phylogenetic analysis of RPS23, a key protein in the ribosomal decoding center, uncovered a lysine residue almost universally conserved across all domains of life, which is replaced by an arginine in a small number of hyperthermophilic archaea. When introduced into eukaryotic RPS23 homologs, this mutation leads to accurate translation, as well as heat shock resistance and longer life, in yeast, worms, and flies. Furthermore, we show that anti-aging drugs such as rapamycin, Torin1, and trametinib reduce translation errors, and that rapamycin extends further organismal longevity in RPS23 hyperaccuracy mutants. This implies a unified mode of action for diverse pharmacological anti-aging therapies. These findings pave the way for identifying novel translation accuracy interventions to improve aging. Loss of proteostasis is a fundamental process driving aging. Proteostasis is affected by the accuracy of translation, yet the physiological consequence of having fewer protein synthesis errors during multi-cellular organismal aging is poorly understood. Our phylogenetic analysis of RPS23, a key protein in the ribosomal decoding center, uncovered a lysine residue almost universally conserved across all domains of life, which is replaced by an arginine in a small number of hyperthermophilic archaea. When introduced into eukaryotic RPS23 homologs, this mutation leads to accurate translation, as well as heat shock resistance and longer life, in yeast, worms, and flies. Furthermore, we show that anti-aging drugs such as rapamycin, Torin1, and trametinib reduce translation errors, and that rapamycin extends further organismal longevity in RPS23 hyperaccuracy mutants. This implies a unified mode of action for diverse pharmacological anti-aging therapies. These findings pave the way for identifying novel translation accuracy interventions to improve aging. IntroductionIn stark contrast to the well-established effect of DNA mutations on multi-cellular organismal aging and disease (Garinis et al., 2008Garinis G.A. van der Horst G.T. Vijg J. Hoeijmakers J.H. DNA damage and ageing: new-age ideas for an age-old problem.Nat. Cell Biol. 2008; 10: 1241-1247Crossref PubMed Scopus (281) Google Scholar), 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., 2017Ke Z. Mallik P. Johnson A.B. Luna F. Nevo E. Zhang Z.D. Gladyshev V.N. Seluanov A. Gorbunova V. Translation fidelity coevolves with longevity.Aging Cell. 2017; 16: 988-993Crossref PubMed Scopus (40) Google Scholar; Kramer et al., 2010Kramer E.B. Vallabhaneni H. Mayer L.M. Farabaugh P.J. A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae.RNA. 2010; 16: 1797-1808Crossref PubMed Scopus (82) Google Scholar; Salas-Marco and Bedwell, 2005Salas-Marco J. Bedwell D.M. Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough.J. Mol. Biol. 2005; 348: 801-815Crossref PubMed Scopus (85) Google Scholar; Stansfield et al., 1998Stansfield I. Jones K.M. Herbert P. Lewendon A. Shaw W.V. Tuite M.F. Missense translation errors in Saccharomyces cerevisiae.J. Mol. Biol. 1998; 282: 13-24Crossref PubMed Scopus (60) Google Scholar). This is several orders of magnitude higher compared to DNA mutations, which are estimated at 1.4 × 10−8 per nucleotide site per generation for base substitutions in humans (Lynch et al., 2016Lynch M. Ackerman M.S. Gout J.F. Long H. Sung W. Thomas W.K. Foster P.L. Genetic drift, selection and the evolution of the mutation rate.Nat. Rev. Genet. 2016; 17: 704-714Crossref PubMed Scopus (333) Google Scholar). Proteostasis disruption is a critical factor underlying aging and age-related diseases, with translation being one of its key determinants (Hipp et al., 2019Hipp M.S. Kasturi P. Hartl F.U. The proteostasis network and its decline in ageing.Nat. Rev. Mol. Cell Biol. 2019; 20: 421-435Crossref PubMed Scopus (403) Google Scholar; Labbadia and Morimoto, 2015Labbadia J. Morimoto R.I. The biology of proteostasis in aging and disease.Annu. Rev. Biochem. 2015; 84: 435-464Crossref PubMed Scopus (718) Google Scholar; López-Otín et al., 2013López-Otín C. Blasco M.A. Partridge L. Serrano M. Kroemer G. The hallmarks of aging.Cell. 2013; 153: 1194-1217Abstract Full Text Full Text PDF PubMed Scopus (7279) Google Scholar; Steffen and Dillin, 2016Steffen K.K. Dillin A. A ribosomal perspective on proteostasis and aging.Cell Metab. 2016; 23: 1004-1012Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). 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., 1997Gallant J. Kurland C. Parker J. Holliday R. Rosenberger R. The error catastrophe theory of aging. Point counterpoint.Exp. Gerontol. 1997; 32: 333-346Crossref PubMed Scopus (25) Google Scholar), 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., 2018Anisimova A.S. Alexandrov A.I. Makarova N.E. Gladyshev V.N. Dmitriev S.E. Protein synthesis and quality control in aging.Aging (Albany N.Y.). 2018; 10: 4269-4288Crossref PubMed Scopus (57) Google Scholar). 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 (von der Haar et al., 2017von der Haar T. Leadsham J.E. Sauvadet A. Tarrant D. Adam I.S. Saromi K. Laun P. Rinnerthaler M. Breitenbach-Koller H. Breitenbach M. et al.The control of translational accuracy is a determinant of healthy ageing in yeast.Open Biol. 2017; 7: 160291Crossref PubMed Scopus (14) Google Scholar). 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., 2018Suhm T. Kaimal J.M. Dawitz H. Peselj C. Masser A.E. Hanzén S. Ambrožič M. Smialowska A. Björck M.L. Brzezinski P. et al.Mitochondrial translation efficiency controls cytoplasmic protein homeostasis.Cell Metab. 2018; 27: 1309-1322.e6Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Additionally, slowing down translation elongation by eEF2K-mediated inhibition of eEF2 resulted in improved translation fidelity in mammalian cells in vitro (Xie et al., 2019Xie J. de Souza Alves V. von der Haar T. O’Keefe L. Lenchine R.V. Jensen K.B. Liu R. Coldwell M.J. Wang X. Proud C.G. Regulation of the elongation phase of protein synthesis enhances translation accuracy and modulates lifespan.Curr. Biol. 2019; 29: 737-749.e5Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). There is tantalizing evidence from rodent cells, where a correlation exists between translation accuracy and maximum lifespan of different species (Ke et al., 2017Ke Z. Mallik P. Johnson A.B. Luna F. Nevo E. Zhang Z.D. Gladyshev V.N. Seluanov A. Gorbunova V. Translation fidelity coevolves with longevity.Aging Cell. 2017; 16: 988-993Crossref PubMed Scopus (40) Google Scholar). 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, 1969Rosset R. Gorini L. A ribosomal ambiguity mutation.J. Mol. Biol. 1969; 39: 95-112Crossref PubMed Scopus (160) Google Scholar). 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, 2005Ogle J.M. Ramakrishnan V. Structural insights into translational fidelity.Annu. Rev. Biochem. 2005; 74: 129-177Crossref PubMed Scopus (448) Google Scholar; Zaher and Green, 2009Zaher H.S. Green R. Fidelity at the molecular level: lessons from protein synthesis.Cell. 2009; 136: 746-762Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Major error contributing factors are misacylation of tRNAs and peptidyl transfer to the mismatched tRNA at the ribosomal A-site (Ogle et al., 2003Ogle J.M. Carter A.P. Ramakrishnan V. Insights into the decoding mechanism from recent ribosome structures.Trends Biochem. Sci. 2003; 28: 259-266Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar; Reynolds et al., 2010Reynolds N.M. Lazazzera B.A. Ibba M. Cellular mechanisms that control mistranslation.Nat. Rev. Microbiol. 2010; 8: 849-856Crossref PubMed Scopus (110) Google Scholar; Zaher and Green, 2009Zaher H.S. Green R. Fidelity at the molecular level: lessons from protein synthesis.Cell. 2009; 136: 746-762Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). We hypothesized that improving fidelity of protein synthesis could be an anti-aging intervention in multi-cellular 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 discussionA single substitution in the ribosomal decoding center, RPS23 K60R, reduces stop-codon readthrough translation errors and is evolutionarily conserved in certain archaeaStructural 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., 2017Loveland A.B. Demo G. Grigorieff N. Korostelev A.A. Ensemble cryo-EM elucidates the mechanism of translation fidelity.Nature. 2017; 546: 113-117Crossref PubMed Scopus (99) Google Scholar; Rodnina et al., 2017Rodnina M.V. Fischer N. Maracci C. Stark H. Ribosome dynamics during decoding.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372: 20160182Crossref PubMed Scopus (52) Google Scholar; Schmeing and Ramakrishnan, 2009Schmeing T.M. Ramakrishnan V. What recent ribosome structures have revealed about the mechanism of translation.Nature. 2009; 461: 1234-1242Crossref PubMed Scopus (506) Google Scholar). Indeed, the most well-described hyperaccuracy mutants found in E. coli contain mutations in E. coli’s RPS23 homolog S12 (Agarwal et al., 2011Agarwal D. Gregory S.T. O’Connor M. Error-prone and error-restrictive mutations affecting ribosomal protein S12.J. Mol. Biol. 2011; 410: 1-9Crossref PubMed Scopus (42) Google Scholar; Funatsu and Wittmann, 1972Funatsu G. Wittmann H.G. Ribosomal proteins. 33. Location of amino-acid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin.J. Mol. Biol. 1972; 68: 547-550Crossref PubMed Scopus (175) Google Scholar; Ogle et al., 2003Ogle J.M. Carter A.P. Ramakrishnan V. Insights into the decoding mechanism from recent ribosome structures.Trends Biochem. Sci. 2003; 28: 259-266Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar; Sharma et al., 2007Sharma D. Cukras A.R. Rogers E.J. Southworth D.R. Green R. Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome.J. Mol. Biol. 2007; 374: 1065-1076Crossref PubMed Scopus (99) Google Scholar). 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., 2010Kramer E.B. Vallabhaneni H. Mayer L.M. Farabaugh P.J. A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae.RNA. 2010; 16: 1797-1808Crossref PubMed Scopus (82) Google Scholar; Salas-Marco and Bedwell, 2005Salas-Marco J. Bedwell D.M. Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough.J. Mol. Biol. 2005; 348: 801-815Crossref PubMed Scopus (85) Google Scholar) (Figure 1D). Measurements of stop codon readthrough, which is a common type of translation error (Dunn et al., 2013Dunn J.G. Foo C.K. Belletier N.G. Gavis E.R. Weissman J.S. Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster.eLife. 2013; 2: e01179Crossref PubMed Scopus (234) Google Scholar), 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., 2017Deliu L.P. Ghosh A. Grewal S.S. Investigation of protein synthesis in Drosophila larvae using puromycin labelling.Biol. Open. 2017; 6: 1229-1234Crossref PubMed Scopus (24) Google Scholar). 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-propargyl-puromycin 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 eIF2α (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., 2011Wohlgemuth I. Pohl C. Mittelstaet J. Konevega A.L. Rodnina M.V. Evolutionary optimization of speed and accuracy of decoding on the ribosome.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2011; 366: 2979-2986Crossref PubMed Scopus (98) Google Scholar), 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 delayedNext, 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., 2016Balchin D. Hayer-Hartl M. Hartl F.U. In vivo aspects of protein folding and quality control.Science. 2016; 353: aac4354Crossref PubMed Scopus (737) Google Scholar; Drummond and Wilke, 2008Drummond D.A. Wilke C.O. Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution.Cell. 2008; 134: 341-352Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar). Interestingly, propensity for misfolding of erroneous proteins is known to be a major selective pressure driving more accurate protein synthesis (Drummond and Wilke, 2008Drummond D.A. Wilke C.O. Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution.Cell. 2008; 134: 341-352Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar). 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., 2013Pechmann S. Willmund F. Frydman J. The ribosome as a hub for protein quality control.Mol. Cell. 2013; 49: 411-421Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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., 2013Pechmann S. Willmund F. Frydman J. The ribosome as a hub for protein quality control.Mol. Cell. 2013; 49: 411-421Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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, 1984Tuite M.F. McLaughlin C.S. The effects of paromomycin on the fidelity of translation in a yeast cell-free system.Biochim. Biophys. Acta. 1984; 783: 166-170Crossref PubMed Scopus (23) Google Scholar), 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., 2005Rea S.L. Wu D. Cypser J.R. Vaupel J.W. Johnson T.E. A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans.Nat. Genet. 2005; 37: 894-898Crossref PubMed Scopus (305) Google Scholar) and endoplasmic reticulum (ER) stress (Ron and Walter, 2007Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4773) Google Scholar), respectively (Figures 2F, 2G, S4A, and S4B). Induction of Phsp-16.2::GFP, which is shown to correlate with longevity (Rea et al., 2005Rea S.L. Wu D. Cypser J.R. Vaupel J.W. Johnson T.E. A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans.Nat. Genet. 2005; 37: 894-898Crossref PubMed Scopus (305) Google Scholar), 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 dose-dependent 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.Figure 2The RPS23 K60R mutants in S. pombe, C. elegans, and Drosophila have enhanced thermotolerance and are developmentally delayedShow full caption(A) Archaea with arginine (R) instead of lysine (K) in the highly conserved KQPNSA region of RPS23 have higher optimal temperatures (p < 0.0001; two-tailed unpaired t test; K variants, n = 118; R variants, n = 55). Optimal growth temperatures extrapolated from in vitro culture measurements of population doubling rates at different temperatures. Data for K and R archaea were obtained from the literature (Table S1).(B) S. pombe RPS23 K60R mutant is heat shock resistant. Ten-fold serial dilutions of overnight cultures spotted and heat stressed at 39°C.(C) The RPS23 K60R mutation significantly protects C. elegans against the effects of heat shock at 37°C. The survival plot shows the combined survival recovery after heat shock stress of three independent biological replicates (total, n = 153 for wild-type; n = 160 for RPS23 K60R; log-rank test, p < 0.0001).(D) Fly RPS23 K60R mutants are heat shock resistant (39°C; n = 100 for wild-type and RPS23 K60R; log-rank test, p < 0.0001; representative of three independent trials).(E) Paromomycin reduces worm survival upon heat shock stress at 37°C. The survival plot shows the combined survival recovery after stress of three independent biological replicates (n = 247 for wild-type control and n = 244 for wild-type pre-treated with 2 mM paromomycin; log-rank test, p < 0.0001).(F) The RPS23 K60R mutation increases the heat shock response measured by Phsp-16.2::GFP upon heat stress. Each image panel on the left shows 10 individual anesthetized worms. Each condition on the right represents 3 independent biological replicates with a total of 33–40 worms. Two-way ANOVA with Tukey’s multiple comparison test, p < 0.0001.(G) An RPS23 K60R mutation significantly protects against the effects of paromomycin on UPRER activation. Each image panel shows 10 individual anesthetized worms. Each condition on the right represents 3 independent biological replicates with a total of 35–50 worms. Two-way ANOVA with Tukey’s multiple comparison test, p = 0.0227 and p < 0.0001.(H) Decreased growth and smaller colonies of the RPS23 K60R S. pombe mutant grown at optimal 32°C. Represented are 10-fold serial dilutions spotted on a YES media plate.(I) Representative growth profiles in microfermentator of RPS23 K60R S. pombe mutant compared to control at 32°C. Light and darker colored curves represent two independent biological repeats.(J) Developmental delay of worms with RPS23 K60R mutation. Percentage of animals at defined developmental stages is shown at defined times post parental egg lay. L1–L4 development stages; YA, young adults; GA, gravid adults. Each condition represents 3 independent biological replicates with a total of 50–54 worms.(K) RPS23 K60R mutant flies are developmentally delayed. Wild type, n = 18 vials; RPS23 K60R, n = 15 vials.∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; n.s., not significant; mean ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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., 2007Marygold S.J. Roote J. Reuter G. Lambertsson A. Ashburner M. Millburn G.H. Harrison P.M. Yu Z. Kenmochi N. Kaufman T.C. et al.The ribosomal protein genes and Minute loci of Drosophila melanogaster.Genome Biol. 2007; 8: R216Crossref PubMed Scopus (244) Google Scholar). 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 healthCollapse of proteostasis is often linked to aging and represents one of its hallmarks (Labbadia and Morimoto, 2015Labbadia J. Morimoto R.I. The biology of proteostasis in aging and disease.Annu. Rev. Biochem. 2015; 84: 435-464Crossref PubMed Scopus (718) Google Scholar; López-Otín et al., 2013López-Otín C. Blasco M.A. Partridge L. Serrano M. Kroemer G. The hallmarks of aging.Cell. 2013; 153: 1194-1217Abstract Full Text Full Text PDF PubMed Scopus (7279) Google Scholar). Therefore, we asked if increased translation fidelity could promote longer life in both single and multi-cellular organisms. Notably, we observed a lifespa" @default.
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• W3199431618 title "Increased fidelity of protein synthesis extends lifespan" @default.
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