Increased fidelity of protein synthesis extends lifespan

Abstract

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.

Keywords: RPS23; aging; archaea; mTOR; protein synthesis; proteostasis; ribosome; translation; translation accuracy; translation fidelity.

Graphical abstract

Figure 1

A mutation in the RPS23 (uS12) of the ribosomal decoding center, present in certain thermophilic and hyperthermophilic archaea, improves translation accuracy when introduced to Drosophila (RPS23 K60R) (A) Structure of 80S ribosome from rabbit (Oryctolagus cuniculus) (Juszkiewicz et al., 2018). (B) A close-up view of the decoding center showing RPS23, lysine residue RPS23 K60, tRNA, and mRNA. (C) Phylogenetic tree of the RPS23 protein sequences from Archaea and Eukarya domains, without branch lengths. Escherichia coli is used as outgroup of the tree. The different colors in the outside ring represent the three domains of life, Bacteria, Archaea, and Eukarya. The organism name color denotes amino acid variation of lysine of the conserved KQPNSA region of the RPS23; the organisms in blue have K (lysine) and in orange have R (arginine) residue. The phyla of the organisms with the R variation are represented in the orange outer ring. R variation is the only evolutionarily selected K alternative and is found in some Archaea. (D) Schematic representation of dual luciferase reporters used to assay translation errors in Drosophila in vivo. (E) Translation fidelity measurements in vivo in young (10-day-old) and old (60-day-old) Drosophila. Stop codon readthrough errors in wild-type flies increase with age (p < 0.0001; one-way ANOVA, Tukey’s post hoc test). Fewer STOP codon readthrough errors in old RPS23 K60R mutants versus old wild-type flies (p < 0.0001; one-way ANOVA, Tukey’s post hoc test). No change in stop codon readthrough between young and old RPS23 K60R flies (p = 0.7653; one-way ANOVA, Tukey’s post hoc test). Wild-type young, n = 24; wild-type old, n = 12; RPS23 K60R young, n = 42; RPS23 K60R old, n = 18; from two independent experiments. (F) Translation measurements by ex vivo puromycin incorporation and western blotting in S. pombe RPS23 K60R mutants and controls during stationary phase (p = 0.011; two-tailed unpaired t tests; n = 6). Quantification of anti-puromycin levels over anti-actin is presented. (G) RPS23 K60R worm mutants do not differ in translation rates compared to N2 controls (p = 0.502; two-tailed paired t test; n = 4). Representative blot of control and puromycin-treated young adult wild-type and RPS23 K60R mutant worms. Quantification of anti-puromycin levels over anti-tubulin. (H) The level of de novo protein synthesis was not altered in adult flies with the RPS23 K60R mutation (p = 0.54; two-tailed unpaired t test; n = 16). Quantification of anti-puromycin levels over anti-H3 is presented. (I) Wing imaginal discs in which anterior compartment consists of cells heterozygous for RPS23 K60R mutation, while the posterior compartment consists of wild-type and RPS23 K60R homozygote clones. O-propargyl-puromycin incorporation comparison among genetically different clones shows that RPS23 K60R mutation does not affect translation. hh-GAL4 > UAS-flp stands for hedgehog-GAL4 driven flipase. A, anterior; P, posterior. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001; n.s., not significant; mean ± SEM.

Figure 2

The RPS23 K60R mutants in S. pombe, C. elegans, and Drosophila have enhanced thermotolerance and are developmentally delayed (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 UPR^ER 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.

Figure 3

Yeast, worms, and flies with an RPS23 K60R mutation live longer and are healthier (A) S. pombe chronological lifespan analysis shows RPS23 K60R mutants live longer compared to controls (p < 0.001; log-rank test). (B) Lifespan analyses showing all three C. elegans CRISPR-Cas9 RPS23 K60R lines are longer lived compared to wild-type controls (p < 0.0001; log-rank test; n = 99–144). (C) Drosophila lifespan analyses of three independent CRISPR-Cas9 RPS23 K60R lines show they are longer lived compared to both wild-type controls (p < 0.0001; log-rank; n∼150). (D) The RPS23 K60R flies show delayed senescence of negative geotaxis or climbing during aging (two-way ANOVA with Sidak’s multiple comparison test for weeks 1–5; p = 0.3378, p = 0.0051, p = 0.0513, p = 0.001 and p = 0.6409, respectively; n = 10 vials each containing 15 flies). (E) Measurements of the production of live progeny per worm show delayed fertility patterns in RPS23 K60R mutants compared to controls. Multiple unpaired t tests with false discovery rate (FDR) were applied: day 1, q = 0.000014; day 2, q = 0.003167; day 3, q = 0.000237; day 4, q = 0.0017783; day 5, q = 0.049286; RPS23 K60R, n = 12; wild type, n = 10. (F) Fly fecundity measurements for RPS23 K60R mutants and control flies (day 35, q = 0.112219; day 42, q = 0.00002; day 49, q = 0.000049; multiple unpaired t tests with FDR were applied; n = 10 vials of 15 flies). (D and E) Data shown as mean ± SEM.

Figure 4

Anti-aging drugs reduce translation errors in Drosophila S2R+ cells (A) A scheme of the dual luciferase reporter used to measure stop codon readthrough. (B) The reporter was validated by treating the Drosophila S2R+ cells with the error-inducing drug paromomycin (p = 0.0041 and p < 0.0001; control [n = 8] compared to 500 and 1,000 μM paromomycin [n = 6]; one-way ANOVA, Tukey’s post hoc test). (C) Rapamycin-treated S2R+ cells have fewer stop codon readthrough translational errors compared to control cells treated with the respective solvent carrier ethanol (EtOH) (p = 0.0349 and p = 0.0082 for 10 and 100 nM rapamycin versus control; one-way ANOVA, Tukey’s post hoc test; n = 18). (D) 5 nM Torin1 reduces stop codon readthrough translational errors in S2R+ cells (p = 0.0112 for 5 nM Torin1 and p = 0.3184 for 20 nM Torin1 versus DMSO-treated controls; one-way ANOVA, Tukey’s post hoc test; n∼26). (E) Trametinib reduces stop codon readthrough errors (p = 0.0313 and p = 0.0011 for 5 and 10 nM trametinib, respectively, compared to DMSO-treated controls; one-way ANOVA, Tukey’s post hoc test; n∼26). (F) A scheme of the dual luciferase reporter and control reporter used to measure misincorporation translational errors. (G) The misincorporation reporter was validated by treating the S2R+ cells overnight with the error-inducing drug paromomycin (p = 0.0073 and p < 0.0001 for control [n = 17] compared to 500 [n = 10] and 1,000 μM [n = 12] paromomycin; one-way ANOVA, Tukey’s post hoc test). (H) S2R+ cells treated with 10 nM rapamycin have fewer misincorporation translational errors compared to the respective solvent ethanol-treated control cells (p = 0.004 and p = 0.5532 for 10 and 100 nM rapamycin, respectively; one-way ANOVA; n∼35). (I) Torin1 reduces misincorporation translational errors (p = 0.0005 and p = 0.015 for 50 and 100 nM Torin1, respectively, compared to DMSO treatment; n∼56). (J) Trametinib-treated S2R+ cells have fewer misincorporation translational errors (p = 0.044 and p = 0.049 for 5 and 10 nM compared to DMSO; n∼33; one-way ANOVA, Tukey’s post hoc test). (K) Rapamycin extends chronological lifespan in S. pombe wild-type (log-rank test, p < 0.001) and RPS23 K60R mutant (log-rank test, p = 0.0267). RPS23 K60R is longer lived than wild-type control in the absence of rapamycin (log-rank test, p < 0.001), but not in presence of 109 nM rapamycin (log-rank test, p = 0.63). (L) Rapamycin treatment at 25 μM extends lifespan of C. elegans wild-type worms (p < 0.001; log-rank test; n = 341 and 315 for wild type without and with 25 μM rapamycin). Rapamycin did not extend lifespan of RPS23 K60R mutant (p = 0.4469; log-rank test; n = 283 and 321 for RPS23 K60R without and with 25 μM rapamycin). The survival plot shows the combined survival of 3 independent biological replicates. (M) 100 μM rapamycin treatment extends lifespan of wild-type (p < 0.0001) and RPS23 K60R flies (p < 0.0001). RPS23 K60R is longer lived than wild type (p < 0.0001), albeit not in presence of rapamycin (p = 0.63; log-rank test; n∼150). (N) Schematic representation of the effect of RPS23 K60R hyperaccuracy mutant on lifespan, heat shock stress, and development. Translation accuracy levels that are evolutionarily optimal for fitness are detrimental to organismal longevity.