Reduced mRNA translation delays aging, but the underlying mechanisms remain underexplored. Mutations in both DAF-2 (IGF-1 receptor) and RSKS-1 (ribosomal S6 kinase/S6K) cause synergistic lifespan extension in C. elegans. To understand the roles of translational regulation in this process, we performed polysomal profiling and identified translationally regulated ribosomal and cytochrome c (CYC-2.1) genes as key mediators of longevity. cyc-2.1 knockdown significantly extends lifespan by activating the intestinal mitochondrial unfolded protein response (UPR mt ), mitochondrial fission, and AMP-activated kinase (AMPK). The germline serves as the key tissue for cyc-2.1 to regulate lifespan, and germline-specific cyc-2.1 knockdown non-autonomously activates intestinal UPR mt and AMPK. Furthermore, the RNA-binding protein GLD-1-mediated translational repression of cyc-2.1 in the germline is important for the non-autonomous activation of UPR mt and synergistic longevity of the daf-2 rsks-1 mutant. Altogether, these results illustrate a translationally regulated non-autonomous mitochondrial stress response mechanism in the modulation of lifespan by insulin-like signaling and S6K.

To study how insulin-like signaling interacts with the TOR pathway to modulate aging, we previously constructed a daf-2 rsks-1 double mutant and observed a synergistic longevity phenotype. Further analysis demonstrated an AMP-activated kinase (AMPK)-mediated positive feedback regulation of the DAF-16 transcriptional factor mechanism in the daf-2 rsks-1 mutant (). However, genes that are translationally regulated in the daf-2 rsks-1 mutant and their roles in aging have not been determined. Because both daf-2 and rsks-1 have profound impacts on mRNA translation, we hypothesized that translational regulation plays important roles in the significantly prolonged longevity of daf-2 rsks-1 mutant animals. By genome-wide translational state analysis and genetic screens, we identified ribosomal protein genes and cyc-2.1, which encodes one of the worm cytochrome c orthologs, as negative regulators of longevity. The inhibition of cyc-2.1 results in a robust lifespan extension that requires UPR, AMPK, and mitochondrial fission in the intestine. Germline is the key tissue for cyc-2.1 to regulate longevity, and inhibition of cyc-2.1 in the germline initiates a cell non-autonomous response that activates UPRand AMPK in the intestine. Based on the translational profiling, we identified the RNA-binding protein GLD-1 as a critical translational repressor of cyc-2.1 in the germline. The synergistic lifespan extension of the daf-2 rsks-1 mutant can be suppressed by inhibiting GLD-1 or key transcriptional regulators of UPR. Therefore, the insulin-like signaling and TOR pathway-mediated tissue-specific translational repression of cytochrome c induce a cell non-autonomous mitochondrial stress response to promote longevity.

Serving as the key organelle in energy homeostasis, mitochondria play important but complex roles in aging. Mitochondrial dysregulation has been regarded as one of the major hallmarks of aging. However, mild perturbation of the mitochondrial electron transport chain (ETC) leads to significant lifespan extension in many species (). Inhibition of mitochondrial ETC genes triggers the mitochondrial unfolded protein response (UPR) via transcriptional regulators such as DVE-1, UBL-5, and ATFS-1 (). Perturbation of mitochondrial ETC functions in neurons releases a pro-longevity cue named mitokine to induce UPRin the intestine, a distal metabolic tissue in worms, and ensures lifespan extension (). Further studies have identified the neurotransmitter serotonin, neuropeptide FLP-2, and retromer-dependent Wnt signaling as the endocrine mediators of the neuron to intestine non-autonomous mitochondrial stress response (). The trans-tissue mitochondrial stress response requires epigenetic modifications that ensure selective gene expression and prolonged longevity, and the epigenetic regulatory mechanisms are conserved in mammals ().

Inhibition of the TOR pathway significantly extends lifespan in many species (). One important function of TOR is to regulate gene expression at the mRNA translation level through the ribosomal S6 kinase (S6K) and the translational initiation factor 4E-binding protein (4E-BP), both of which have been shown to play important roles in aging (). Deletion mutants of rsks-1, which encodes the C. elegans ribosomal S6K ortholog, lead to significant changes in development, lipid metabolism, reproduction, and longevity (). Previous studies have identified multiple mediators of the prolonged longevity of the rsks-1 mutant (). However, genes translationally regulated by RSKS-1 that influence lifespan and the underlying molecular mechanisms remain to be characterized.

With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging.

Aging can be genetically modulated by perturbation of insulin/insulin-like signaling (IIS), target of rapamycin (TOR) pathway, and mitochondrial functions (). These genetic manipulations often lead to significant changes in gene expression at both transcriptional and translational levels. Inhibition of DAF-2, the C. elegans ortholog of the insulin growth factor 1 (IGF-1) receptor, doubles adult lifespan by activating the DAF-16 (FOXO) transcriptional factor to regulate downstream genes involved in stress resistance, detoxification, and metabolism (). Quantitative proteomics and polysomal profiling studies revealed that the daf-2 mutant also shows altered mRNA translation in many genes, and translational regulation plays important roles in significantly prolonged longevity and extended survival during heat stress ().

In summary, we have performed genome-wide translational state analysis via polysomal profiling coupled with RNA-seq and identified genes that are regulated at the translational level in the significantly long-lived daf-2 rsks-1 mutant. Mechanistically, we demonstrated that the GLD-1 RNA-binding protein is upregulated at the protein level in the germline of daf-2 rsks-1 mutant. Elevated GLD-1 leads to translational repression of CYC-2.1 in the germline, which non-autonomously activates UPRand AMPK in the intestine and significantly extends lifespan ( Figure 6 G). These results highlight the importance of translational regulation of a highly conserved mitochondrial gene in the significantly prolonged longevity exerted by inhibiting both insulin-like signaling and S6K.

To test whether GLD-1 is upregulated in the daf-2 rsks-1 mutant, we first performed CRISPR/Cas9-based genome editing experiments to knock in the mKate2 red fluorescent protein coding sequence to the 3′ of gld-1. Compared with the wild-type control, GLD-1::mKate2 protein levels are significantly increased in the germline of daf-2 rsks-1 mutant animals ( Figures 6 A and 6B ). To examine whether GLD-1 is involved in the translational regulation of cyc-2.1, we applied gld-1 RNAi to the daf-2 rsks-1 mutant and found that CYC-2.1 protein levels are significantly elevated in the gonad, but not in the intestine, when compared with the control RNAi-treated animals ( Figures 6 C and 6D). Consistently, RNAi knockdown of gld-1 significantly decreases UPRin the intestine of daf-2 rsks-1 mutant animals ( Figure 6 E). Although gld-1 RNAi treatment has little effect on lifespan in the wild-type and daf-2 mutant backgrounds, knockdown of gld-1 significantly decreases lifespan of the rsks-1 mutant and daf-2 rsks-1 double mutant ( Figure 6 F; Table S3 ). Altogether, these results demonstrate that GLD-1 functions as a translational repressor of cyc-2.1 in the germline to non-autonomously activate intestinal UPRand extend lifespan in the daf-2 rsks-1 mutant.

(G) Model depicting the translational repression of CYC-2.1 by GLD-1 in the germline non-autonomously activates UPR mt and AMPK in the intestine via germline-produced mitokine (gMitokine) signaling, which leads to significant lifespan extension in the daf-2 rsks-1 mutant.

(E) qRT-PCR of UPR mt markers (median with range) using RNAs extracted from dissected intestinal tissues of the daf-2 rsks-1 mutant treated with the control versus gld-1 RNAi based on three biological replicates ( ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, two-tailed t tests).

(C and D) Immunoblots (C) and quantification (D) of CYC-2.1::3× FLAG and tubulin using proteins extracted from dissected gonadal and intestinal tissues of N2 and daf-2 rsks-1 mutant animals. Data are represented as mean ± SEM based on three biological replicates (ns, ∗ p < 0.05, p = 0.7063, two-tailed t tests).

(A and B) Representative photographs (A) and quantification (B) of GLD-1::mKate2 expression in the germline of N2 and daf-2 rsks-1 mutant animals. Data are represented as mean ± SEM ( ∗∗∗∗ p < 0.0001, two-tailed t test). Scale bar, 50 μm.

Translational Repression of cyc-2.1 by GLD-1 Contributes to the UPR mt Activation and Lifespan Extension in the daf-2 rsks-1 Mutant

Figure 6 Translational Repression of cyc-2.1 by GLD-1 Contributes to the UPR mt Activation and Lifespan Extension in the daf-2 rsks-1 Mutant

Specific translational regulations in many cases are mediated by RNA-binding proteins and their association with 5′ UTRs or 3′ UTRs . GLD-1, a K homology (KH) RNA-binding protein, is regulated by GLP-1/Notch signaling to negatively regulate target genes’ translation in the germline (). Our previous studies showed that a glp-1 gain-of-function mutation, which decreases GLD-1 expression, suppresses the significantly extended lifespan of daf-2 rsks-1 (). The genome-wide translational state analysis indicates that gld-1 mRNAs have elevated ribosomal loading in the daf-2 rsks-1 mutant ( Table S1 ). These results suggest that the germline-specific translational repressor GLD-1 might be involved in regulating CYC-2.1 protein levels in the germline of the daf-2 rsks-1 mutant.

GLD-1-Mediated Translational Repression of CYC-2.1 Is Important for the UPR mt Activation and Significantly Prolonged Longevity of the daf-2 rsks-1 Mutant

Previous studies showed that transcriptional factors DVE-1 and ATFS-1, as well as UBL-5, a small ubiquitin-like factor that serves as the co-factor for DVE-1, are the key mediators of UPR). Inhibition of dve-1 by RNAi shortens N2 lifespan, whereas ubl-5 or atfs-1 RNAi treatment does not affect N2 lifespan ( Figure 5 D; Table S3 ). The prolonged longevity of daf-2 rsks-1 mutant animals can be significantly decreased by dve-1, ubl-5, or atfs-1 RNAi ( Figure 5 E; Table S3 ). Knockdown of these key transcriptional regulators of UPRsignificantly decreases the lifespan extension produced by the daf-2 rsks-1 double mutant ( Figure 5 F). Therefore, the daf-2 rsks-1 mutant shows germline reduction of CYC-2.1 and intestinal activation of UPR, which is required for significantly prolonged longevity.

cyc-2.1 was identified as one of the genes that show significantly decreased ribosomal loading in the daf-2 rsks-1 mutant compared with the wild-type N2 from the translational profiling analysis ( Table S1 ). To examine whether CYC-2.1 is repressed at the protein level in the daf-2 rsks-1 mutant, we used a CRISPR/Cas9-based genome editing approach to knock in a 3× FLAG tag to the C terminus of CYC-2.1 for immunoblots using anti-FLAG antibodies. CYC-2.1 protein levels are significantly reduced in the gonad, but not in the intestine, of the daf-2 rsks-1 mutant ( Figures 5 A and 5B ). qRT-PCR measurement of UPRmarkers using dissected tissues demonstrates that the daf-2 rsks-1 mutant has UPRactivation in the intestine, but not in the gonad ( Figure 5 C).

(F) The daf-2 rsks-1 double mutations induced changes in mean lifespan upon control, dve-1, ubl-5, or atfs-1 RNAi treatment. Data are represented as mean ± SEM based on three biological replicates ( ∗∗∗ p < 0.001, ∗∗ p < 0.01, two-tailed t tests).

(D and E) Survival curves of N2 (D) and the daf-2 rsks-1 mutant (E) treated with the control, dve-1, ubl-5, or atfs-1 RNAi.

(C) qRT-PCR of UPR mt markers (median with range) using RNAs extracted from dissected gonadal and intestinal tissues of N2 and daf-2 rsks-1 mutant animals based on three biological replicates (ns, ∗∗∗ p < 0.001, ∗∗ p < 0.01, p > 0.05, two-tailed t tests).

(A and B) Immunoblots (A) and quantification (B) of CYC-2.1::3× FLAG and tubulin using proteins extracted from dissected gonadal and intestinal tissues of N2 and daf-2 rsks-1 mutant animals. Data are represented as mean ± SEM based on three biological replicates (ns, ∗∗ p < 0.01, p = 0.0880, two-tailed t tests).

Translational Repression of CYC-2.1 in the Germline and Non-autonomous Activation of UPR mt in the Intestine Play an Important Role in Regulating the Synergistic Lifespan Extension by daf-2 rsks-1

Figure 5 Translational Repression of CYC-2.1 in the Germline and Non-autonomous Activation of UPR mt in the Intestine Play an Important Role in Regulating the Synergistic Lifespan Extension by daf-2 rsks-1

RNAi knockdown of cyc-2.1 leads to the transcriptional upregulation of several ATFS-1 targets, including drp-1, which encodes a dynamin-related protein that is required for mitochondrial fission (). Using a transgenic line that expresses the TOMM-20 mitochondria outer membrane protein fused with the mKate2 fluorescent protein in the intestine (), we found that knockdown of cyc-2.1 significantly increases intestinal mitochondrial fragmentation ( Figures 4 A and 4B ). Blocking the mitochondrial fission by a drp-1 knockout mutant significantly suppresses cyc-2.1 knockdown-induced AMPK activation ( Figures 4 C and 4D). cyc-2.1 RNAi treatment extends lifespan in wild-type animals by 66%, whereas the mean lifespan extension is significantly decreased to 42% in the drp-1 mutant ( Figures 4 E and 3 F; Table S3 ). Therefore, DRP-1-mediated changes in mitochondrial dynamics serve as the key mechanism for cytochrome c reduction-induced lifespan extension.

(F) cyc-2.1 RNAi-induced changes in mean lifespan relative to the control RNAi treatments in N2 and drp-1 mutant backgrounds. Data are represented as mean ± SEM based on three biological replicates ( ∗ p < 0.05, two-tailed t test).

(C and D) Immunoblots (C) and quantification (D) of phospho-AAK-2 and actin in N2 and drp-1 mutant animals treated with control or cyc-2.1 RNAi. Data are represented as mean ± SEM based on three biological replicates (ns, ∗ p < 0.05, p = 0.3103, two-tailed t tests).

C. elegans has six macrophage-like coelomocytes that take up soluble macromolecules in the body cavity via endocytosis. Coelomocytes have direct physical contact with the gonad and intestine. CUP-4, an ion channel, is required for endocytosis in coelomocytes (). When treated with the germline-specific cyc-2.1 RNAi, the cup-4 mutant cannot activate UPR Figure 3 I) and AMPK ( Figures 3 J and 3K) in the intestine or extend lifespan ( Figure 3 L; Table S3 ). Altogether, these results indicate the existence of gonad to intestine signaling that regulates UPR, AMPK, and lifespan in response to cytochrome c reduction in the germline.

We next tested whether tissue-specific knockdown of cyc-2.1 could activate UPRand AMPK. Although both germline and intestinal-specific cyc-2.1 RNAi treatments significantly activate UPRmarkers in the intestine ( Figure 3 F), only germline, not intestinal, cyc-2.1 RNAi treatment leads to increased phospho-AAK-2 levels in the intestine ( Figures 3 G and 3H). Because there is no direct physical contact between gonad and intestine, these results suggest the existence of endocrine-like signaling for cell non-autonomous regulation.

To better understand whether cyc-2.1 RNAi activates UPRin a tissue-specific manner, we performed qRT-PCR experiments to measure mRNA levels of several ATFS-1 direct target genes, including hsp-6, dnj-10, timm-17, and drp-1, transcription levels of which are elevated upon mitochondrial ETC perturbation (). Gonadal and intestinal tissues were dissected from wild-type animals treated with either control or cyc-2.1 RNAi for qRT-PCR assays. Because the gonad is 95% germline and 5% somatic gonad, and the germline and somatic gonad cannot be further dissected, we used the gonadal tissue as a proxy for the germline in subsequent experiments. Surprisingly, global cyc-2.1 RNAi treatment significantly activates UPRmarkers in the intestine, but not in the germline ( Figure 3 B), although the latter is the tissue in which cyc-2.1 functions to regulate lifespan. Consistently, global cyc-2.1 RNAi treatment significantly activates AMPK in the intestine, but not in the gonad ( Figures 3 C and 3D). The aak-2 deletion mutant carrying a single-copy aak-2 transgene driven by the vha-6 intestine-specific promoter shows a 45% lifespan extension upon cyc-2.1 RNAi treatment ( Figure 3 E; Table S3 ). The intestinal AAK-2 is therefore sufficient to extend lifespan substantially upon cyc-2.1 RNAi knockdown.

To determine the key tissue in which cyc-2.1 functions to regulate lifespan, we performed tissue-specific RNAi experiments to knockdown cyc-2.1 in the germline, intestine, epidermis, and muscles. Spatially restricted RNAi knockdown was achieved by tissue-specific promoters driving transgene rescue of mutations in rde-1, which is essential for the RNAi machinery to be functional (). Knockdown of cyc-2.1 in the germline significantly extends lifespan ( Figure 3 A; Figure S3 A; Table S3 ), whereas knockdown of cyc-2.1 in the intestine, epidermis, or muscles does not extend lifespan ( Figure 3 A; Figures S3 B–S3D; Table S3 ). The importance of germline in cyc-2.1 RNAi-induced lifespan extension is supported by evidence that knockdown of cyc-2.1 in the germline-less glp-4(ts) mutant () only results in mild lifespan extension by 9% ( Figure S3 E). Altogether, these results demonstrate that germline is the key tissue in which CYC-2.1 functions to regulate lifespan.

(J and K) Immunoblots (J) and quantification (K) of phospho-AAK-2 and tubulin using proteins extracted from dissected intestinal tissues of the cup-4 mutant treated with germline-specific control or cyc-2.1 RNAi. Data are represented as mean ± SEM based on three biological replicates (ns, p = 0.3957, two-tailed t tests).

(I) qRT-PCR of UPR mt markers (median with range) using RNAs extracted from dissected intestinal tissues of the cup-4 mutant treated with germline-specific control or cyc-2.1 RNAi based on three biological replicates (ns, ∗ p < 0.05, p > 0.05, two-tailed t tests).

(G and H) Immunoblots (G) and quantification (H) of phospho-AAK-2 and tubulin using proteins extracted from dissected intestinal tissues of animals treated with germline- or intestine-specific control versus cyc-2.1 RNAi. Data are represented as mean ± SEM based on three biological replicates (ns, ∗∗ p < 0.001, p = 0.6141, two-tailed t tests).

(F) qRT-PCR of UPR mt markers (median with range) using RNAs extracted from dissected intestinal tissues of animals treated with germline- or intestine-specific control versus cyc-2.1 RNAi based on three biological replicates ( ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, two-tailed t tests).

(E) Survival curves of the aak-2 mutant carrying an aak-2 transgene driven by the intestine-specific vha-6 promoter treated with the control or cyc-2.1 RNAi (p < 0.0001, log-rank test).

(C and D) Immunoblots (C) and quantification (D) of phospho-AAK-2 and actin using proteins extracted from dissected gonadal and intestinal tissues of N2 treated with global control or cyc-2.1 RNAi. Data are represented as mean ± SEM based on three biological replicates (ns, ∗∗ p < 0.01, p = 0.2436, two-tailed t tests).

(B) qRT-PCR of UPR mt markers hsp-6, dnj-10, timm-17, and drp-1 (median with range) using RNAs extracted from dissected gonadal and intestinal tissues of N2 treated with global control or cyc-2.1 RNAi based on three biological replicates (ns, ∗∗∗ p < 0.001, p > 0.05, two-tailed t tests).

(A) Tissue-specific cyc-2.1 RNAi-induced changes in the mean lifespan relative to the control RNAi treatments. Data are represented as mean ± SEM based on three biological replicates.

Inhibition of cyc-2.1 in the Germline Extends Lifespan by Cell-Non-autonomous Activation of UPR mt and AMPK in the Intestine

Figure 3 Inhibition of cyc-2.1 in the Germline Extends Lifespan by Cell-Non-autonomous Activation of UPR mt and AMPK in the Intestine

Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma.

To characterize the relationship between UPRand AMPK in animals treated with cyc-2.1 RNAi, we first measured phospho-AAK-2 levels in the atfs-1 deletion mutant treated with either control or cyc-2.1 RNAi. Immunoblots and quantification results indicate that unlike in the N2 background ( Figures 2 E and 2F), cyc-2.1 RNAi does not increase phospho-AAK-2 levels in the atfs-1 mutant ( Figures 2 H and 2I). We then crossed the hsp-6 promoter::gfp reporter into the aak-2 deletion mutant. In the absence of AAK-2, cyc-2.1 RNAi still significantly activates the hsp-6::gfp reporter ( Figure 2 J), as well as the endogenous hsp-6 transcription in the intestine ( Figure 2 K). Altogether, these results demonstrate that AMPK functions downstream of UPRactivation to promote lifespan extension produced by the cyc-2.1 RNAi treatment.

It has been reported that paraquat, a ROS (reactive oxygen species) generator, and hypomorphic mutations in mitochondrial ETC genes, such as isp-1, extend C. elegans lifespan by activating AMPK (). The synergistic lifespan extension by daf-2 rsks-1 also requires AMPK (). We performed immunoblots to measure the levels of phosphorylated AAK-2 (AMPKα), which serve as an indicator of AMPK activation, in control or cyc-2.1 RNAi-treated animals. Knockdown of cyc-2.1 significantly increases phospho-AAK-2 levels compared with the control ( Figures 2 E and 2F). Consistently, cyc-2.1 RNAi fails to extend lifespan of the aak-2 deletion mutant ( Figure 2 G; Table S3 ). Therefore, inhibition of cyc-2.1 activates UPRand AMPK to extend lifespan.

To examine the effect of cyc-2.1 knockdown on the mitochondrial stress response, we applied cyc-2.1 RNAi to transgenic animals carrying a gfp reporter driven by the hsp-6 promoter, which has been widely used to monitor UPRactivation. Inhibition of cyc-2.1 significantly activates hsp-6p::gfp reporter expression ( Figure 2 B). Because the hsp-6p::gfp reporter is mainly expressed in the intestine, we performed micro-dissection to isolate the intestinal tissue of wild-type animals treated with the control or cyc-2.1 RNAi for qRT-PCR of the endogenous hsp-6 mRNA. Consistent with the hsp-6p::gfp reporter results, knockdown of cyc-2.1 significantly increases hsp-6 mRNA levels compared with the control RNAi treatment ( Figure 2 C). Previous studies have identified ATFS-1 as one of the key transcription factors that mediate the UPRactivation (). The cyc-2.1 RNAi-induced longevity phenotype is suppressed by a deletion mutant of atfs-1 ( Figure 2 D; Table S3 ). Therefore, activated mitochondrial stress response plays an essential role in cytochrome c knockdown-induced lifespan extension.

Cytochrome c functions in the mitochondrial ETC by transferring electrons from complex III to complex IV. Previous studies demonstrated that inhibition of certain mitochondrial ETC components extends lifespan, and the underlying mechanisms involve CEP-1/p53 (), the intrinsic apoptosis pathway (), and UPR). Mutations in the C. elegans p53 ortholog CEP-1 or a key component of the apoptosis pathway CED-4 significantly suppress the lifespan extension by mitochondrial ETC mutants (). However, cyc-2.1 RNAi treatment significantly extends lifespan in cep-1 or ced-4 knockout mutant ( Figures S2 A and S2B), suggesting a different mechanism.

Among all genes tested, cyc-2.1, which encodes one of the two highly conserved cytochrome c orthologs, showed the strongest lifespan extension upon RNAi knockdown ( Table S2 ). cyc-2.1 was originally identified as a lifespan determinant from an RNAi screen for enhanced oxidative stress resistance (). Knockdown of cyc-2.1 robustly extends lifespan in the wild-type, rsks-1 mutant, and daf-2 mutant backgrounds, but it does not further extend lifespan of the daf-2 rsks-1 double mutant ( Figure 2 A; Table S3 ). These results suggest that reduced CYC-2.1 might serve as the key mechanism in mediating the synergistic effect of daf-2 rsks-1 on longevity.

(K) qRT-PCR of hsp-6 (median with range) using RNAs extracted from dissected intestinal tissues of the aak-2 mutant treated with control or cyc-2.1 RNAi based on three biological replicates ( ∗∗∗∗ p < 0.0001, two-tailed t test).

(H and I) Immunoblots (H) and quantification (I) of phospho-AAK-2 and actin in the atfs-1 deletion mutant treated with control or cyc-2.1 RNAi. Data are represented as mean ± SEM based on four biological replicates (ns, p = 0.5239, two-tailed t test).

(E and F) Immunoblots (E) and quantification (F) of phospho-AAK-2 (AMPKα) and actin in N2 animals treated with control or cyc-2.1 RNAi. Ratio of band intensity of phospho-AAK-2 to actin was normalized to the control RNAi-treated animals. Data are represented as mean ± SEM based on eight biological replicates ( ∗∗ p = 0.0041, two-tailed t test).

(C) qRT-PCR of hsp-6 (median with range) using RNAs extracted from dissected intestinal tissues of N2 treated with control or cyc-2.1 RNAi based on three biological replicates ( ∗∗∗∗ p < 0.0001, two-tailed t test).

Numerous studies have demonstrated that genes differentially expressed in long-lived mutants are key regulators of lifespan. We thus hypothesized that genes translationally downregulated in the long-lived daf-2 rsks-1 mutant are likely to be negative regulators of longevity, inhibition of which in the wild-type background could extend lifespan. Thus, we performed an RNAi-based genetic screen to individually knockdown those 115 translationally downregulated genes in N2 to test their effects on lifespan. To facilitate the survival assays, the primary screen was performed at 25°C using the spe-9; rrf-3 double mutant, which shows enhanced RNAi sensitivity, temperature-sensitive sterility, and normal lifespan. Intriguingly, 39 of the 115 RNAi treatments during development led to larval arrest, suggesting that genes translationally downregulated in the daf-2 rsks-1 mutant are enriched with developmentally essential genes. We then performed RNAi treatments against these 39 genes only during adulthood to test their lifespan phenotypes. After the re-test in the wild-type background at 20°C, we identified 24 genes, inhibition of which leads to significant lifespan extension ( Table S2 ). Among them, 17 genes are essential ones that encode various ribosomal subunits. These results highlight the importance of developmentally essential genes and translationally regulated ribosomal biogenesis in lifespan determination.

To validate whether mRNAs with differential ribosome loading are regulated at the mRNA translational level, we compared expression of rps-0, rps-3, rpl-5, and rpl-25.2 at both mRNA and protein levels between N2 and daf-2 rsks-1 mutant animals. These genes were chosen based on the availability of antibodies () to detect their protein products. Consistent with the RNA-seq data ( Table S1 ), mRNA levels of rps-0, rps-3, rpl-5, and rpl-25.2 show no significant changes between N2 and daf-2 rsks-1 mutant animals ( Figure 1 D), whereas protein products of these genes are significantly decreased in the daf-2 rsks-1 mutant compared with N2 ( Figures 1 E and 1F). Previous quantitative proteomics studies also showed that the abundance of these ribosomal proteins is decreased in the daf-2 mutant compared with N2 (). Altogether, these results indicate that polysomal profiling is a valid approach to quantitatively assess mRNA translation, which allowed us to identify differentially translated mRNAs in the daf-2 rsks-1 mutant.

To characterize the roles of mRNA translation in the significantly prolonged longevity of daf-2 rsks-1, we performed genome-wide translational state analysis via polysomal profiling coupled with RNA sequencing (RNA-seq) using wild-type N2 and daf-2 rsks-1 mutant animals ( Figure 1 A). Day 4 adult animals were collected for extraction of total mRNAs and translated mRNAs (≥2 ribosomes/transcript) for quantification via RNA-seq ( Figure 1 A). Gene set enrichment analysis (GSEA) was performed to compare RNA-seq results with our previous microarray studies (). Transcriptionally up- and downregulated gene lists both showed significant concordance with the microarray results (false discovery rate [FDR] < 0.001), with normalized enrichment scores of 2.84 and 3.19, respectively ( Figure S1 ). Changes in translation were determined by comparing the ratio of polysome-associated mRNAs to total mRNAs between N2 and daf-2 rsks-1 mutant animals. This is called the differential polysome association ratio (DPAR). Altogether, we identified 167 transcripts with differential translation but no changes at total mRNA levels. Among them, 52 genes are upregulated and 115 genes are downregulated in the daf-2 rsks-1 mutant ( Figure 1 B; Table S1 ). Gene Ontology (GO) enrichment analysis of biological processes revealed meiotic cell cycle, cell cycle, and organelle fission are the top three terms in the upregulated genes, whereas translation, ribosome biogenesis, and developmental process are the top three terms in the downregulated genes ( Figure 1 C).

(E and F) Immunoblots (E) and quantification (F) of RPS-0, RPS-3, RPL-5, RPL-25.2, and tubulin protein levels in N2 and the daf-2 rsks-1 mutant. The ratio of band intensity of ribosomal proteins to tubulin was normalized to N2. Data are represented as mean ± SEM based on three biological replicates ( ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01, two-tailed t tests).

(D) qRT-PCR of rps-0, rps-3, rpl-5, and rpl-25.2 mRNA levels (median with range) in N2 and the daf-2 rsks-1 mutant based on three biological replicates (not significant [ns], p > 0.05, two-tailed t tests).

(C) Top Gene Ontology (GO) terms for genes differentially expressed at the translational level in the daf-2 rsks-1 mutant.

(A) Experimental design of the genome-wide translational state analysis. On the left are representative polysome profiles of wild-type N2 and the daf-2 rsks-1 double mutant. The total and translated mRNA fractions were used for the genome-wide transcriptional and translational state analysis, as depicted by the workflow on the right.

Discussion

Fontana et al., 2010 Fontana L.

Partridge L.

Longo V.D. Extending healthy life span—from yeast to humans. Kenyon, 2010 Kenyon C.J. The genetics of ageing. Chen et al., 2013 Chen D.

Li P.W.-L.

Goldstein B.A.

Cai W.

Thomas E.L.

Chen F.

Hubbard A.E.

Melov S.

Kapahi P. Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans. Highly conserved IIS and the TOR pathway play an important role in aging across species (). To examine how these pathways interact with each other to modulate aging, we previously constructed a daf-2 rsks-1 double mutant that carries loss-of-function mutations in the DAF-2/IGF-1 receptor and TOR effector RSKS-1/S6K. The double mutant shows a synergistic effect, rather than an additive effect, on longevity, suggesting active interactions between these two important aging-related pathways. Functional genomics studies via transcriptome profiling helped to identify AMPK-mediated positive feedback regulation of the DAF-16/FOXO transcription factor mechanism in the daf-2 rsks-1 mutant (). However, RSKS-1, which serves as a key regulator of mRNA translation, has not been well characterized for its roles in the significantly extended longevity of daf-2 rsks-1 mutant animals.

Hansen et al., 2007 Hansen M.

Taubert S.

Crawford D.

Libina N.

Lee S.-J.

Kenyon C. Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Kapahi et al., 2004 Kapahi P.

Zid B.M.

Harper T.

Koslover D.

Sapin V.

Benzer S. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Kapahi et al., 2010 Kapahi P.

Chen D.

Rogers A.N.

Katewa S.D.

Li P.W.-L.

Thomas E.L.

Kockel L. With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Pan et al., 2007 Pan K.Z.

Palter J.E.

Rogers A.N.

Olsen A.

Chen D.

Lithgow G.J.

Kapahi P. Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans. Rogers et al., 2011 Rogers A.N.

Chen D.

McColl G.

Czerwieniec G.

Felkey K.

Gibson B.W.

Hubbard A.

Melov S.

Lithgow G.J.

Kapahi P. Life span extension via eIF4G inhibition is mediated by posttranscriptional remodeling of stress response gene expression in C. elegans. It has been well documented that inhibition of translation delays aging (). One hypothesis is that reduced global translation helps organisms to maintain better protein homeostasis, dysregulation of which leads to aging and age-related pathologies. A linked hypothesis suggests that the anti-aging effect is achieved by the translational regulation of key modulators of aging. We reasoned that identification of genes that are translationally regulated in the daf-2 rsks-1 mutant and characterization of the translational regulation should help in gaining better mechanistic insights into the significantly extended longevity of the daf-2 rsks-1 double mutant.

mt in the intestine ( Berendzen et al., 2016 Berendzen K.M.

Durieux J.

Shao L.-W.

Tian Y.

Kim H.-E.

Wolff S.

Liu Y.

Dillin A. Neuroendocrine Coordination of Mitochondrial Stress Signaling and Proteostasis. Durieux et al., 2011 Durieux J.

Wolff S.

Dillin A. The cell-non-autonomous nature of electron transport chain-mediated longevity. Shao et al., 2016 Shao L.-W.

Niu R.

Liu Y. Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response. mt and AMPK in the intestine. Although intestine-specific cyc-2.1 RNAi also activates UPRmt, it fails to activate AMPK and extend lifespan (mt is required, but not sufficient, for the lifespan extension produced by cyc-2.1 knockdown. Because there is no direct contact between the germline and the intestine in C. elegans, we propose that reduced cytochrome c in the germline might produce an endocrine-like signaling, named gMitokine (germline-produced mitokine), to activate UPRmt in the distal tissue for prolonged survival. The germline to intestine regulation can be blocked by the cup-4 mutant, which disrupts endocytosis in coelomocytes (mt-mediated anti-aging mechanisms. Previous studies showed that perturbation of mitochondrial ETC in C. elegans neurons provokes a pro-longevity signal to non-autonomously activate UPRin the intestine (). In this study, we found that inhibition of cyc-2.1 in the germline non-autonomously activates UPRand AMPK in the intestine. Although intestine-specific cyc-2.1 RNAi also activates UPR, it fails to activate AMPK and extend lifespan ( Figures 3 A and 3F–3H; Figure S3 B; Table S3 ). These results indicate that activation of UPRis required, but not sufficient, for the lifespan extension produced by cyc-2.1 knockdown. Because there is no direct contact between the germline and the intestine in C. elegans, we propose that reduced cytochrome c in the germline might produce an endocrine-like signaling, named gMitokine (germline-produced mitokine), to activate UPRin the distal tissue for prolonged survival. The germline to intestine regulation can be blocked by the cup-4 mutant, which disrupts endocytosis in coelomocytes ( Figures 3 I–3L). Altogether, our findings demonstrate a cell non-autonomous signaling that is distinctive from the neuron to intestine mitokine pathways. Further characterization of this germline to intestine signal transduction process, especially identifying the gMitokine and its downstream effectors, will help in better understanding UPR-mediated anti-aging mechanisms.

Berman and Kenyon, 2006 Berman J.R.

Kenyon C. Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Hsin and Kenyon, 1999 Hsin H.

Kenyon C. Signals from the reproductive system regulate the lifespan of C. elegans. The germline tissue plays an important role in C. elegans aging. Germline-less worms showed significant lifespan extension that depends on the DAF-16 FOXO transcription factor and DAF-12 nuclear hormone receptor. The prolonged longevity of germline-less animals is not due to sterility; rather, it is caused by diminished pro-aging signaling from the germline (). However, the molecular identity of aging signals produced by germ cells has not been fully determined. We speculate that cyc-2.1 knockdown and germline deficiency function through different mechanisms to regulate lifespan, because unlike the long-lived germline-less animals, the cyc-2.1 RNAi-treated animals show significantly prolonged longevity independent of DAF-16 or DAF-12 ( Figure S3 F).

Chen et al., 2013 Chen D.

Li P.W.-L.

Goldstein B.A.

Cai W.

Thomas E.L.

Chen F.

Hubbard A.E.

Melov S.

Kapahi P. Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans. Han et al., 2017 Han S.

Schroeder E.A.

Silva-García C.G.

Hebestreit K.

Mair W.B.

Brunet A. Mono-unsaturated fatty acids link H3K4me3 modifiers to C. elegans lifespan. mt is involved in this process, in future studies. It has been shown that the germline is the key tissue for RSKS-1 to regulate lifespan. Knockdown of rsks-1 in the germline of daf-2 mutant produces a synergistic effect on longevity (). The histone H3 lysine 3 trimethylation (H3K3me3) deficiency-induced lifespan extension is also mediated by downregulation of RSKS-1 in the germline, which leads to increased accumulation of mono-unsaturated fatty acids (MUFAs) in the distal intestine tissue (). It will be interesting to test whether inhibition of cyc-2.1 in the germline affects lipid metabolism in the intestine, and if so, whether UPRis involved in this process, in future studies.

In conclusion, genome-wide translational state analysis allowed us to identify a series of translational regulation of lifespan-determinant genes in the significantly long-lived daf-2 rsks-1 mutant. Functional studies revealed that RNA-binding protein GLD-1-mediated translational repression of cytochrome c in the germline leads to non-autonomous activation of UPRmt and AMPK in the intestine, which is indispensable for the significantly extended lifespan. In the future, it will be important to understand how the translational regulation is achieved and how the germline signals the distal metabolic tissue upon mitochondrial perturbation at the molecular level.