Translational Regulation of Non-autonomous Mitochondrial Stress Response Promotes Longevity

Abstract

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.

Keywords: AMPK; C. elegans; UPR(mt); aging; daf-2 rsks-1; germline; mRNA translation.

Figure 1.. Identification of Genes that Are Translationally Regulated 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. (B) Cluster of genes differentially expressed in the daf-2 rsks-1 mutant compared with N2. (C) Top Gene Ontology (GO) terms for genes differentially expressed at the translational level in the daf-2 rsks-1 mutant. (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). (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). See also Table S1.

Figure 2.. Knockdown of cyc-2.1 Significantly Extends Lifespan by Activating UPR^mt and AMPK

(A) Survival curves of the wild-type N2, rsks-1, daf-2, and daf-2 rsks-1 mutant animals treated with control or cyc-2.1 RNAi. (B) Representative photographs of hsp-6p::gfp expression in animals treated with control or cyc-2.1 RNAi. Scale bar, 200 μm. (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). (D) Survival curves of the atfs-1 deletion mutant treated with control or cyc-2.1 RNAi (p = 0.2355, log-rank test). (E and F) Immunoblots (E) and quantification (F) of phospho-AAK-2 (AMPKα) and actin in N2 animalstreated 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). (G) Survival curves of the aak-2 deletion mutant treated with control or cyc-2.1 RNAi (p = 0.9647, log-rank 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). (J) Representative photographs of hsp-6p::gfp expression in aak-2 deletion mutant animals treated with control or cyc-2.1 RNAi. Scale bar, 200 μm. (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) . See also Table S3.

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

(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. (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). (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). (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). (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). (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). (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). (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). (L) Survival curves of the cup-4 mutant treated with germline-specific control or cyc-2.1 RNAi (p = 0.8629, log-rank test). See also Figure S3 and Table S3.

Figure 4.. cyc-2.1 Knockdown-Induced Mitochondria Fragmentation Plays an Important Role in AMPK Activation and Lifespan Extension

(A and B) Representative photographs (A) and quantification (B) of intestinal mitochondrial morphology in animals treated with control or cyc-2.1 RNAi (p < 0.0001, χ² test). Scale bar, 10 μm. (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). (E) Survival curves of N2 and the drp-1 mutant treated with control or cyc-2.1 RNAi. (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). See also Table S3.

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

(A and B) Immunoblots (A) and quantification (B) of CYC-2.1::3x 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). (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). (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. (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). See also Table S3.

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

(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. (C and D) Immunoblots (C) and quantification (D) of CYC-2.1::3x 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). (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). (F) Survival curves of the wild-type N2, rsks-1, daf-2, and daf-2 rsks-1 mutant animals treated with control or gld-1 RNAi. (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. See also Table S3.