Inhibiting RNA Polymerase III engages TOR signalling to control development and improves C. elegans late-life health and longevity.
Abstract
Organismal growth and lifespan are inextricably linked. Target of Rapamycin (TOR) signalling regulates protein production for growth and development, but if reduced, extends lifespan across species. Reduction in the enzyme RNA polymerase III, which transcribes tRNAs and 5S rRNA, also extends longevity. Here, we identify a temporal genetic relationship between TOR and Pol III in Caenorhabditis elegans, showing that they collaborate to regulate progeny production and lifespan. Interestingly, the lifespan interaction between Pol III and TOR is only revealed when TOR signaling is reduced, specifically in adulthood, demonstrating the importance of timing to control TOR regulated developmental versus adult programs. In addition, we show that Pol III acts in C. elegans muscle to promote both longevity and healthspan and that reducing Pol III even in late adulthood is sufficient to extend lifespan. This demonstrates the importance of Pol III for lifespan and age-related health in adult C. elegans.
Introduction
In eukaryotic cells, three multi-subunit RNA polymerase (Pol) enzymes, Pol I, II, III, transcribe the nuclear genome. Each polymerase synthesises a distinct set of genes: Pol II transcribes all coding genes to generate mRNA, whereas polymerases Pol I and III only transcribe non-coding genes (1). RNA Pol III comprises 17 subunits and is specialised for the transcription of short, abundant, non-coding RNA transcripts. Its main gene products are tRNAs and 5S ribosomal RNA (rRNA), which are abundantly transcribed and required for protein synthesis (2). Other Pol III transcripts include small nuclear (snRNAs) and small nucleolar (snoRNAs), both of which are implicated in RNA processing, the 7SK snRNAs that plays role in regulating transcription, as well as other small RNAs including Y RNA, L RNA, vault RNA, and U6 spliceosomal RNA (3, 4) Pol III-mediated transcription regulates a wide range of biological processes including cell and organismal growth (5, 6, 7, 8), the cell cycle (9), differentiation (8, 10, 11), development (12), regeneration (13) and cellular responses to stress (14). Pol III subunits have also been implicated in a wide variety of disease states and lifespan (15, 16, 17).
Reducing the activity of Pol III by partially down-regulating its individual subunits can significantly extend lifespan in multiple model organisms including baker’s yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster (18). In C. elegans, rpc-1 encodes the largest Pol III subunit and its down-regulation by RNAi increases lifespan (18). In D. melanogaster, lifespan is increased in heterozygous females with a single functional copy of gene encoding for Pol III-specific subunit C53. Pol III activity in the fly gut limits survival and can drive ageing, specifically from the gut stem-cell compartment. Importantly, in flies, Pol III knockdown is downstream of the target of Rapamycin (TOR) pathway, an evolutionarily conserved regulator of longevity (18, 19)
The TOR kinase is a serine/threonine kinase that regulates growth and development chiefly by modulating protein synthesis in response to changes in nutrients and other cues (19) (Fig 1A). TOR can be part of two complexes, and it is specifically the TOR complex 1 (TORC1) that drives growth, development, and anabolic metabolism (19, 20). In C. elegans, complete loss of let-363, the gene which encodes the TOR ortholog (CeTOR), is lethal, whereas depletion of let-363/CeTOR by RNAi or pharmacologically extends lifespan (21) (Fig 1A).
Figure 1. CeTOR signalling and RNA Polymerase III interact to control reproduction.
(A) Schematic showing the C. elegans CeTORC1 complex and its potential downstream outcomes in response to nutrient cues. (B) rpc-1 RNAi leads to L3 stage developmental arrest. This RNAi treatment typically leads to a 60% reduction in rpc-1 mRNA levels (18). Scale bar indicates 1 mm. (C) Schematic showing timings of the rpc-1 and CeTOR interventions: for rpc-1 and let-363/CeTOR RNAi worms were raised on OP50 and moved to the appropriate RNAi at the L4 stage; loss-of-function mutation in raga-1(ok386) is present throughout life, from the embryonic stage. (D, E, F, G) Interaction between the CeTOR pathway and rpc-1 for brood size. Combined data for three biological replicates are shown. EV, Empty Vector. Statistical comparisons were made by One-way ANOVA. P-value: *P < 0.05, **P < 0.005, ***P < 0.0005; ns, non-significant.
Here we show that CeTOR and Pol III act together to control development and adult lifespan. Reducing CeTOR signaling from embryonic stages together with Pol III inhibition demonstrated their genetic interaction to control progeny production, but their ability to collaborate and regulate lifespan is only revealed when CeTOR signaling is reduced specifically in adulthood. This demonstrates the critical temporal importance of TOR signaling throughout life, how this alters its interactions with key transcriptional regulators, and the impact of this on developmental versus longevity programs. In addition, we show that Pol III acts in C. elegans muscle to promote longevity, healthspan and body-wall mitochondrial networks, and that late-life Pol III knockdown is sufficient to extend lifespan. Overall, our data uncover a mechanism which describes the interactions of TORC1 and Pol III in reproduction and longevity, and the importance of Pol III for lifespan and age-related health in adult C. elegans.
Results
RNA polymerase III is important for C. elegans reproduction
RNA polymerase III is essential for growth and development (6, 7, 22). Indeed, when we reduced Pol III mRNA expression by feeding C. elegans bacteria expressing a dsRNA for rpc-1 from the embryonic stage, the worms exhibited growth defects and more than 90% of worms were arrested at L2-L3 larval stage, whereas all animals in control RNAi developed to adults within 3 d (Fig 1B). This confirms the requirement of Pol III for reproduction has not been examined. To determine the effect of Pol III knockdown on progeny production, we raised the worms to early adulthood on the standard laboratory food source Escherichia coli OP50-1, transferring to rpc-1 RNAi at the L4 larval stage (Fig 1C). We found that C. elegans treated with rpc-1 RNAi at this late larval stage developed into adults but displayed a dramatic reduction in their total brood size compared to control (∼40% P = 2.98 × 10-6, Fig 1D). The reduction in brood size is likely independent of spermatogenesis as rpc-1 RNAi did not diminish the total number of sperm in self-fertilized hermaphrodites (Fig S1A and B). Nor did rpc-1 RNAi treatment lead to any change in embryonic viability, or age-specific fecundity and embryos hatched into larvae within a similar time frame as WT (Figs S1C and S2A). This suggests that Pol III inhibition from late larval development is sufficient to reduce progeny production without affecting spermatogenesis or embryo maturation ex-utero. Taken together, these data show that Pol III is required for normal organismal growth and reproductive processes.
Figure S1. Knockdown of RNA polymerase III does not diminish sperm count.
(A) rpc-1 RNAi treatment because early adulthood does not diminish the sperm count in wild-type hermaphrodite C. elegans. Representative images of DAPI stained sperm nuclei are shown at 60X and 100X magnifications. Arrows indicate spermatheca. Scale bars indicate 10 μm. (B) Quantification of distinct sperm nuclei as observed upon DAPI staining in control versus rpc-1 RNAi treated animals N = 10. Statistical comparisons were made by using two-tailed t test. (C) rpc-1 RNAi treatment does not lead to excess embryonic lethality. Total hatched larvae are compared with eggs laid. Statistical comparisons were made by two-tailed t test. ns, non-significant.
Figure S2. RNA polymerase III and let-363/CeTOR knockdown diminish brood size without affecting fecundity.
(A, B, C) rpc-1 RNAi and let-363/CeTOR RNAi treatments when either undiluted or diluted 1:1 with empty vector control: (A) do not affect age-specific fecundity, (B) cause a significant decrease in brood size(s), and (C) do not alter embryonic viability. NB: At 25°C (the temperature that we did these experiments) very few animals suffered internal hatching and those that did were taken of the analysis. Combined data for three biological replicates are shown. Statistical comparisons were made by one-way ANOVA. P-value: ***P < 0.0001; **P < 0.005; *P < 0.05, na, not applicable, ns, non-significant.
TOR signaling interacts genetically with RNA polymerase III to control C. elegans reproduction
Reproduction and embryonic development in worms is an intense anabolic process requiring high levels of protein translation (23, 24). The TORC1 pathway acts as a master switch for governing cellular growth, whereas Pol III transcribes two of the most important non-coding RNA families (tRNAs and rRNAs) required for protein synthesis (2). To test whether Pol III could act downstream of TORC1 to control reproduction, we examined the epistatic relationship between rpc-1 knockdown and C. elegans TORC1 (CeTORC1) using progeny production as a readout. In C. elegans, the gene encoding RagA (a RAS-related GTP-binding protein) is raga-1/RagA, which acts as a positive regulator of the CeTORC1 kinase (25) (Fig 1A). Animals carrying the raga-1(ok386) mutation have constitutively low CeTORC1 signaling throughout development and adulthood (26) (Fig 1C). raga-1(ok386) mutants develop normally, but adults have a reduced brood size compared with WT (Fig 1E, P = 4.81 × 10-6). However, treatment of raga-1(ok386) mutants with rpc-1 RNAi resulted in a further reduction in progeny numbers compared with either raga-1/RagA mutation or rpc-1 RNAi alone (Fig 1E, P = 7.91 × 10-11 and P = 6.67 × 10-8, respectively).
The raga-1(ok386) strain harbors a mutation predicted to remove the entire gene, and is considered null. However, TORC1 is controlled by multiple inputs, and we were concerned that the raga-1(ok386) mutation was not sufficient to completely reduce CeTORC1 signaling. To address this, we depleted the C. elegans TOR kinase itself using RNAi. C. elegans fed let-363/CeTOR RNAi from the L4 stage had a reduced brood size compared with control (Fig 1F, P = 4.9 × 10-4). However, combining let-363/CeTOR RNAi with rpc-1 RNAi was non-additive compared with either rpc-1 or let-363/CeTOR RNAi (Fig 1F, P = 0.57 and P = 0.62, respectively). This was not because of an artifact of performing double RNAi as diluting either let-363/CeTOR or rpc-1 RNAi by 50% with EV control achieved the same fecundity-related phenotypes as undiluted RNAi (Fig S2A–C). Thus, this implies that Pol III and TOR act in the same pathway to regulate C. elegans reproduction. To explore this epistatic relationship further, we examined progeny production in raga-1(ok386) mutants treated with let-363/CeTOR RNAi. Depletion of let-363/CeTOR from early adulthood resulted in a further reduction in raga-1(ok386) brood size (Fig 1G, P = 1.31 × 10-5), indicative that the initial raga-1/RagA mutation did not achieve complete TORC1 reduction. However, this was non-additive with rpc-1 RNAi (Fig 1G, P = 0.80). Together, our data suggest that CeTORC1 and Pol III act in the same pathway to affect organismal reproduction.
TOR and RNA polymerase III interact genetically and temporally to control adult lifespan
TOR inhibition extends lifespan in a range of model organisms from yeast to mammals (27). In C. elegans, depletion of CeTOR signaling either by raga-1/RagA loss-of-function mutation or let-363/CeTOR RNAi extends lifespan (26, 28). Given that Pol III and TORC1 act epistatically in respect to progeny production (Fig 1E–G), we wanted to test the genetic interaction between CeTOR signaling and Pol III for C. elegans adult lifespan. As above, we reduced CeTOR signaling at two different time points: from embryonic stages using raga-1/RagA mutation and from the L4 stage using let-363/CeTOR RNAi, where it consistently initiating Pol III knockdown at the L4 stage (Fig 1C). We found that although raga-1(ok386) mutants are long-lived, the addition of rpc-1 RNAi further extended this lifespan, indicating that CeTOR and Pol III are acting in parallel (Fig 2A, Table 1). To rule out the possibility that raga-1/RagA mutation is incomplete and thus masking an epistatic relationship between TOR and Pol III, we examined the simultaneous knock down of raga-1/RagA with let-363/CeTOR with and without rpc-1 RNAi (Fig 1A and C). In our hands, raga-1 mutation and let-363/CeTOR RNAi were not additive; however, the addition of rpc-1 RNAi did further increase the raga-1/RagA; let-363/CeTOR lifespan (Fig 2B, Table 1). This supports our conclusion that CeTOR signaling engages additional, Pol III independent, mediators to control lifespan if knocked out during development (Fig 2C, Table 1).
Figure 2. CeTOR signalling and RNA Polymerase III interact temporally to control lifespan.
(A) rpc-1 RNAi and raga-1(ok386) additively increases lifespan. (B) let-363/CeTOR RNAi and raga-1(ok386) mutation extend lifespan and are additive with rpc-1 RNAi. (C) let-363/CeTOR RNAi interacts with rpc-1 RNAi to extend lifespan. (A, B, C) Diluted and non-diluted let-363/CeTOR or rpc-1 RNAi treatments extend lifespan equally (Table 1). One representative experiment is shown, refer to Table 1 for data on all replicates and statistical analysis.
Table 1.
Lifespan data for Fig 2.
| Figure | Trial | Strain | Genotype | Mean lifespan (days) | Extension (%) | Extension (%) | P-value (Log-rank) vs | N dead (total) |
|---|---|---|---|---|---|---|---|---|
| raga-1 control | WT control | |||||||
| 2C | 1 | WT | Control RNAi | 10.83 | 93 | |||
| 2C | WT | rpc-1 RNAi | 11.72 | 8.22 | WT<0.001 | 91 | ||
| 2C | WT | let-363/CeTOR RNAi | 11.52 | 6.37 | WT<0.05 | 95 | ||
| WT rpc-1:NS | ||||||||
| 2B, 2C | WT | rpc-1 RNAi diluted (1:1 with control) | 11.73 | 8.31 | WT<0.001 | 91 | ||
| WT rpc-1:NS | ||||||||
| WT let-363/ceTOR:NS | ||||||||
| 2B, 2C | WT | let-363/CeTOR RNAi diluted (1:1 with control) | 11.84 | 9.33 | WT<0.001 | 72 | ||
| WT rpc-1:NS | ||||||||
| WT let-363/ceTOR:NS | ||||||||
| WT rpc-1:NS | ||||||||
| 2B, 2C | WT | rpc-1 RNAi + let-363/CeTOR RNAi | 12.4 | 14.50 | WT<0.001 | 84 | ||
| WT rpc-1<0.05 | ||||||||
| WT let-363/ceTOR<0.05 | ||||||||
| WT rpc-1 :NS | ||||||||
| WT let-363/ceTOR :NS | ||||||||
| 2A | raga-1(ok386) | Control RNAi | 20.38 | 70.83 | WT<0.001 | 90 | ||
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 <0.001 | ||||||||
| WT let-363/ceTOR <0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| 2A | raga-1(ok386) | rpc-1 RNAi | 21.9 | 7.46 | 83.57 | WT<0.001 | 81 | |
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 diluted<0.001 | ||||||||
| WT let-363/ceTOR diluted <0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1<0.05 | ||||||||
| 2B | raga-1(ok386) | let-363/CeTOR RNAi | 22.03 | 8.10 | 84.66 | WT<0.001 | 80 | |
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 diluted<0.001 | ||||||||
| WT let-363/ceTOR diluted<0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1 rpc-1:NS | ||||||||
| 2B | raga-1(ok386) | rpc-1 RNAi diluted (1:1 with control) | 21.84 | 7.16 | 83.07 | WT<0.0001 | 81 | |
| WT rpc-1<0.0001 | ||||||||
| WT let-363/ceTOR<0.0001 | ||||||||
| WT rpc-1 diluted<0.0001 | ||||||||
| WT let-363/ceTOR diluted <0.0001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.0001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1 rpc-1:NS | ||||||||
| raga-1 ceTOR:NS | ||||||||
| 2B | raga-1(ok386) | rpc-1 RNAi + let-363/CeTOR RNAi | 23.14 | 13.60 | 113.94 | WT<0.0001 | 82 | |
| WT rpc-1<0.0001 | ||||||||
| WT let-363/ceTOR<0.0001 | ||||||||
| WT rpc-1 diluted<0.0001 | ||||||||
| WT let-363/ceTOR diluted <0.0001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.0001 | ||||||||
| raga-1 control<0.05 | ||||||||
| raga-1 rpc-1 dil.<0.05 | ||||||||
| raga-1 ceTOR dil.<0.05 | ||||||||
| WT rpc-1<0.0001 | ||||||||
| 2 | WT | Control RNAi | 10.55 | 88 | ||||
| WT | rpc-1 RNAi | 11.77 | 10.37 | WT<0.001 | 93 | |||
| WT | let-363/CeTOR RNAi | 11.89 | 11.27 | WT<0.05 | 100 | |||
| WT rpc-1:NS | ||||||||
| WT | rpc-1 RNAi diluted (1:1 with control) | 11.51 | 8.34 | WT<0.001 | 89 | |||
| WT rpc-1:NS | ||||||||
| WT let-363/ceTOR:NS | ||||||||
| WT | let-363/CeTOR RNAi diluted (1:1 with control) | 11.40 | 7.46 | WT<0.001 | 81 | |||
| WT rpc-1:NS | ||||||||
| WT let-363/ceTOR:NS | ||||||||
| WT rpc-1:NS | ||||||||
| WT | rpc-1 RNAi + let-363/CeTOR RNAi | 12.43 | 15.12 | WT<0.001 | 94 | |||
| WT rpc-1<0.05 | ||||||||
| WT let-363/ceTOR<0.05 | ||||||||
| WT rpc-1 :NS | ||||||||
| WT let-363/ceTOR :NS | ||||||||
| raga-1(ok386) | Control RNAi | 19.98 | 89.38 | WT<0.001 | 78 | |||
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 <0.001 | ||||||||
| WT let-363/ceTOR <0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1(ok386) | rpc-1 RNAi | 22.01 | 10.11 | 108.62 | WT<0.001 | 91 | ||
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 diluted<0.001 | ||||||||
| WT let-363/ceTOR diluted <0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1(ok386) | rpc-1 RNAi diluted (1:1 with control) | 21.88 | 9.52 | 108.34 | WT<0.001 | 94 | ||
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 diluted<0.001 | ||||||||
| WT let-363/ceTOR diluted<0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1 rpc-1:NS | ||||||||
| raga-1(ok386) | CeTOR RNAi diluted (1:1 with control) | 22.21 | 11.16 | 110.52 | WT<0.0001 | 96 | ||
| WT rpc-1<0.0001 | ||||||||
| WT let-363/ceTOR<0.0001 | ||||||||
| WT rpc-1 diluted<0.0001 | ||||||||
| WT let-363/ceTOR diluted <0.0001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.0001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1 rpc-1:NS | ||||||||
| raga-1 ceTOR:NS | ||||||||
| raga-1(ok386) | rpc-1 RNAi + let-363/CeTOR RNAi | 23.08 | 15.51 | 118. | WT<0.0001 | 91 | ||
| WT rpc-1<0.0001 | ||||||||
| WT let-363/ceTOR<0.0001 | ||||||||
| WT rpc-1 diluted<0.0001 | ||||||||
| WT let-363/ceTOR diluted <0.0001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.0001 | ||||||||
| raga-1 control<0.05 | ||||||||
| raga-1 rpc-1 dil.<0.05 | ||||||||
| raga-1 ceTOR dil.<0.05 | ||||||||
| 3 | WT | Control RNAi | 10.69 | 67 | ||||
| WT | rpc-1 RNAi | 11.72 | 11.09 | WT<0.001 | 74 | |||
| WT | let-363/CeTOR RNAi | 11.88 | 12.60 | WT<0.05 | 78 | |||
| WT rpc-1:NS | ||||||||
| WT | rpc-1 RNAi diluted (1:1 with control) | 11.67 | 10.61 | WT<0.001 | 81 | |||
| WT rpc-1:NS | ||||||||
| WT let-363/ceTOR:NS | ||||||||
| WT | let-363/CeTOR RNAi diluted (1:1 with control) | 11.63 | 10.23 | WT<0.001 | 83 | |||
| WT rpc-1:NS | ||||||||
| WT let-363/ceTOR:NS | ||||||||
| WT rpc-1:NS | ||||||||
| WT | rpc-1 RNAi + let-363/CeTOR RNAi | 11.97 | 13.45 | WT<0.001 | 76 | |||
| WT rpc-1<0.05 | ||||||||
| WT let-363/ceTOR<0.05 | ||||||||
| WT rpc-1 :NS | ||||||||
| WT let-363/ceTOR :NS | ||||||||
| raga-1(ok386) | Control RNAi | 19.11 | 81.13 | WT<0.001 | 69 | |||
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 <0.001 | ||||||||
| WT let-363/ceTOR <0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1(ok386) | rpc-1 RNAi | 22.15 | 15.90 | 109.95 | WT<0.001 | 71 | ||
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 diluted<0.001 | ||||||||
| WT let-363/ceTOR diluted <0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1(ok386) | rpc-1 RNAi diluted (1:1 with control) | 21.56 | 12.82 | 104.36 | WT<0.001 | 67 | ||
| WT rpc-1<0.001 | ||||||||
| WT let-363/ceTOR<0.001 | ||||||||
| WT rpc-1 diluted<0.001 | ||||||||
| WT let-363/ceTOR diluted<0.001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1 rpc-1:NS | ||||||||
| raga-1(ok386) | CeTOR RNAi diluted (1:1 with control) | 21.38 | 11.87 | 102.65 | WT<0.0001 | 77 | ||
| WT rpc-1<0.0001 | ||||||||
| WT let-363/ceTOR<0.0001 | ||||||||
| WT rpc-1 diluted<0.0001 | ||||||||
| WT let-363/ceTOR diluted <0.0001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.0001 | ||||||||
| raga-1<0.05 | ||||||||
| raga-1 rpc-1:NS | ||||||||
| raga-1 ceTOR:NS | ||||||||
| raga-1(ok386) | rpc-1 RNAi + let-363/CeTOR RNAi | 23.78 | 24.43 | 125.40 | WT<0.0001 | 78 | ||
| WT rpc-1<0.0001 | ||||||||
| WT let-363/ceTOR<0.0001 | ||||||||
| WT rpc-1 diluted<0.0001 | ||||||||
| WT let-363/ceTOR diluted <0.0001 | ||||||||
| WT rpc-1 + let-363/CeTOR<0.0001 | ||||||||
| raga-1 control<0.05 | ||||||||
| raga-1 rpc-1 dil.<0.05 | ||||||||
| raga-1 ceTOR dil.<0.05 |
All biological trials are indicated.
In fruit flies, RNA Pol III acts downstream of TOR to regulate lifespan. RNA Pol III is activated when TORC1 signalling is inhibited with the use of rapamycin (18). In addition, the lifespan increases incurred by rapamycin treatment and Pol III knockdown (both from adulthood) are non-additive, indicating that Pol III mediates the effects of TOR for longevity (18). To test whether a similar scenario exists in worms, we knocked down CeTOR signaling only from the L4 stage using let-363/CeTOR RNAi and examined its epistatic interaction with Pol III. Interestingly, although both knockdown of let-363/CeTOR or rpc-1 from the L4 stage extended lifespan, there was no additive effect when the RNAi treatments were given simultaneously. This indicates that Pol III mediates the effects of CeTOR signaling in adulthood to control lifespan in C. elegans (Fig 2C, Table 1). Taken together, our data suggest that the genetic interaction between CeTOR signaling and RNA Pol III is dependent on the timing of CeTOR reduction, and whereas reducing CeTOR signaling during development engages mediators in addition to Pol III to control lifespan, CeTOR reduction specifically in adulthood could act through Pol III to promote longevity.
RNA polymerase III does not interact with global protein synthesis, insulin or germline signalling to control lifespan
Reducing CeTOR signaling during development engages mediators distinct from Pol III to affect lifespan (Fig 1C). These additional mediators may work downstream of CeTOR to drive organismal development. To identify these, we tested the genetic interaction of Pol III with candidate mediators implicated in protein synthesis or developmental processes. The ribosomal S6 Kinase (S6K) is a direct target of the TOR kinase and regulates global translation (29, 30). In C. elegans, rsks-1 encodes for S6K and animals lacking this gene are long-lived (31) (Fig S3A, Table S1). We found that this longevity was additive, with that incurred by rpc-1 RNAi treatment, suggesting that Pol III and S6K do not interact to extend lifespan (Fig S3A, Table S1). In mammals, another TOR target is the translational repressor, eukaryotic initiation factor 4E-binding protein (4eBP) (29, 32). C. elegans does not encode 4eBP, but animals with mutations in the translation initiation factors, ife-2/eIF4E and ppp-1/eIF2Bgamma, exhibit disruptions in protein translation (33). However, although deletion of either ife-2 or ppp-1 increased C. elegans lifespan, this was additive with rpc-1 RNAi in both instances (Fig S3B and C, Table S1). In addition to CeTOR signalling, lifespan is also controlled by other signaling pathways implicated in development and growth (34). Mutation of the insulin receptor extends lifespan across species and has been implicated in protein synthesis and turnover in C. elegans (35, 36). In C. elegans, daf-2 encodes the insulin receptor and daf-2 loss-of-function mutants are long-lived (37) (Fig S4A, Table S2). However, knockdown of Pol III using rpc-1 RNAi from the L4 stage was additive with daf-2 lifespan, suggesting that Pol III-does not interact with insulin signalling for lifespan. Loss of germline signalling also consistently increases lifespan in model organisms (38). The C. elegans mutants glp-1(e2141ts) and glp-4 (bn2) are both sterile and live significantly longer than wild-types (39, 40). However, both of these mutations were additive with rpc-1 RNAi (Fig S4B and C, Table S2) implying that Pol III-inhibition induced longevity is independent of germline signaling. Taken together, these data show that Pol III knockdown does not seem to affect the lifespan incurred by reducing global protein synthesis, although this does not rule out the possibility that Pol III affects protein synthesis by other mechanisms not interact with global translation mechanisms.
Figure S3. RNA polymerase III does not genetically interact with global translation modulators.
(A, B, C) rpc-1 RNAi additively increases lifespan with rsks-1(ok1255), ppp-1 (syb7781), and ife-2(ok306) mutants. One representative experiment is shown, refer to Table S1 for data on all replicates and statistical analysis.
Figure S4. RNA polymerase III does not genetically interact with insulin or germline signalling.
(A, B, C) rpc-1 RNAi additively increases lifespan with daf-2(m577), glp-1(e2141), and glp-4(bn2) mutants. One representative experiment is shown, refer to Table S2 for data on all replicates and statistical analysis.
Table S1 Lifespan data for indicated figures including all biological trials. (50.2KB, docx)
Table S2 Lifespan data for indicated figures including all biological trials. (50KB, docx)
RNA polymerase III knockdown acts in specific tissues to extend C. elegans lifespan
In both C. elegans and D. melanogaster the longevity phenotype caused by Pol III knockdown appears to be mediated in a tissue-specific manner (18). A detailed contribution of individual tissues has not been tested in worms, so to characterize the expression pattern of Pol III in C. elegans, we examined the expression of a rpc-1::gfp::3xflag translational reporter in wild type adult animals (41). We found RPC-1::GFP in a wide variety of tissues including nuclei of muscle, intestine, hypodermis, neurons, and germline cells (Fig 3A). Treatment of this reporter strain with rpc-1 RNAi, however, reduced expression of RPC-1::GFP in all of these tissues (Fig S5), indicating that these tissues could contribute to the Pol III-inhibition longevity phenotype.
Figure 3. RPC-1 acts tissue specifically to control lifespan.
(A) RPC-1::GFP expression in L4 C. elegans. In each labelled tissue, RPC-1::GFP is localized in the nuclei. Representative images shown. We also noted similar expression patterns throughout adulthood (see also Fig S5). Scale bar indicates 50 μm. (B, C, D, E, F) rpc-1 RNAi specifically in body-wall muscle extends lifespan, but rpc-1 knockdown in the intestine, hypodermis, neurons or germline does not extend lifespan. The intestine-specific, hypodermis-specific, germline-specific and muscle-specific RNAi rescue rde-1 in the rde-1(ne300) strain is under control of the mtl-2, col-62, sun-1, and myo-3 promoters, respectively. (B, C, D, E, F) One representative experiment is shown, refer to Table 2 for data on all replicates and statistical analysis.
Figure S5. Tissue specific expression and function of RNA Polymerase III.
Representative images showing RPC-1::GFP expression in C. elegans fed either control or rpc-1 RNAi in day 5 adults. RPC-1::GFP expression is reduced on worms fed with rpc-1 RNAi in each tissue examined. Scale bar indicates 10 μm.
To explore the requirement of these individual tissues for Pol III-knockdown mediated longevity, we used strains harboring a mutation in the dsRNA processing-pathway gene rde-1. The knockout mutant rde-1(ne300) is strongly resistant to dsRNA induced via the feeding RNAi method, with transgenic rescue of RDE-1 in different tissues allowing tissue-specific knockdown using RNAi (42). We knocked-down rpc-1 using RNAi from early adulthood in the muscle, intestine, hypodermis, neurons and germline, and measured their lifespan. Interestingly, rpc-1 RNAi, when knocked-down specifically in the body-wall muscles, extended the median lifespan by a modest 8%, thus indicating a role for this tissue in mediating Pol III knockdown longevity (Fig 3B, Table 2). However, we found that rpc-1 RNAi in the hypodermis, germline, neurons or intestine could not increase lifespan compared with control (Fig 3C–F, Table 2). Taken together, this suggests that Pol III acts in C. elegans muscle to inhibit lifespan under normal conditions.
Table 2.
Lifespan data for indicated figures including all biological trials.
| Figure | Trial | Strain | Genotype | Tissue affected by RNAi | Mean lifespan (days) | P-value (Log-rank) vs | N dead |
|---|---|---|---|---|---|---|---|
| 3B | 1 | WT | control RNAi | Body-wall muscle | 10.32 | 139 | |
| WT | rpc-1 RNAi | 11.3 | WT cont.<0.05 | 98 | |||
| WM118 | control RNAi | 9.12 | 93 | ||||
| WM118 | rpc-1 RNAi | 9.93 | WM118:Cont<0.05 | 100 | |||
| 2 | WT | control RNAi | Body-wall muscle | 10.11 | 78 | ||
| WT | rpc-1 RNAi | 10.98 | WT cont.<0.05 | 99 | |||
| WM118 | control RNAi | 10.13 | 101 | ||||
| WM118 | rpc-1 RNAi | 9.99 | WM118:Cont<0.05 | 84 | |||
| 3 | WT | control RNAi | Body-wall muscle | 10.486 | 88 | ||
| WT | rpc-1 RNAi | 11.589 | WT cont.<0.05 | 68 | |||
| WM118 | control RNAi | 10.22 | 66 | ||||
| WM118 | rpc-1 RNAi | 11.01 | WM118:Cont<0.05 | 78 | |||
| 3C | 1 | WT | control RNAi | Intestine | 10.51 | 111 | |
| WT | rpc-1 RNAi | 11.48 | WT cont.<0.05 | 106 | |||
| IG1839 | control RNAi | 10.72 | 62 | ||||
| IG1839 | rpc-1 RNAi | 11.01 | IG1839:Cont RNAi:NS | 80 | |||
| 2 | WT | control RNAi | Intestine | 10.77 | 68 | ||
| WT | rpc-1 RNAi | 11.85 | WT cont.<0.05 | 52 | |||
| IG1839 | control RNAi | 10.33 | 72 | ||||
| IG1839 | rpc-1 RNAi | 10.39 | IG1839:Cont RNAi:NS | 79 | |||
| 3 | WT | control RNAi | Intestine | 10.486 | 88 | ||
| WT | rpc-1 RNAi | 11.389 | WT cont.<0.05 | 68 | |||
| IG1839 | control RNAi | 11.692 | 92 | ||||
| IG1839 | rpc-1 RNAi | 11.395 | IG1839:Cont RNAi:NS | 78 | |||
| 3D | 1 | WT | control RNAi | Hypodermis | 10.71 | 111 | |
| WT | rpc-1 RNAi | 11.65 | WT cont.<0.05 | 93 | |||
| NR222 | control RNAi | 12.8 | 108 | ||||
| NR222 | rpc-1 RNAi | 12.65 | NR222:Cont RNAi :NS | 118 | |||
| 2 | WT | control RNAi | Hypodermis | 10.71 | 111 | ||
| WT | rpc-1 RNAi | 11.65 | WT cont.<0.05 | 106 | |||
| NR222 | control RNAi | 12.1 | 83 | ||||
| NR222 | rpc-1 RNAi | 12.08 | NR222:Cont RNAi :NS | 79 | |||
| 3 | WT | control RNAi | Hypodermis | 10.56 | 72 | ||
| WT | rpc-1 RNAi | 11.37 | WT cont.<0.05 | 56 | |||
| NR222 | control RNAi | 12.78 | 90 | ||||
| NR222 | rpc-1 RNAi | 12.1 | NR222:Cont RNAi :NS | 94 | |||
| 3E | 1 | WT | control RNAi | Neurons | 10.71 | 111 | |
| WT | rpc-1 RNAi | 11.59 | WT cont.<0.05 | 106 | |||
| TU3401 | control RNAi | 10.08 | 167 | ||||
| TU3401 | rpc-1 RNAi | 10.43 | TU3401:Cont RNAi:NS | 122 | |||
| 2 | WT | control RNAi | Neurons | 10.11 | 91 | ||
| WT | rpc-1 RNAi | 11.25 | WT cont.<0.05 | 77 | |||
| TU3401 | control RNAi | 9.98 | 78 | ||||
| TU3401 | rpc-1 RNAi | 9.90 | TU3401 :Cont RNAi:NS | 78 | |||
| 3F | 1 | WT | control RNAi | Germline | 10.57 | 139 | |
| WT | rpc-1 RNAi | 11.53 | WT cont.<0.05 | 98 | |||
| DCL569 | control RNAi | 10.62 | 168 | ||||
| DCL569 | rpc-1 RNAi | 9.93 | DCL569:Cont RNAi:NS | 121 | |||
| 2 | WT | control RNAi | Germline | 10.57 | 111 | ||
| WT | rpc-1 RNAi | 11.53 | WT cont.<0.05 | 106 | |||
| DCL569 | control RNAi | 9.93 | 99 | ||||
| DCL569 | rpc-1 RNAi | 10.12 | DCL569:Cont RNAi:NS | 101 | |||
| S8 | 1 | WT | control RNAi | Intestine | 10.98 | 88 | |
| WT | rpc-1 RNAi | 12.12 | WT cont.<0.05 | 72 | |||
| VP303 | control RNAi | 9.76 | 82 | ||||
| VP303 | rpc-1 RNAi | 8.89 | VP303:Cont RNAi<0.05 | 69 | |||
| 2 | WT | control RNAi | Intestine | 10.11 | 91 | ||
| WT | rpc-1 RNAi | 11.25 | WT cont.<0.05 | 77 | |||
| VP303 | control RNAi | 9.90 | 71 | ||||
| VP303 | rpc-1 RNAi | 10.98 | VP303:Cont RNAi<0.05 | 78 |
Late-life knockdown of RNA polymerase III is sufficient to extend lifespan and improve age-related health
In mice, the inhibition of TOR by rapamycin has been shown to extend lifespan even when fed late in life (43). Given that the TOR-Pol III axis is important for adult lifespan (Fig 2C, Table 1), we asked whether Pol III knockdown in late adulthood could also extend longevity in C. elegans. To test this, we raised worms on E. coli OP50-1 until day 5 of adulthood and then transferred them to either control or rpc-1 RNAi plates. Excitingly, compared with control fed animals, worms on rpc-1 RNAi lived longer, suggesting that Pol III knockdown in late adulthood effectively extends longevity (Fig 4A, Table 3). Indeed, this late-life knockdown of Pol III resulted in a lifespan almost identical to that incurred by L4 RNAi treatment (Fig S6, Table 3, Filer et al, 2017 (18)). Overall, these results show that Pol III knockdown in late adulthood is sufficient to extend C. elegans lifespan, strengthening our earlier finding that the beneficial longevity effects of Pol III are post-reproduction.
Figure 4. Reducing RNA polymerase III in late life extends lifespan and improves health.
(A) rpc-1 RNAi in late-adulthood extends lifespan. One representative experiment is shown, refer to Table 3 for data on all replicates and statistical analysis. (B) rpc-1 RNAi improves C. elegans thrashing ability in liquid as they age. Measurements taken on days 1, 4, 7, and 11 of adulthood. Combined data from three trials shown, n = < 60 worms per group. t test against age-matched control: **P < 0.005; ns, non-significant. (C) rpc-1 RNAi improves normal C. elegans movement in late life. (D) rpc-1 RNAi specifically in muscle improves normal C. elegans movement in late life. Note that the strain used for muscle specific RNAi also carries a rol-6 marker; therefore, the average movement is decreased compared with those exhibiting normal sinusoidal movement. (E) rpc-1 RNAi improves mitochondrial network organization in body-wall muscles. N = <55 per condition over three independent experiments. Scoring system is shown in Fig S7. Scale bar indicates 20 μm. In (C, D) One representative experiment is shown. n = <20 worms per group. Statistical comparisons were made by One-way ANOVA. P-value: ***P < 0.0001; **P < 0.005; *P < 0.05; ns, non-significant. In (E) control versus rpc-1, RNAi was compared using a Chi-square test P = 0.033.
Table 3.
Lifespan data for indicated figures including all biological trials.
| Figure | Trial | Strain | Genotype | Mean lifespan (days) | P-value (Log-rank) vs | N dead |
|---|---|---|---|---|---|---|
| 4A | 1 | WT | control RNAi (since day 5) | 9.95 | 106 | |
| WT | rpc-1 RNAi (since day 5) | 11.35 | WT cont.<0.0005 | 189 | ||
| 2 | WT | control RNAi (since day 5) | 10.02 | 89 | ||
| WT | rpc-1 RNAi (since day 5) | 11.15 | WT cont.<0.005 | 91 | ||
| 3 | WT | control RNAi (since day 5) | 9.13 | 92 | ||
| WT | rpc-1 RNAi (since day 5) | 11.20 | WT cont.<0.005 | 86 | ||
| S6 | 1 | WT | control RNAi (since L4) | 9.89 | 59 | |
| WT | rpc-1 RNAi (since L4) | 11.23 | WT cont.<0.005 | 59 | ||
| 2 | WT | control RNAi (since L4) | 9.97 | 62 | ||
| WT | rpc-1 RNAi (since L4) | 11.89 | WT cont.<0.005 | 66 |
Figure S6. rpc-1 RNAi from L4 larval stage extends lifespan to a similar extent as late-life rpc-1 RNAi.
rpc-1 RNAi from L4 larval stage extends lifespan similarly to RNAi induced on day fifth adulthood (Fig 4A). One representative experiment is shown, refer to Table 3 for data on all replicates and statistical analysis.
Healthspan is an important indicator of youthfulness and virility and, ideally, an increase in lifespan is complemented by improvement in organismal healthspan (44). We investigated whether Pol III knockdown could also enhance the health and fitness of worms across their lives by testing their ability to move in liquid media using a thrashing assay. As expected, thrashing ability decreased with age but compared with controls, worms on rpc-1 RNAi moved better at later time points (day 7 and 11 of adulthood) than controls, suggesting improved fitness and better late-life health (Fig 4B). We then compared the ability of C. elegans with move independently on food over their lifespan by videoing their natural movement on E. coli seeded plates. Similarly to the thrashing assay, C. elegans moved around on plates less as they aged, but older worms treated with rpc-1 RNAi moved better compared with controls (Fig 4C, day 5 adults P = 0.0003, day 8 adults P = 0.002 compared with age-matched controls). Together with the thrashing data, this supports a role for Pol III in mediating life-long health and overall lifespan.
Given that Pol III knockdown in muscle is sufficient to promote longevity, we also tested whether muscle-specific rpc-1 RNAi improved age-related fitness. We found that indeed rpc-1 knockdown in the muscle was sufficient to improve overall body movement on day 5 of adulthood (Fig 4D, P = 0.001 compared with control). This supports our finding that rpc-1 is acting on muscles to control age-related health.
It is well reported that ageing contributes to disorganization of the sarcomeres in the body-wall muscle, a phenomenon called sarcopenia which is also a general hallmark of mammalian ageing (45, 46). It has also been shown that changes in muscle mitochondrial structure are strongly correlated with the decline of both sarcomeric structure and speed of movement (47). To further explore the underlying dynamics that might contribute to the improved muscle function in rpc-1 knockdown animals, we examined the mitochondrial structures using a marker of body-wall muscle mitochondria. Interestingly, knock down of rpc-1 improved the organization of mitochondrial networks in older worms compared with control (Fig 4E P = 0.033). Taken together, these results suggest that knockdown of Pol III in adulthood leads to significant increase in lifespan because of improvement in muscle-specific health parameters.
Discussion
RNA polymerase III and TOR signalling interact temporally to control development and lifespan
Both TOR signalling and RNA Polymerase III are important regulators of anabolic processes, and control a wide array of cellular functions including reproduction and lifespan (16). Here we show that TOR and Pol III interact in a time-dependent manner, and that knockdown of each component individually, sequentially or in parallel influences the outcome of both reproductive and longevity processes (Fig 5). In C. elegans, we manipulated CeTOR signalling using both genetic mutants (that constitutively reduce CeTOR signalling throughout life) and let-363/CeTOR RNAi (which reduces CeTOR signalling the late L4 stage). We show that the genetic relationship between CeTOR signaling and Pol III is timing dependent. The requirement and collaboration of TOR and Pol III to support developmental processes, and the fact that their reduction in adulthood extends lifespan supports the antagonistic pleiotropy theory of ageing; whereby, certain genes are switched “on” to promote the anabolic programs leading to growth, sexual maturity and reproductive fitness, and “hyperfunction” of these genes in adulthood leads to senescence and ageing (48).
Figure 5. Temporal genetic interactions between TORC1 and RNA Polymerase III.
Model summarizing the interactions between CeTORC1 and RNA Polymerase III required for development, reproduction and lifespan. This relationship is highly temporal: Reducing CeTOR signalling specifically during adulthood requires RNA Pol III for reproduction and lifespan, whereas CeTORC1 reduction during the embryonic and larval period requires other mediators to promote reproduction and longevity. Data supporting this model are shown in Figs 1 and 2 and S1 and S2.
The embryonic lethality of rpc-1 and let-363/CeTOR meant that these genes must be knocked down specifically in adulthood (49, 50). Thus, our epigenetic approach meant that artifacts or reduced RNAi efficacy relating to use of combinatorial RNAi could arise (51). However, we observe the same fecundity and lifespan phenotypes for each gene whether it was knocked down individually or diluted 1:1 with control RNAi (Fig S2A–C and Table 1). In fact, these phenotypes were striking, both supporting our methodology and demonstrating the sensitivity of C. elegans to RNAi knockdown of these crucial genes.
The contrasting effects on fitness and longevity as observed by knockdown of Pol III in early development as opposed to late adulthood are similar to those observed for other signaling pathways including mTOR and insulin. Knockdown of daf-2 from early to mid-adulthood also promotes longevity whereas its knockdown from early larval stages reduces fecundity and reproductive fitness (52). Late-life interventions targeting IGF-1 receptor have also been shown to improve lifespan and healthspan in mice (53). However, whereas the lifespan of daf-2 worms cannot be further extended by let-363/CeTOR inhibition, suggesting a common mechanistic pathway (28), we did not find evidence for a genetic interaction with the C. elegans insulin receptor daf-2 (Fig S4A). In fact, daf-2 mutation and rpc-1 RNAi were additives in our hands, implying independent downstream effectors of Pol III and DAF-2. Our similar results with germline-signaling mutants (Fig S4B and C), which increase longevity by reducing germline activity, lead us to speculate that this could be in part due to the common transcriptional denominator DAF-16, which integrates signals both from the IGF-1/DAF-2 axis and germline-loss driven longevity (38, 54, 55), whereas Pol III knockdown bypasses DAF-16 requirements entirely.
RNA polymerase III inhibition as a modulator of late-life health and lifespan
Studies in a wide variety of model organisms show that mTOR inhibition increases longevity and improves organismal health, spurring interest in mTOR inhibitors for slowing down human ageing (56). However, this can occur at the cost of development, e.g., treating female flies with rapamycin increased lifespan, but significantly reduced brood size (57, 58). In addition, clinical studies have shown that chronic exposure to mTOR inhibitors can result in serious side effects including immunosuppression and metabolic dysfunction, making them unattractive anti-ageing therapies (27). Consequently, transient knockdown of mTOR genetically, or by inhibitors, is a fast emerging paradigm for targeting mTOR complexes. Studies targeting mTORC1 by rapamycin even late in life have shown to extend lifespan in mice models, without major side effects (43, 59). Here we show that whereas Pol III knockdown during early larval stages leads to developmental abnormalities and larval arrest (Fig 1B), Pol III knockdown post-reproduction on the fifth day of adulthood, extends lifespan, suggesting that, similar to TOR, even late perturbations in Pol III activity are sufficient to promote longevity (Fig 4A). Importantly, we also show that Pol III inhibition is sufficient to improve age-related decline in movement (Fig 4C and D), which has consistently been correlated with fitness and improved lifespan in worms (60, 61, 62). Overall, our results make RNA Pol III a promising target molecule for ameliorating post-reproductive age-related decline.
RNA polymerase III acts in muscle to drive ageing
It is known that aging progresses heterogeneously and various tissue-specific responses have been identified to perturb organismal aging. Earlier studies have shown that knockdown of Pol III in the fly gut improves gut barrier function and is sufficient to ameliorate age-related impairments in gut health which may ultimately promote longevity (18). In C. elegans, multiple studies have shown tissue-specific requirements for longevity genes (55, 63) and Pol III RNAi mediated longevity can also be mediated via the intestine (18). The broad expression of rpc-1::GFP (Figs 3A and S5), led us to explore its longevity and health functions in other tissues and found that muscle-specific knockdown of rpc-1 was sufficient to extend lifespan (Fig 3B).
Sarcopenia, which leads to debilitating conditions among the elderly, is characterised by low skeletal muscle mass or strength and is believed to be the most frequent cause of disability and a major risk factor of health-related conditions (64, 65). In C. elegans, even reducing translation in body-wall muscle during development shortens lifespan and increases reproduction (66). Conversely, multiple studies, in both worms and mammals, have shown that improving body-wall muscle function can improve overall health and longevity (67, 68, 69), with muscle mitochondrial organisation being a critical contributing factor. For example, loss of RAGA-1/RagA in the nervous system increases lifespan via maintaining mitochondrial networks in muscle (70). Here we show that reducing Pol III/rpc-1 preserves both mitochondrial organisation (Fig 4E) and suppresses the age-related decline observed in older worms (Figs 4D and S7). This strongly supports a role for Pol III in maintaining muscle function with age, with the potential to be used as a novel target molecule for interventions aimed at reducing sarcopenia in human ageing.
Figure S7. Representative myo-3p::mitGFP images for scoring mitochondrial morphology.
Representative images used as reference for scoring organization of mitochondrial network in the body wall muscle of myo-3p::mitGFP animals. Scoring markers were described previously (75). Scale bar indicates 5 μm.
Previously, we showed that rpc-1 RNAi in the intestine-specific RNAi strain VP303 significantly extends lifespan as reported in Filer et al, 2017 (18) (Fig S8). This observation is in contrast to our published data wherein rpc-1 RNAi extends the lifespan of a different intestine-specific RNAi strain rde-1(ne219). However, this difference is likely attributed to the allele-specific differences between rde-1(ne219) and rde-1(ne300); ne219 retains significant RNAi processing capacity and is not completely null; whereas, ne300 deletion allele is completely resistant to RNAi (Watts et al (42)). Thus, the rde-1(ne300) strain we initially tested is likely to be “leaky” and RNAi knockdown might not be limited to the gut of the worms.
Figure S8. Pol III knockdown in “leaky-gut” strain extends lifespan.
rpc-1 RNAi in the intestine-specific RNAi strain VP303 significantly extends lifespan. One representative experiment is shown, refer to Table 2 for data on all replicates and statistical analysis.
Lowering translation or mTORC1 activity in adulthood consistently increases lifespan (20), reduces brood size and improves motility (31, 71). Surprisingly though, suppressing CeTORC1 specifically in body-wall muscle increased reproduction and slowed motility, indicating a role for the muscle in CeTOR-mediated activity (72). We could not test if muscle-specific reduction in Pol III during development altered lifespan owing to the strong larval arrest phenotype of rpc-1 RNAi but suspect that such an intervention may perturb global protein translation and remodel inter-tissue communication via one or many of the small-RNAs transcribed by Pol III.
Exploring additional mediators of the Pol III response
It has been well documented that inhibition of translation delays ageing and evidence suggests that reducing global translation can maintain protein homeostasis, dysregulation of which leads to aging and senescence (71, 73, 74). It is likely that Pol III reduction leads to a decrease in two major RNA types, tRNAs and 5s rRNA, subsequently down-regulating global protein translation. Hence, we were surprised to find that rpc-1 RNAi induced longevity was additive with C. elegans mutants of other key translation mediators (Fig S3). This implies a more nuanced role of Pol III in maintaining a healthy proteome, possibly via improving protein folding or reducing insoluble protein loads, which would be important for lifespan and age-related disease. In the future, addressing the impact of Pol III inhibition on protein levels and quality should prove interesting.
Materials and Methods
C. elegans maintenance and strains
C. elegans were routinely grown and maintained on Nematode Growth Media (NGM) seeded with E. coli OP50-1. The wild-type strain was Bristol N2. The following strains were used:VC222 raga-1(ok386),RB1206 rsks-1(ok1255), RB579 ife-2 (ok306), ppp-1(syb7781), DR1567 daf-2, CB4037 glp-1(e2141), SJ4103 myo-3p::mitGFP, SS104 glp-4(bn2), IG1839 frSi17 II; frIs7 IV; rde-1(ne300) V (intestine-specific RNAi), DCL569 mkcSi13 II; rde-1(mkc36) V (germline-specific RNAi). WM118 rde-1(ne300) V; neIs9 X (muscle-specific RNAi). TU3401 sid-1(pk3321) V; uIs69 V (neuronal-specific RNAi), NR222 rde-1(ne219) V; kzIs9 (hypodermis-specific RNAi), VP303 rde-1(ne219) V; kbIs7 (intestine-specific RNAi). NB: The raga-1(ok386) allele harbors a 1,242 bp deletion at the raga-1/RagA locus that removes almost the entire coding region of the gene.
Lifespan assay
Longevity assays were performed as previously described (Filer et al, 2017 (18)). Briefly, synchronous L1 animals were placed on NGM plates seeded with E. coli OP50-1 until they reached L4. From L1 to L4 larval stage, animals were maintained at 20°C. At L4 stage, worms were shifted to plates seeded with E. coli HT1115 carrying appropriate RNAi clones. These RNAi clones were obtained from the Ahringer RNAi library. The plates were supplemented with 50 μM FuDR solution and lifespans were carried out at 25°C. For combinatorial RNAi knockdown, the two respective overnight RNAi cultures were mixed 1:1 before seeding the plates. In these experiments, controls were also diluted 1:1 with HT115 expressing empty pL4440. Live, dead and censored worms were counted every 2–3 d in the worm populations by scoring movement with gentle prodding when necessary. Data were analyzed and statistics performed using the online freeware OASIS (http://sbi.postech.ac.kr/oasis/surv/).
Brood size measurements
Animals were grown on E. coli OP50-1 plates at 20°C till L4 larval stage. At L4, ∼15 animals were placed on individual NGM plates seeded with the appropriate RNAi. For combinatorial RNAi, see protocol in lifespan section. Progeny production was assessed at 25°C and each parent animal moved to a fresh plate daily until egg-laying ceased. The total number of eggs and larvae were counted after 24 h. Plates in which the animals went missing or showed intestinal bursting were not included in the final analysis. NB: At 25°C (the temperature that we did these experiments) very few animals suffered internal hatching and those that did were taken out of the analysis.
Thrashing assay
Animals were raised to L4 for lifespan assays. At L4, the animals were moved to control RNAi or rpc-1 RNAi seeded plates. On days 4, 7, and 11 of adulthood, worms were put in the isotonic M9 buffer and allowed to acclimatize for 1 min before measuring the number of side-to-side mid-body movements per minute. Fifteen animals were analyzed per condition.
Microscopy
C. elegans carrying the rpc-1::gfp::3Xflag reporter were grown in fed conditions to the L4 stage. myo-3::mitGFP imaging, animals were imaged on day fifth of adulthood with rpc-1 RNAi treatment initiated on day 1. When imaging, animals were immobilized on 2% agarose pads using 0.06% Levamisole and imaged immediately using a Zeiss Confocal Ultra at 40X zoom.
Speed measurement
C. elegans were raised and maintained as for the lifespan assays. At each timepoints, 20–30 worms were moved to a thinly seeded 60 mm plate and were allowed to acclimatize for 10 min. Their individual movements were then recorded using Wormtracker (MBF Bioscience) for 2 min with 60 fpm settings. Average speeds for individual animals were calculated using MS Excel.
Spermatocyte measurement
Hermaphrodite C. elegans were raised and maintained for brood size assays. At the early L4 stage, 30–40 worms were moved to RNAi plates. After 24 h (day 1 of adulthood), worms were washed with 0.01% Tween PBS to remove excess bacteria, fixed in methanol for 5 min. Worms were washed once again with 0.1% Tween PBS. DAPI dye was added immediately and kept for 10 min. Worms were washed once again with 0.1% Tween PBS and mounted on 2.5% agarose pads for visualization. DIC and DAPI images were acquired using a Olympus IX 81 microscope. Individually, distinct spermatocytes for each worm were counted.
Supplementary Material
Acknowledgements
This work was funded by a BBSRC NI award BB/R003629/1 to JMA Tullet and BBSRC award BB/S014365/1 to JMA Tullet, N Alic and C Selman. Some strains were obtained from Caenorhabditis Genetics Center, Minnesota, USA, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD01044). ppp-1(syb7781) was a kind gift from the Denzel laboratory (Cologne, Germany).
Author Contributions
Y Malik: investigation and writing—original draft.
I Goncalves Silva: investigation.
RR Diazgranados: investigation.
C Selman: formal analysis, funding acquisition, and writing—original draft.
N Alic: formal analysis, funding acquisition, and writing—original draft.
JMA Tullet: conceptualization, supervision, methodology, project administration, and writing—original draft, review, and editing.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
Data Availability
All relevant data are within the manuscript and its Supporting Information files.
References
- 1.Cook DA, Slater RJ (1993) Eukaryotic nuclear RNA polymerases (EC 2.7.7.6). Methods Mol Biol 16: 59–72. 10.1385/0-89603-234-5:59 [DOI] [PubMed] [Google Scholar]
- 2.Turowski TW, Tollervey D (2016) Transcription by RNA polymerase III: Insights into mechanism and regulation. Biochem Soc Trans 44: 1367–1375. 10.1042/BST20160062 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Arimbasseri AG, Maraia RJ (2016) RNA Polymerase III Advances: Structural and tRNA functional views. Trends Biochem Sci 41: 546–559. 10.1016/j.tibs.2016.03.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Dieci G, Fiorino G, Castelnuovo M, Teichmann M, Pagano A (2007) The expanding RNA polymerase III transcriptome. Trends Genet 23: 614–622. 10.1016/j.tig.2007.09.001 [DOI] [PubMed] [Google Scholar]
- 5.Goodfellow SJ, White RJ (2007) Regulation of RNA polymerase III transcription during mammalian cell growth. Cell Cycle 6: 2323–2326. 10.4161/cc.6.19.4767 [DOI] [PubMed] [Google Scholar]
- 6.Marshall L, Rideout EJ, Grewal SS (2012) Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila. EMBO J 31: 1916–1930. 10.1038/EMBOJ.2012.33 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Rideout EJ, Marshall L, Grewal SS (2012) Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNA iMet synthesis and systemic insulin signaling. Proc Natl Acad Sci U S A 109: 1139–1144. 10.1073/pnas.1113311109 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Dumay-Odelot H, Durrieu-Gaillard S, da Silva D, Roeder RG, Teichmann M (2010) Cell growth- and differentiation-dependent regulation of RNA polymerase III transcription. Cell Cycle 9: 3687–3699. 10.4161/cc.9.18.13203 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.White RJ, Gottlieb TM, Downes CS, Jackson SP (1995) Cell Cycle Regulation of RNA Polymerase III Transcription Inactivation of the TATA-binding protein-containing complex TFIIIB contributes to the mitotic repression of RNA polymerase III transcription, both in frogs and in humans. Mol Cell Biol 15: 81–84. 10.1128/MCB.15.12.6653 [DOI] [Google Scholar]
- 10.McQueen C, Hughes GL, Pownall ME (2019) Skeletal muscle differentiation drives a dramatic downregulation of RNA polymerase III activity and differential expression of Polr3g isoforms. Dev Biol 454: 74–84. 10.1016/j.ydbio.2019.06.001 [DOI] [PubMed] [Google Scholar]
- 11.Wang X, Gerber A, Chen W-Y, Roeder RG (2020) Functions of paralogous RNA polymerase III subunits POLR3G and POLR3GL in mouse development. Proc Natl Acad Sci U S A 117: 15702–15711. 10.1073/pnas.1922821117 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Schmitt BM, Rudolph KLM, Karagianni P, Fonseca NA, White RJ, Talianidis I, Odom DT, Marioni JC, Kutter C (2014) High-resolution mapping of transcriptional dynamics across tissue development reveals a stable mRNA-tRNA interface. Genome Res 24: 1797–1807. 10.1101/gr.176784.114 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Yeganeh M, Praz V, Carmeli C, Villeneuve D, Rib L, Guex N, Herr W, Delorenzi M, Hernandez N, CycliX consortium (2019) Differential regulation of RNA polymerase III genes during liver regeneration. Nucleic Acids Res 47: 1786–1796. 10.1093/nar/gky1282 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Gouge J, Satia K, Guthertz N, Widya M, Thompson AJ, Cousin P, Dergai O, Hernandez N, Vannini A (2015) Redox signaling by the RNA polymerase III TFIIB-related factor Brf2. Cell 163: 1375–1387. 10.1016/j.cell.2015.11.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Yeganeh M, Hernandez N (2020) RNA polymerase III transcription as a disease factor. Genes Dev 34: 865–882. 10.1101/gad.333989.119 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Kulaberoglu Y, Malik Y, Borland G, Selman C, Alic N, Tullet JMA (2021) RNA polymerase III, ageing and longevity. Front Genet 12: 705122. 10.3389/fgene.2021.705122 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Javidnia S, Cranwell S, Mueller SH, Selman C, Tullet JMA, Kuchenbaecker K, Alic N (2022) Mendelian randomization analyses implicate biogenesis of translation machinery in human aging. Genome Res 32: 258–265. 10.1101/gr.275636.121 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Filer D, Thompson MA, Takhaveev V, Dobson AJ, Kotronaki I, Green JWM, Heinemann M, Tullet JMA, Alic N (2017) RNA polymerase III limits longevity downstream of TORC1. Nature 552: 263–267. 10.1038/nature25007 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Liu GY, Sabatini DM (2020) mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol 21: 246. 10.1038/s41580-020-0219-y [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Saxton RA, Sabatini DM (2017) mTOR signaling in growth, metabolism, and disease. Cell 168: 960–976. 10.1016/J.CELL.2017.02.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Blackwell TK, Sewell AK, Wu Z, Han M (2019) TOR signaling in Caenorhabditis elegans development, metabolism, and aging. Genetics 213: 329–360. 10.1534/genetics.119.302504 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Vannini A, Cramer P (2012) Conservation between the RNA polymerase I, II, and III transcription initiation machineries. Mol Cell 45: 439–446. 10.1016/j.molcel.2012.01.023 [DOI] [PubMed] [Google Scholar]
- 23.Pazdernik N, Schedl T (2013) Introduction to germ cell development in Caenorhabditis elegans. Adv Exp Med Biol 757: 1–16. 10.1007/978-1-4614-4015-4_1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Wu Y, Li M, Yang M, Li M, Yang M, Wu Y (2021) Post-translational modifications in oocyte maturation and embryo development. Front Cell Dev Biol 9: 645318. 10.3389/fcell.2021.645318 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Jewell JL, Russell RC, Guan K-L (2013) Amino acid signalling upstream of mTOR. Nat Rev Mol Cell Biol 14: 133–139. 10.1038/nrm3522 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Schreiber MA, Pierce-Shimomura JT, Chan S, Parry D, McIntire SL (2010) Manipulation of behavioral decline in Caenorhabditis elegans with the rag GTPase Raga-1. PLoS Genet 6: 1000972. 10.1371/journal.pgen.1000972 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Lamming DW, Ye L, Sabatini DM, Baur JA (2013) Rapalogs and mTOR inhibitors as anti-aging therapeutics. J Clin Invest 123: 980–989. 10.1172/JCI64099 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Müller F (2003) Influence of TOR kinase on lifespan in C. elegans. Nature 426: 620. 10.1038/426620a [DOI] [PubMed] [Google Scholar]
- 29.Magnuson B, Ekim B, Fingar DC (2012) Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J 441: 1–21. 10.1042/BJ20110892 [DOI] [PubMed] [Google Scholar]
- 30.Yang M, Lu Y, Piao W, Jin H (2022) The translational regulation in mTOR pathway. Biomolecules 12: 802. 10.3390/biom12060802 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Pan KZ, Palter JE, Rogers AN, Olsen A, Chen D, Lithgow GJ, Kapahi P (2007) Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans. Aging Cell 6: 111–119. 10.1111/j.1474-9726.2006.00266.x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Choo AY, Yoon S-O, Kim SG, Roux PP, Blenis J (2008) Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci U S A 105: 17414–17419. 10.1073/pnas.0809136105 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Derisbourg MJ, Wester LE, Baddi R, Denzel MS, Wester LE (2021) Mutagenesis screen uncovers lifespan extension through integrated stress response inhibition without reduced mRNA translation. Nat Commun 12: 1678. 10.1038/s41467-021-21743-x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Taormina G, Ferrante F, Vieni S, Grassi N, Russo A, Mirisola MG (2019) Longevity: Lesson from model organisms. Genes (Basel) 10: 518. 10.3390/genes10070518 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Depuydt G, Shanmugam N, Rasulova M, Dhondt I, Braeckman BP (2016) Increased protein stability and decreased protein turnover in the Caenorhabditis elegans ins/IGF-1 daf-2 mutant. J Gerontol A Biol Sci Med Sci 71: 1553–1559. 10.1093/GERONA/GLV221 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.White MF (2014) IRS2 integrates insulin/IGF1 signalling with metabolism, neurodegeneration and longevity. Diabetes Obes Metab 16: 4–15. 10.1111/dom.12347 [DOI] [PubMed] [Google Scholar]
- 37.Murphy CT, Hu PJ (2013) Insulin/insulin-like growth factor signaling in C. elegans. In WormBook: The Online Review of C. elegans Biology [Internet], 2005–2018. Pasadena, CA: WormBook. Available at: https://www.ncbi.nlm.nih.gov/books/NBK179230/ [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Antebi A (2013) Regulation of longevity by the reproductive system. Exp Gerontol 48: 596–602. 10.1016/j.exger.2012.09.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Rastogi S, Borgo B, Pazdernik N, Fox P, Mardis ER, Kohara Y, Havranek J, Schedl T (2015) Caenorhabditis elegans glp-4 encodes a valyl aminoacyl tRNA synthetase. G3 (Bethesda) 5: 2719–2728. 10.1534/g3.115.021899 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Kodoyianni V, Maine EM, Kimble J (1992) Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans. Mol Biol Cell 3: 1199–1213. 10.1091/mbc.3.11.1199 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Pham K, Masoudi N, Leyva-Díaz E, Hobert O (2021) A nervous system-specific subnuclear organelle in Caenorhabditis elegans. Genetics 217: 1–17. 10.1093/genetics/iyaa016 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Watts JS, Harrison HF, Omi S, Guenthers Q, Dalelio J, Pujol N, Watts JL (2020) New strains for tissue-specific RNAi studies in Caenorhabditis elegans. G3 (Bethesda) 10: 4167–4176. 10.1534/g3.120.401749 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, Carter CS, et al. (2009) Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460: 392–395. 10.1038/NATURE08221 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Crimmins EM (2015) Lifespan and healthspan: Past, present, and promise. Gerontologist 55: 901–911. 10.1093/geront/gnv130 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Herndon LA, Schmeissner PJ, Dudaronek JM, Brown PA, Listner KM, Sakano Y, Paupard MC, Hall DH, Driscoll M (2002) Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans. Nature 419: 808–814. 10.1038/nature01135 [DOI] [PubMed] [Google Scholar]
- 46.Larsson L, Degens H, Li M, Salviati L, Lee YIl, Thompson W, Kirkland JL, Sandri M (2019) Sarcopenia: Aging-Related loss of muscle mass and function. Physiol Rev 99: 427–511. 10.1152/PHYSREV.00061.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Gaffney CJ, Pollard A, Barratt TF, Constantin-Teodosiu D, Greenhaff PL, Szewczyk NJ (2018) Greater loss of mitochondrial function with ageing is associated with earlier onset of sarcopenia in C. elegans. Aging 10: 3382–3396. 10.18632/AGING.101654 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Gems D (2022) The hyperfunction theory: An emerging paradigm for the biology of aging. Ageing Res Rev 74: 101557. 10.1016/J.ARR.2021.101557 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Long X, Spycher C, Han ZS, Rose AM, Müller F, Avruch J (2002) TOR deficiency in C. elegans causes developmental arrest and intestinal atrophy by inhibition of mRNA translation. Curr Biol 12: 1448–1461. 10.1016/S0960-9822(02)01091-6 [DOI] [PubMed] [Google Scholar]
- 50.Roy D, Kahler DJ, Yun C, Hubbard EJA (2018) Functional interactions between rsks-1/S6K, glp-1/notch, and regulators of Caenorhabditis elegans fertility and germline stem cell maintenance. G3 (Bethesda) 8: 3293–3309. 10.1534/G3.118.200511 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Gönczy P, Echeverri C, Oegema K, Coulson A, Jones SJM, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E, et al. (2000) Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408: 331–336. 10.1038/35042526 [DOI] [PubMed] [Google Scholar]
- 52.Dillin A, Crawford DK, Kenyon C (2002) Timing requirements for insulin/IGF-1 signaling in C. elegans. Science 298: 830–834. 10.1126/science.1074240 [DOI] [PubMed] [Google Scholar]
- 53.Mao K, Quipildor GF, Tabrizian T, Novaj A, Guan F, Walters RO, Delahaye F, Hubbard GB, Ikeno Y, Ejima K, et al. (2018) Late-life targeting of the IGF-1 receptor improves healthspan and lifespan in female mice. Nat Commun 9: 2394. 10.1038/s41467-018-04805-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Qin Z, Hubbard EJA (2015) Non-autonomous DAF-16/FOXO activity antagonizes age-related loss of C. elegans germline stem/progenitor cells. Nat Commun 6: 7107. 10.1038/NCOMMS8107 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Libina N, Berman JR, Kenyon C (2003) Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan. Cell 115: 489–502. 10.1016/S0092-8674(03)00889-4 [DOI] [PubMed] [Google Scholar]
- 56.Kennedy BK, Lamming DW (2016) The mechanistic target of rapamycin: The grand ConducTOR of metabolism and aging. Cell Metab 23: 990–1003. 10.1016/J.CMET.2016.05.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Schmeisser K, Parker JA (2019) Pleiotropic effects of mTOR and autophagy during development and aging. Front Cell Dev Biol 7: 192. 10.3389/fcell.2019.00192 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Bjedov I, Toivonen JM, Kerr F, Slack C, Jacobson J, Foley A, Partridge L (2010) Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab 11: 35–46. 10.1016/J.CMET.2009.11.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Zhang Y, Bokov A, Gelfond J, Soto V, Ikeno Y, Hubbard G, Diaz V, Sloane L, Maslin K, Treaster S, et al. (2014) Rapamycin extends life and health in C57BL/6 mice. J Gerontol A Biol Sci Med Sci 69: 119–130. 10.1093/gerona/glt056 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60.Hahm JH, Kim S, Diloreto R, Shi C, Lee SJV, Murphy CT, Nam HG (2015) C. elegans maximum velocity correlates with healthspan and is maintained in worms with an insulin receptor mutation. Nat Commun 6: 8919. 10.1038/NCOMMS9919 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61.Pincus Z, Smith-Vikos T, Slack FJ (2011) MicroRNA predictors of longevity in Caenorhabditis elegans. PLoS Genet 7: e1002306. 10.1371/JOURNAL.PGEN.1002306 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Huang C, Xiong C, Kornfeld K (2004) Measurements of age-related changes of physiological processes that predict lifespan of Caenorhabditis elegans. Proc Natl Acad Sci U S A 101: 8084–8089. 10.1073/PNAS.0400848101 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63.Zhao Y, Zhang B, Marcu I, Athar F, Wang H, Galimov ER, Chapman H, Gems D (2021) Mutation of daf-2 extends lifespan via tissue-specific effectors that suppress distinct life-limiting pathologies. Aging Cell 20: e13324. 10.1111/ACEL.13324 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.Visser M, Schaap LA (2011) Consequences of sarcopenia. Clin Geriatr Med 27: 387–399. 10.1016/J.CGER.2011.03.006 [DOI] [PubMed] [Google Scholar]
- 65.Janssen I, Baumgartner RN, Ross R, Rosenberg IH, Roubenoff R (2004) Skeletal muscle cutpoints associated with elevated physical disability risk in older men and women. Am J Epidemiol 159: 413–421. 10.1093/AJE/KWH058 [DOI] [PubMed] [Google Scholar]
- 66.Dridi H, Forrester F, Umanskaya A, Xie W, Reiken S, Lacampagne A, Marks A (2022) Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans. Elife 11: e75529. 10.7554/ELIFE.75529 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 67.Laranjeiro R, Harinath G, Hewitt JE, Hartman JH, Royal MA, Meyer JN, Vanapalli SA, Driscoll M (2019) Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration. Proc Natl Acad Sci U S A 116: 23829–23839. 10.1073/pnas.1909210116 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 68.Demontis F, Piccirillo R, Goldberg AL, Perrimon N (2013) The influence of skeletal muscle on systemic aging and lifespan. Aging Cell 12: 943–949. 10.1111/ACEL.12126 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 69.McLeod M, Breen L, Hamilton DL, Philp A (2016) Live strong and prosper: The importance of skeletal muscle strength for healthy ageing. Biogerontology 17: 497–510. 10.1007/S10522-015-9631-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 70.Zhang Y, Lanjuin A, Chowdhury SR, Mistry M, Silva-García CG, Weir HJ, Lee CL, Escoubas CC, Tabakovic E, Mair WB (2019) Neuronal TORC1 modulates longevity via ampk and cell nonautonomous regulation of mitochondrial dynamics in C. Elegans. Elife 8: e49158. 10.7554/ELIFE.49158 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 71.Hansen M, Taubert S, Crawford D, Libina N, Lee S-J, Kenyon C (2007) Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell 6: 95–110. 10.1111/j.1474-9726.2006.00267.x [DOI] [PubMed] [Google Scholar]
- 72.Howard AC, Mir D, Snow S, Horrocks J, Sayed H, Ma Z, Rogers AN (2021) Anabolic function downstream of TOR controls trade-offs between longevity and reproduction at the level of specific tissues in C. elegans. Front Aging 2: 47. 10.3389/FRAGI.2021.725068 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 73.Rogers AN, Chen D, McColl G, Czerwieniec G, Felkey K, Gibson BW, Hubbard A, Melov S, Lithgow GJ, Kapahi P (2011) Life span extension via eIF4G inhibition is mediated by posttranscriptional remodeling of stress response gene expression in C. elegans. Cell Metab 14: 55–66. 10.1016/J.CMET.2011.05.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 74.Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S (2004) Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol 14: 885–890. 10.1016/J.CUB.2004.03.059 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 75.Tataridas-Pallas N, Thompson MA, Howard A, Brown I, Ezcurra M, Wu Z, Silva IG, Saunter CD, Kuerten T, Weinkove D, et al. (2021) Neuronal SKN-1B modulates nutritional signalling pathways and mitochondrial networks to control satiety. PLoS Genet 17: e1009358. 10.1371/JOURNAL.PGEN.1009358 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1 Lifespan data for indicated figures including all biological trials. (50.2KB, docx)
Table S2 Lifespan data for indicated figures including all biological trials. (50KB, docx)
Data Availability Statement
All relevant data are within the manuscript and its Supporting Information files.