Timing of TORC1 inhibition dictates Pol III involvement in longevity in Caenorhabditis elegans

  1. Yasir Malik
  2. Isabel Goncalves Silva
  3. Rene Rivera Diazgranados
  4. Colin Selman
  5. Nazif Alic
  6. Jennifer M. A. Tullet

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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 of 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 C. 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 vs adult programs. Additionally, 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 .

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    The manuscript reports effects on brood size, lifespan and healthspan upon manipulation of C. elegans genes encoding RagA, TOR and Pol III orthologs, as well as other well-characterized lifespan-affecting genes. The results point to complex relationships among TOR and Pol III that are not fully resolved, suggest a role for rpc-1 Pol III that is additive with well-characterized lifespan pathways, indicate a late-life requirement for rpc-1 Pol III to limit lifespan, and, contrary to a previous publication, suggest a muscle requirement for rpc-1 Pol III for lifespan limitation.

    Major comments regarding key conclusions:

    The work demonstrates that brood size is reduced upon rpc-1 Pol III RNAi feeding from the L4 stage. However, no further analysis is provided to show how later aspects of reproduction impair brood. Minimally, ruling out effects on spermatogenesis would be important since sperm number limits self-fertile brood size. It is also unclear from the methods whether the brood size results include embryonic lethality (post-reproduction). Internal hatching, if it occurred, could also affect interpretation of the results. A change in the reproductive period should be noted if it occurred.

    The reviewer is correct that it is important to address the role of Pol III more thoroughly in relation to reproduction.

    • The brood size experiments we present simply record the number of hatched progeny. To develop this analysis further we will present the age-specific fecundity data that we generated whilst doing these assays to demonstrate the impact of Pol III on the reproductive period. In addition, we will quantify and present data on the total brood size (dead eggs and hatched progeny) to address whether Pol III also impact embryonic development.
    • At 25oC (the temperature that we did these experiments) very few animals suffered internal hatching and those that did were taken out of the analysis – therefore this is unlikely to skew the results.
    • The question as to whether Pol III limits egg or sperm function (or later developmental roles) is also interesting and is not yet addressed. To examine this we will: Quantify brood size (dead eggs and hatched progeny) in elegans +/- Pol III RNAi that have been exposed to males during the reproductive period compared to those that reproduce solely as hermaphrodites.

    The authors claim that, similar to the relationship previously concluded from aging studies, rpc-1 acts downstream of TORC1. However, this claim is not well supported. In an effort to circumvent early lethality caused by loss of let-363 ("CeTOR"), they use a mutation in raga-1 RagA and demonstrate a further reduction in brood with rpc-1 RNAi. If raga-1(ok386) were a null this result would demonstrate a relationship that is at least partially parallel, not linear. By contrast, double RNAi with let-363 was "non-additive", suggesting a more linear relationship. However, interpretation of these experiments requires (1) that the raga-1 mutation is null and affects only TORC1 signaling, (2) evidence that the double RNAi worked well (e.g., qPCR; see Ahringer et al. 2006 review regarding issues with multi-RNAi), and (3) failure to consider alternative effects of loss of let-363 (e.g., TORC2). Negative results with RNAi are particularly problematic in the absence of convincing evidence that the RNAi worked well. Moreover, results in Figure 1G are difficult to interpret since the initial values are low. Here and elsewhere the genetics descriptions are unconventional, hampering interpretation. For example, what is meant by a mutation being "incomplete"? That it acts as a hypomorph?

    We understand the concerns of the reviewer:

    For reference, this has been used in several other studies, e.g. doi.org/10.7554/eLife.49158

    • We agree that double RNAi can be challenging. Appropriate controls were used here e.g. each RNAi diluted 50:50 with control RNAi in the single treatments and phenotypes were observed in each case (either brood size or lifespan). However, to address the precise knockdown of rpc-1 and let-363 obtained with RNAi we will perform qPCR in response to single and double RNAi treatment (both in WT and raga-1 mutant elegans).
    • In addition, we will attempt to measure S6Kinase phosphorylation, a downstream readout of TORC1 signalling in response to raga-1 mutation or let-363 RNAi treatment with and without rpc-1 A phosphor S6 Kinase antibody is commercially available and has been used successfully in C. elegans - doi.org/10.7554/eLife.31268
    • Our apologies that the nomenclature was confusing. The CeTOR RNAi nomenclature was ’borrowed’ from other papers describing this tool e.g. org/10.7554/eLife.31268 and doi: 10.1371/journal.pgen.1000972. Here, to make our work clearer, we will change ceTOR to let-363 TOR RNAi and raga-1 to raga-1 RagA in the manuscript – as suggested by the reviewer (see below). The description of ‘incomplete’ mutations will also be amended, and informed by our proposed qPCR analysis.

    Another claim is that rpc-1 Pol III limits adult lifespan downstream of TOR. These results are not convincing. The two treatments (raga-1 mutation as "embryonic" and L4 stage "CeTOR" let-363 RNAi as late) are not directly comparable for reasons noted above, and the double RNAi problem hampers interpretation.

    Our lifespan data points out that the longevity increase upon Pol III knockdown is additive with TOR/let-363, suggesting a mechanism independent of TOR. Indeed, due to lack of ideal reagents, we were forced to try the double RNAi knockdown approach for TOR/let-363 and Pol III/ rpc-1. To make the data interpretation easier, and rule out the possibility of confounding background RNAi to the maximum possible extent, we have included appropriate RNAi controls. Wherever double RNAi has been used, the effect on the phenotype by 50% dilution of target RNAi with empty-vector control, has also been shown independently and used for the statistical comparison with combinatorial RNAi. Our results have shown that diluting let-363 RNAi and rpc-1 RNAi both to 50%, is enough to impart lifespan increase when initiated from L4 stage.

    The nomenclature might be easier to follow if the authors state the actual C. elegans genes manipulated (e.g., let-363 TOR versus raga-1 RagA) rather than using "CeTOR" as a catch-all since these genes are not identical in action.

    Thank you for this suggestion. We will implement this in the manuscript where appropriate.

    Based on genetic interactions (rsks-1, ife-2, ppp-1, daf-2 and germline loss) they show that rpc-1 RNAi further extends the long lifespan conferred by each of the mutant alleles tested, as well as germline loss induced by two different mutant conditions. These results, though negative, are important. The statement that rpc-1 does not affect global protein synthesis is somewhat overstated without additional experimental support.

    We thank the reviewer for supporting our inclusion of ‘negative data’. We agree that our statement relating to protein synthesis is overstated given the data presented. We will soften this to: “rpc-1 does not seem to affect the lifespan incurred by reducing global protein synthesis, although this does not rule out the possibility that Pol III affect protein synthesis by other mechanisms”.

    Extending and challenging their own previous work showing an intestinal focus of activity for rpc-1 in limiting longevity (Filer et al., 2017), and noting that RPC-1::GFP detection can be knocked down by RNAi in several tissues, they use a tissue restricted rde-1 expression approach (or sid-1 for neurons) to test the contribution of intestine, hypodermis, neurons, muscle and germline. This new analysis points to a role for the muscle. This result is intriguing and warrants further experiments. To shore up tissue-specific claims the authors could consider (1) additional drivers for intestine and muscle rde-1 in the RNAi experiments, or, ideally, a different approach such as tissue-specific protein degradation (again with multiple drivers), (2) a sufficiency experiment for muscle (wild-type muscle expression in the mutant to demonstrate reversal of the phenotype, or rescue of RNAi defects with an RNAi-insensitive reagent expressed in muscle).

    Thank for you appreciating the work we have done here and suggesting further experiments. To take your points one at a time: (1) We have already used the most robust tissue-specific alleles generated and reported in the C. elegans literature so far. It would be a significant amount of work to generate new rde-1 driven tissue specific alleles to double check the Pol III levels/ rpc-1 knockdown response in certain tissues, and we feel this is beyond the scope of this project. Suggestion (2) is interesting and would require us to generate a muscle specific rpc-1 strain. However, there are issues with this approach. Firstly, it would require that we have a rpc-1 mutant to rescue – which we don’t as it is embryonically lethal and secondly it would not be possible to do this experiment using RNAi as the RNAi would then knock down the muscle construct.

    The possible explanation for the differences in rde-1 results from the previous work should not be buried in the legends of Figure 3 and Figure S3. Perhaps this leaky background hypothesis should be directly tested (e.g., using the RPC-1::GFP to examine whether residual expression exists in ne219 but not in ne300)? In any case, legend to Figure S3 needs editing: The ne219 background is not itself "intestine-specific", as implied, and the last sentence of Figure S3 legend should be "Thus, the rde-1(ne219)...", right?

    The differences between the different tissue-specific strains is interesting. On reflection we agree with the reviewer that it should be included in the main text. We will describe the differences between the two rde-1 alleles ne219 and ne300 in the appropriate section in the manuscript and state our results.

    Finally, they show that late-adult rpc-1 RNAi extends lifespan over control RNAi and that, by several movement assays, healthspan is improved upon L4 rpc-1 RNAi, even when RNAi is active in muscle (based on WM118).
    The most significant new results are that rpc-1(RNAi) affects brood size, can extend lifespan (though modestly) after day 5 of adulthood, and that muscle may be involved rather than intestine.

    Additional comments:

    Text throughout should clarify TOR vs presumed TORC1. Methods are insufficient. Important aspects of the lifespan methods and raw data are missing - e.g. exact numbers of worms censored. Exact information regarding statistical analysis is lacking (e.g., which tests, corrections for multiple testing). References should be given for all strains. For the rde-1 strains, it would be helpful to include, in addition to the transgene alleles, the actual promoters used to claim tissue specificity. Note, worms do not have "skeletal" muscle, as implied in the discussion. Figure 5 was not helpful for this reviewer. Figure legend to S3A is confusing: the intestinal signal appears stronger or at least equal, not weaker, in the rpc-1 RNAi background. Were these images collected using the exact same exposure settings?

    To address this we will:

    • Standardise genetic notation throughout the manuscript (see specific comments above)
    • Provide more detail on the transgenic alleles used e.g. promoters driving rde-1.
    • The majority of strains were obtained from the CGC but wherever appropriate we will also supply a reference.
    • Expand and revise Material and Methods section to appropriately describe all the statistical analyses performed.
    • Revise lifespan methods to include censoring detail and lifespan Tables to include information on censored animals.
    • Remove the reference to ‘skeletal muscle’ and replace with ‘body wall muscle’.
    • Once we have generated new data on the specific knockdowns and downstream targets achieved with let-363 TOR RNAi and raga-1 RagA mutation, as well as on the brood size/dead eggs effects, we will incorporate this information into Fig. 5A for better clarity and readability.
    • We can see on reflection that Figure S3A is confusing, mainly due to the gut autofluorescence in both the control and rpc-1 RNAi conditions. We will amend this figure to make this clear and include a selection of close up images of each tissue to make it easier to see the tissue specific knockdown by RNAi.

    Reviewer #1 (Significance):

    See above. Study will be of interest to aging community.

    Reviewer #2 (Evidence, reproducibility and clarity):

    The study by Malik and Silva et al describes results of the study investigating the role of RNA Polymerase III in regulating fecundity and lifespan in C. elegans. The authors show that knockdown of Pol III, similar to mTOR suppression, is detrimental for reproduction. Likewise, suppression of either Pol III or mTOR in adult animals extends lifespan via apparently the same pathway. In contrast, Pol III knockdown has an additive effect on lifespan in combination with other established genetic lifespan-extending approaches suggesting that they are working via different mechanisms. Furthermore, using the tissue-specific knockdown of Pol III the authors found that suppression Pol III expression is the muscle, but not other major worm tissues, is sufficient for its lifespan extending effect. Finally, the lifespan extension is also observed when Pol III knockdown is initiated late in adulthood. The overall conclusion is that suppression of Pol III expression late in animal life, particularly in the muscle, is a potential strategy to extend life- and health-span. Overall, the study is well-designed, the tools and results are robust and analysed appropriately. The data presentation is excellent, and the manuscript is clearly written. Addressing the points below will help to improve the clarity further.

    We thank the reviewer for their very positive response to our study and are pleased that they found the data convincing. We are extremely pleased that the reviewer agrees with the design and tools used in this study. We can address all of the review’s comments – as discussed below.

    Major:

    Significant amount of GFP signal is still present in RNAi treated animals, what is the tissue that maintains particularly high levels of expression (Fig. 3A) and how does it affect the conclusions? What is the level of Pol III reduction in different tissues? Could different efficiency of knockdown explain the tissue-specific effect of Pol III downregulation on lifespan? It would be important to show (and, if possible, to quantify) the knockdown efficiency in different tissues using the available reporter

    • This experiment had originally been done to test the efficiency of the RNAi, particularly in tissues where rpc-1 RNAi did not impact lifespan. The reviewer is right though, and this information could be analysed further to enhance our study. Figure 3A shows C. elegans expressing the rpc-1::3xflag::gfp reporter. This was used to a) determine the expression pattern of RPC-1 and b) determine the effect of rpc-1 RNAi on this. We noted that RPC-1::GFP is expressed a wide number of tissues and when the reporter strain is treated with rpc-1 RNAi, it is decreased in all tissues. The ‘green’ observed in the RNAi treatment is unfortunately attributable to autofluorescence generated by lysozymes in the C. elegans intestine and masks some of the effects we saw by eye.
    • To establish the tissue-specific efficiency of Pol III knockdown and also address the confounding issue of the autofluorescence we will now use a combination of quantitative and qualitative fluorescent microscopy to measure the percentage RPC-1::GFP knockdown in each tissue relevant to this study.

    Minor:
    Fig. S3B is not cited in the text and the legend for the figure is somewhat confusing, potentially containing errors, this needs to be clarified.

    We thank the reviewers for pointing this out. The legend for this figure will be re-written as a result of the analysis described above and we will cite it in the main text.

    Reviewer #2 (Significance):

    This is the first thorough study of Pol III knockdown as a lifespan extending strategy in C. elegans. In addition to the different laboratory model (previous study of Pol III in ageing primarily focused on Drosophila), this manuscript also offers several novel insights into consequences of Pol III perturbation at phenotypic, as well as mechanistic level in terms of interaction with other longevity pathways. The study will be of interest to those interested in processes underlying longevity and ageing. Considering that this topic is currently in fashion the publication will probably attract attention of not only specialist but also general public.

    We are extremely pleased that the reviewer shares our enthusiasm for this study and that they find the experimental evidence compelling.

    Reviewer #3 (Evidence, reproducibility and clarity):

    Summary: The paper by Yasir Malik et al investigates the genetic interrelationship between TOR signalling and Pol III expression regarding fecundity and longevity in C. elegans. Based on a previous study that defined a role of Pol III downstream of TOR in longevity across various species, this study looks particularly at the relative timing and tissue requirements for TOR and Pol III inhibition in longevity. Data indicate that Pol III acts downstream of TOR in regulating fecundity while there are additive effects regarding survival. The Pol III effect on longevity is based on its role in the muscle. Finally, health-span parameters mirror the survival data.

    Major comments: This is a nice study the relies on genetic interaction to ask how TOR and Pol III interact. I find the observation that Pol III inhibition extends survival when initiated at day 5 of adulthood very exciting. In general, the study would benefit from additional data that back up the genetic observations._We thank the reviewer for appreciating the study and the novel insights it provides about the TOR-Pol III inter-relationship. We can address reviewer’s comments with the a few, limited experiments. Discussed below.

    In Fig. 1, experiments are done to inhibit TOR to varying degrees in order to perform epistasis experiment. Of course these are difficult to interpret without the use of full KOs/loss of function. So while this is a good solution, it would be important to quantify the level to which TOR signalling is inhibited, optimally with biochemical experiments. We fully appreciate the reviewer’s point. A similar concern was raised by reviewer 1. We propose to address this in two ways: 1) by quantifying mRNA levels by qPCR of let-363 in response to either let-363 TOR RNAi; and 2) by determining the extend of TORC1 activity by using a biochemical readout of the pathway’s activity – S6 Kinase phosphorylation using Western blotting as described here: doi.org/10.7554/eLife.31268

    1. General brood size is very low in the WT worms. Normally, one would expect 250-300 offspring per adult worm. It would be helpful if the authors could address this.

    Indeed, as pointed out by the reviewer, the WT worms have a brood size of 250-300 eggs when kept at 20oC. but C. elegans exhibit different brood sizes dependent on temperature and these decline in size with increasing temperature. The experiments shown here were carried out at 25oC, where C. elegans produce less offspring. Our observation is in agreement with other studies of similar nature e.g. doi:10.1371/journal.pone.0112377 and doi.org/10.1371/journal.pone.0145925

    1. Why were lifespan assays performed at 25C? The standard temperature for the worm is 20C and here I think this is very relevant as the TOR pathway is responsive to suboptimal conditions. I wonder if the results are also true for lower temperatures.

    The reviewer raises an interesting point. This study follows from the previous study of Filer et al., Nature 2017 which demonstrated the role of Pol III in ageing. During this study we found and reported that there was a high proportion of intestinal bursting when lifespans were carried out at 20oC, which was ameliorated by carrying out the experiments at 25oC. This was quantified in the original manuscript. To maintain consistency, we continued carrying out Pol III lifespans at this slightly higher temperature. Due to this limitation it is not possible to test the impact of TOR signalling on Pol III at lower temperatures.

    **Minor comments: **

    1. It would help to better delineate the rationale for the experiments in Fig. S1. Experiments here are aimed to find mediators of TOR effects distinct from Pol III. Such distinct mediators would be additive to Pol III (as is the case in the figure) and downstream of TOR.

    Interpreting epistasis analysis is challenging. We were looking for interactors of Pol III using this targeted genetic approach and working on the premise that if two genes interacted then their effects would be non-additive. However, the reviewer is correct that if two genes are doing the same thing independently then their effects may be additive. Although our data does not suggest these mediators interacting with Pol III in the same pathway, it does not rule out the other possibility. When we re-work the manuscript we will explain our rational more clearly and outline the two scenarios.

    Reviewer #3 (Significance):

    Strengths: The study advances our knowledge regarding the timing of the Pol III targeting intervention for survival effects.
    Limitations: The study relies only on genetic data and not all of it is conclusive.

    This study will be interesting for the geroscience community with an eye on TOR inhibition and is relevant to worm biology. I work with C. elegans as a genetic model and I am interested in protein homeostasis, metabolism, health, and longevity.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The paper by Yasir Malik et al investigates the genetic interrelationship between TOR signalling and Pol III expression regarding fecundity and longevity in C. elegans. Based on a previous study that defined a role of Pol III downstream of TOR in longevity across various species, this study looks particularly at the relative timing and tissue requirements for TOR and Pol III inhibition in longevity. Data indicate that Pol III acts downstream of TOR in regulating fecundity while there are additive effects regarding survival. The Pol III effect on longevity is based on its role in the muscle. Finally, health-span parameters mirror the survival data.

    Major comments:

    This is a nice study the relies on genetic interaction to ask how TOR and Pol III interact. I find the observation that Pol III inhibition extends survival when initiated at day 5 of adulthood very exciting. In general, the study would benefit from additional data that back up the genetic observations.

    1. In Fig. 1, experiments are done to inhibit TOR to varying degrees in order to perform epistasis experiment. Of course these are difficult to interpret without the use of full KOs/loss of function. So while this is a good solution, it would be important to quantify the level to which TOR signalling is inhibited, optimally with biochemical experiments.
    2. General brood size is very low in the WT worms. Normally, one would expect 250-300 offspring per adult worm. It would be helpful if the authors could address this.
    3. Why were lifespan assays performed at 25C? The standard temperature for the worm is 20C and here I think this is very relevant as the TOR pathway is responsive to suboptimal conditions. I wonder if the results are also true for lower temperatures.

    Minor comments:

    It would help to better delineate the rationale for the experiments in Fig. S1. Experiments here are aimed to find mediators of TOR effects distinct from Pol III. Such distinct mediators would be additive to Pol III (as is the case in the figure) and downstream of TOR.

    Significance

    Strengths: The study advances our knowledge regarding the timing of the Pol III targeting intervention for survival effects.

    Limitations: The study relies only on genetic data and not all of it is conclusive.

    This study will be interesting for the geroscience community with an eye on TOR inhibition and is relevant to worm biology.

    I work with C. elegans as a genetic model and I am interested in protein homeostasis, metabolism, health, and longevity.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The study by Malik and Silva et al describes results of the study investigating the role of RNA Polymerase III in regulating fecundity and lifespan in C. elegans. The authors show that knockdown of Pol III, similar to mTOR suppression, is detrimental for reproduction. Likewise, suppression of either Pol III or mTOR in adult animals extends lifespan via apparently the same pathway. In contrast, Pol III knockdown has an additive effect on lifespan in combination with other established genetic lifespan-extending approaches suggesting that they are working via different mechanisms. Furthermore, using the tissue-specific knockdown of Pol III the authors found that suppression Pol III expression is the muscle, but not other major worm tissues, is sufficient for its lifespan extending effect. Finally, the lifespan extension is also observed when Pol III knockdown is initiated late in adulthood. The overall conclusion is that suppression of Pol III expression late in animal life, particularly in the muscle, is a potential strategy to extend life- and health-span. Overall, the study is well-designed, the tools and results are robust and analysed appropriately. The data presentation is excellent, and the manuscript is clearly written. Addressing the points below will help to improve the clarity further.

    Major:

    Significant amount of GFP signal is still present in RNAi treated animals, what is the tissue that maintains particularly high levels of expression (Fig. 3A) and how does it affect the conclusions?

    What is the level of Pol III reduction in different tissues? Could different efficiency of knockdown explain the tissue-specific effect of Pol III downregulation on lifespan? It would be important to show (and, if possible, to quantify) the knockdown efficiency in different tissues using the available reporter.

    Minor:

    Fig. S3B is not cited in the text and the legend for the figure is somewhat confusing, potentially containing errors, this needs to be clarified.

    Significance

    This is the first thorough study of Pol III knockdown as a lifespan extending strategy in C. elegans. In addition to the different laboratory model (previous study of Pol III in ageing primarily focused on Drosophila), this manuscript also offers several novel insights into consequences of Pol III perturbation at phenotypic, as well as mechanistic level in terms of interaction with other longevity pathways. The study will be of interest to those interested in processes underlying longevity and ageing. Considering that this topic is currently in fashion the publication will probably attract attention of not only specialist but also general public.

    My expertise is in cellular proteostasis and its perturbation in age-related diseases.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The manuscript reports effects on brood size, lifespan and healthspan upon manipulation of C. elegans genes encoding RagA, TOR and Pol III orthologs, as well as other well-characterized lifespan-affecting genes. The results point to complex relationships among TOR and Pol III that are not fully resolved, suggest a role for rpc-1 Pol III that is additive with well-characterized lifespan pathways, indicate a late-life requirement for rpc-1 Pol III to limit lifespan, and, contrary to a previous publication, suggest a muscle requirement for rpc-1 Pol III for lifespan limitation.

    Major comments regarding key conclusions:

    1. The work demonstrates that brood size is reduced upon rpc-1 Pol III RNAi feeding from the L4 stage. However, no further analysis is provided to show how later aspects of reproduction impair brood. Minimally, ruling out effects on spermatogenesis would be important since sperm number limits self-fertile brood size. It is also unclear from the methods whether the brood size results include embryonic lethality (post-reproduction). Internal hatching, if it occurred, could also affect interpretation of the results. A change in the reproductive period should be noted if it occurred.
    2. The authors claim that, similar to the relationship previously concluded from aging studies, rpc-1 acts downstream of TORC1. However, this claim is not well supported. In an effort to circumvent early lethality caused by loss of let-363 ("CeTOR"), they use a mutation in raga-1 RagA and demonstrate a further reduction in brood with rpc-1 RNAi. If raga-1(ok386) were a null this result would demonstrate a relationship that is at least partially parallel, not linear. By contrast, double RNAi with let-363 was "non-additive", suggesting a more linear relationship. However, interpretation of these experiments requires (1) that the raga-1 mutation is null and affects only TORC1 signaling, (2) evidnce that the double RNAi worked well (e.g., qPCR; see Ahringer et al. 2006 review regarding issues with multi-RNAi), and (3) failure to consider alternative effects of loss of let-363 (e.g., TORC2). Negative results with RNAi are particularly problematic in the absence of convincing evidence that the RNAi worked well. Moreover, results in Figure 1G are difficult to interpret since the initial values are low. Here and elsewhere the genetics descriptions are unconventional, hampering interpretation. For example, what is meant by a mutation being "incomplete"? That it acts as a hypomorph?
    3. Another claim is that rpc-1 Pol III limits adult lifespan downstream of TOR. These results are not convincing. The two treatments (raga-1 mutation as "embryonic" and L4 stage "CeTOR" let-363 RNAi as late) are not directly comparable for reasons noted above, and the double RNAi problem hampers interpretation. The nomenclature might be easier to follow if the authors state the actual C. elegans genes manipulated (e.g., let-363 TOR versus raga-1 RagA) rather than using "CeTOR" as a catch-all since these genes are not identical in action.
    4. Based on genetic interactions (rsks-1, ife-2, ppp-1, daf-2 and germline loss) they show that rpc-1 RNAi further extends the long lifespan conferred by each of the mutant alleles tested, as well as germline loss induced by two different mutant conditions. These results, though negative, are important. The statement that rpc-1 does not affect global protein synthesis is somewhat overstated without additional experimental support.
    5. Extending and challenging their own previous work showing an intestinal focus of activity for rpc-1 in limiting longevity (Filer et al., 2017), and noting that RPC-1::GFP detection can be knocked down by RNAi in several tissues, they use a tissue restricted rde-1 expression approach (or sid-1 for neurons) to test the contribution of intestine, hypodermis, neurons, muscle and germline. This new analysis points to a role for the muscle. This result is intriguing and warrants further experiments. To shore up tissue-specific claims the authors could consider (1) additional drivers for intestine and muscle rde-1 in the RNAi experiments, or, ideally, a different approach such as tissue-specific protein degradation (again with multiple drivers), (2) a sufficiency experiment for muscle (wild-type muscle expression in the mutant to demonstrate reversal of the phenotype, or rescue of RNAi defects with an RNAi-insensitive reagent expressed in muscle). The possible explanation for the differences in rde-1 results from the previous work should not be buried in the legends of Figure 3 and Figure S3. Perhaps this leaky background hypothesis should be directly tested (e.g., using the RPC-1::GFP to examine whether residual expression exists in ne219 but not in ne300)? In any case, legend to Figure S3 needs editing: The ne219 background is not itself "intestine-specific", as implied, and the last sentence of Figure S3 legend should be "Thus, the rde-1(ne219)...", right?
    6. Finally, they show that late-adult rpc-1 RNAi extends lifespan over control RNAi and that, by several movement assays, healthspan is improved upon L4 rpc-1 RNAi, even when RNAi is active in muscle (based on WM118).
    7. The most significant new results are that rpc-1(RNAi) affects brood size, can extend lifespan (though modestly) after day 5 of adulthood, and that muscle may be involved rather than intestine.

    Additional comments

    Text throughout should clarify TOR vs presumed TORC1. Methods are insufficient. Important aspects of the lifespan methods and raw data are missing - e.g. exact numbers of worms censored. Exact information regarding statistical analysis is lacking (e.g., which tests, corrections for multiple testing). References should be given for all strains. For the rde-1 strains, it would be helpful to include, in addition to the transgene alleles, the actual promoters used to claim tissue specificity. Note, worms do not have "skeletal" muscle, as implied in the discussion. Figure 5 was not helpful for this reviewer. Figure legend to S3A is confusing: the intestinal signal appears stronger or at least equal, not weaker, in the rpc-1 RNAi background. Were these images collected using the exact same exposure settings?

    Significance

    See above. Study will be of interest to aging community.

  5. Version published to 10.1101/2022.10.17.512483 on bioRxiv