RNA polymerase III limits longevity downstream of TORC1

Abstract

Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus1. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA2. The historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. Target of rapamycin kinase complex 1 (TORC1) regulates Pol III activity, and is also an important determinant of longevity3. This raises the possibility that Pol III is involved in ageing. Here we show that Pol III limits lifespan downstream of TORC1. We find that a reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting the activity of Pol III in the gut of adult worms or flies is sufficient to extend lifespan; in flies, longevity can be achieved by Pol III inhibition specifically in intestinal stem cells. The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Pol III acts on lifespan downstream of TORC1, and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal mediator of this key nutrient-signalling network for longevity; the growth-promoting anabolic activity of Pol III mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target.

Figure 1: Inhibition of Pol III extends lifespan.
Figure 2: Gut-specific inhibition of Pol III extends lifespan, reduces protein synthesis and increases tolerance to proteostatic stress.
Figure 3: Pol III regulates lifespan downstream of TORC1.
Figure 4: Stem-cell-restricted Pol III inhibition improves age-related dysplasia and gut barrier function.

Accession codes

Primary accessions

ArrayExpress

References

  1. 1

    Roeder, R. G. & Rutter, W. J. Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature 224, 234–237 (1969)

    ADS  CAS  PubMed  Google Scholar 

  2. 2

    Arimbasseri, A. G. & Maraia, R. J. RNA polymerase III advances: structural and tRNA functional views. Trends Biochem. Sci. 41, 546–559 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Kennedy, B. K. & Lamming, D. W. The mechanistic target of rapamycin: the grand conducTOR of metabolism and aging. Cell Metab. 23, 990–1003 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Vannini, A. & Cramer, P. Conservation between the RNA polymerase I, II, and III transcription initiation machineries. Mol. Cell 45, 439–446 (2012)

    CAS  PubMed  Google Scholar 

  5. 5

    Moir, R. D. & Willis, I. M. Regulation of pol III transcription by nutrient and stress signaling pathways. Biochim. Biophys. Acta 1829, 361–375 (2013)

    CAS  PubMed  Google Scholar 

  6. 6

    Grewal, S. S. Why should cancer biologists care about tRNAs? tRNA synthesis, mRNA translation and the control of growth. Biochim. Biophys. Acta 1849, 898–907 (2015)

    CAS  PubMed  Google Scholar 

  7. 7

    Vellai, T. et al. Genetics: influence of TOR kinase on lifespan in C. elegans. Nature 426, 620 (2003)

    ADS  CAS  PubMed  Google Scholar 

  8. 8

    Powers, R. W. III, Kaeberlein, M., Caldwell, S. D., Kennedy, B. K. & Fields, S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 20, 174–184 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Harrison, D. E. et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460, 392–395 (2009)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Bitto, A. et al. Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. eLife 5, e16351 (2016)

    PubMed  PubMed Central  Google Scholar 

  11. 11

    Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature Methods 6, 917–922 (2009)

    CAS  PubMed  Google Scholar 

  12. 12

    Libina, N., Berman, J. R. & Kenyon, C. Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan. Cell 115, 489–502 (2003)

    CAS  PubMed  Google Scholar 

  13. 13

    Piper, M. D., Selman, C., McElwee, J. J. & Partridge, L. Separating cause from effect: how does insulin/IGF signalling control lifespan in worms, flies and mice? J. Intern. Med. 263, 179–191 (2008)

    CAS  PubMed  Google Scholar 

  14. 14

    Espelt, M. V., Estevez, A. Y., Yin, X. & Strange, K. Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C β and γ. J. Gen. Physiol. 126, 379–392 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Poirier, L., Shane, A., Zheng, J. & Seroude, L. Characterization of the Drosophila gene-switch system in aging studies: a cautionary tale. Aging Cell 7, 758–770 (2008)

    CAS  PubMed  Google Scholar 

  16. 16

    Lemaitre, B. & Miguel-Aliaga, I. The digestive tract of Drosophila melanogaster. Annu. Rev. Genet. 47, 377–404 (2013)

    CAS  PubMed  Google Scholar 

  17. 17

    Biteau, B. et al. Lifespan extension by preserving proliferative homeostasis in Drosophila. PLoS Genet. 6, e1001159 (2010)

    PubMed  PubMed Central  Google Scholar 

  18. 18

    Dieci, G., Preti, M. & Montanini, B. Eukaryotic snoRNAs: a paradigm for gene expression flexibility. Genomics 94, 83–88 (2009)

    CAS  PubMed  Google Scholar 

  19. 19

    Laferte, A. et al. The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components. Genes Dev. 20, 2030–2040 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Harding, H. P., Zhang, Y., Bertolotti, A., Zeng, H. & Ron, D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5, 897–904 (2000)

    CAS  PubMed  Google Scholar 

  21. 21

    Nagy, P. et al. Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila. Autophagy 10, 453–467 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Tsokanos, F. F. et al. eIF4A inactivates TORC1 in response to amino acid starvation. EMBO J. 35, 1058–1076 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Fan, X. et al. Rapamycin preserves gut homeostasis during Drosophila aging. Oncotarget 6, 35274–35283 (2015)

    PubMed  PubMed Central  Google Scholar 

  24. 24

    Rera, M., Clark, R. I. & Walker, D. W. Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila. Proc. Natl Acad. Sci. USA 109, 21528–21533.

  25. 25

    Regan, J. C. et al. Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction. eLife 5, e10956 (2016)

    PubMed  PubMed Central  Google Scholar 

  26. 26

    Bjedov, I. et al. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab. 11, 35–46 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Williams, G. C. Pleiotropy, natural-selection, and the evolution of senescence. Evolution 11, 398–411 (1957)

    Google Scholar 

  28. 28

    Verduyn, C., Postma, E., Scheffers, W. A. & Van Dijken, J. P. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501–517 (1992)

    CAS  PubMed  Google Scholar 

  29. 29

    Lee, S. S., Avalos Vizcarra, I., Huberts, D. H., Lee, L. P. & Heinemann, M. Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform. Proc. Natl Acad. Sci. USA 109, 4916–4920 (2012)

    ADS  CAS  PubMed  Google Scholar 

  30. 30

    Huberts, D. H. et al. Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast. Nat. Protoc. 8, 1019–1027 (2013)

    CAS  PubMed  Google Scholar 

  31. 31

    Papagiannakis, A., de Jonge, J. J., Zhang, Z. & Heinemann, M. Quantitative characterization of the auxin-inducible degron: a guide for dynamic protein depletion in single yeast cells. Sci. Rep. 7, 4704 (2017)

    ADS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Gelino, S. et al. Intestinal autophagy improves healthspan and longevity in C. elegans during dietary restriction. PLoS Genet. 12, e1006135 (2016)

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Mathur, D., Bost, A., Driver, I. & Ohlstein, B. A transient niche regulates the specification of Drosophila intestinal stem cells. Science 327, 210–213 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Giannakou, M. E. et al. Long-lived Drosophila with overexpressed dFOXO in adult fat body. Science 305, 361 (2004)

    CAS  PubMed  Google Scholar 

  35. 35

    Niccoli, T. et al. Increased glucose transport into neurons rescues Aβ toxicity in Drosophila. Curr. Biol. 26, 2291–2300 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Rideout, E. J., Marshall, L. & Grewal, S. S. Drosophila RNA polymerase III repressor Maf1 controls body size and developmental timing by modulating tRNAiMet synthesis and systemic insulin signaling. Proc. Natl Acad. Sci. USA 109, 1139–1144 (2012)

    ADS  CAS  PubMed  Google Scholar 

  37. 37

    Bass, T. M. et al. Optimization of dietary restriction protocols in Drosophila. J. Gerontol. A Biol. Sci. Med. Sci. 62, 1071–1081 (2007)

    PubMed  PubMed Central  Google Scholar 

  38. 38

    Alic, N., Hoddinott, M. P., Vinti, G. & Partridge, L. Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor. Aging Cell 10, 137–147 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Hoogewijs, D., Houthoofd, K., Matthijssens, F., Vandesompele, J. & Vanfleteren, J. R. Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Mol. Biol. 9, 10.1186/1471-2199-9-9 (2008)

  40. 40

    Frendewey, D., Dingermann, T., Cooley, L. & Söll, D. Processing of precursor tRNAs in Drosophila. Processing of the 3′ end involves an endonucleolytic cleavage and occurs after 5′ end maturation. J. Biol. Chem. 260, 449–454 (1985)

    CAS  PubMed  Google Scholar 

  41. 41

    Chan, P. P. & Lowe, T. M. GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009)

    CAS  PubMed  Google Scholar 

  42. 42

    Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15, 10.1186/S13059-014-0550-8 (2014)

  44. 44

    Hahn, K. et al. PP2A regulatory subunit PP2A-B′ counteracts S6K phosphorylation. Cell Metab. 11, 438–444 (2010)

    CAS  PubMed  Google Scholar 

  45. 45

    Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6, 275–277 (2009)

    CAS  PubMed  Google Scholar 

  46. 46

    Alic, N. et al. Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling. Mol. Syst. Biol. 7, 10.1038/msb.2011.36 (2011)

  47. 47

    Alic, N. et al. Interplay of dFOXO and two ETS-family transcription factors determines lifespan in Drosophila melanogaster. PLoS Genet. 10, e1004619 (2014)

    PubMed  PubMed Central  Google Scholar 

  48. 48

    O’Brien, L. E., Soliman, S. S., Li, X. & Bilder, D. Altered modes of stem cell division drive adaptive intestinal growth. Cell 147, 603–614 (2011)

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank S. Grewal, B. Ohlstein, L. Partridge and S. Pletcher for fly lines; C. Bouchoux and F. Uhlmann for yeast reagents; G. Juhasz and A. Teleman for antibodies; E. Bolukbasi and L. Partridge for the Flag-tagged dTor construct and S2 cells; M. Hill and D. Ivanov for help with RNA-seq analysis; L. Conder, A. Garaeva, D. Mostapha, G. Phillips and P. van der Poel for technical assistance, and M. Piper, J. Bähler and the IHA members for support, comments and critical reading of the manuscript. Reagents were obtained from Developmental Studies Hybridoma Bank, Vienna Drosophila Resource Centre, Bloomington Stock Center and the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was funded in part by Biotechnology and Biological Sciences Research Council grant BB/M029093/1, Royal Society grant RG140694 and Medical Research Council grant MR/L018802/1 to N.A., and Royal Society grant RG140122 to J.M.A.T. M.H. and V.T. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 642738. D.F. is a recipient of the UCL Impact PhD studentship.

Author information

Affiliations

Authors

Contributions

N.A. conceived the study; D.F. and N.A. made the yeast strains and performed chronological lifespan experiments; V.T. performed and analysed yeast replicative lifespan experiments under the supervision of M.H.; M.A.T. and J.W.M.G. performed and analysed worm experiments under the supervision of J.M.A.T.; D.F., A.J.D., I.K. and N.A. performed and analysed fly experiments under the supervision of N.A.; D.F., M.A.T., J.M.A.T. and N.A. wrote the manuscript with contributions from A.J.D.

Corresponding authors

Correspondence to Jennifer M. A. Tullet or Nazif Alic.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks N. Blewett, R. Maraia and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Inhibition of Pol III in yeast.

a, The growth of strains carrying pADH–OsTir with RPC160–AID, RPB220–AID or the control lacking an AID fusion in the presence or absence of 2.5 mM IAA (single trial). b, Chronological lifespans of the control and RPB220–AID strains treated with 0, 0.125 and 0.25 mM IAA. Top panels show a representative of two experiments, performed in parallel with the RPC160–AID experiment shown in Fig. 1b. The bottom panels show a single experiment; the improved survival of RPB220–AID was also observed at a higher IAA concentration in a second experiment. c, Duration of cell cycle (bottom panels), but not replicative lifespan (top panels), is altered by 1-naphthaleneacetic acid (NAA; analogue of IAA). Both were assessed in the pADH–OsTir RPC160–AID strain on a microfluidics dissection platform. The concentrations of NAA span the dynamic range in which the degree of protein depletion can be efficiently modulated in this setup31. The same control data are shown in each panel for comparison. One experiment was performed for each NAA concentration. For replicative lifespan experiments, 95% c.i. is indicated by shading (or in brackets for median lifespan), together with log-rank P value. A one-sided Mann–Whitney U test was used to test for significant differences in cell-cycle duration. No adjustments were made for multiple comparisons. Dashed lines on bottom panels represent medians. Source data

Extended Data Figure 2 Inhibition of Pol III extends worm lifespan.

a, Lifespan is extended by feeding N2 worms with rpc-1 RNAi at 20 °C in the absence of FUDR (log-rank test, P < 10−3; control and rpc-1 RNAi-treated, n = 100). b, Lifespan is also extended at 25 °C in the presence of FUDR (log-rank test, P = 9 × 10−3; control, n = 60; rpc-1 RNAi, n = 77). c, Summary of each worm lifespan experiment, including the representative trials presented in the figures. P values for log-rank tests are shown. The total number of animals in the trial = dead + censored. In general, fewer worms were censored in control conditions compared to rpc-1 RNAi conditions (mean number of censored N2 worms at 25 °C = 25% (control) and 38% (rpc-1 RNAi); mean number of censored N2 worms at 20 °C = 53% (control) and 73% (rpc-1 RNAi); mean number of censored VP303 at 25 °C = 3% (control) and 4% (rpc-1 RNAi), and mean number of censored VP303 at 20 °C = 37% (control) and 54% (rpc-1 RNAi)), which is likely to be due to an increased number of gut explosions in the rpc-1 RNAi treated worms. 84.9% of censoring events in controls and 85.6% of those in rpc-1 RNAi-treated animals occurred before the 25th percentile of the survival curve. Overall, increasing the temperature to 25 °C reduced censoring without altering our findings. d, Lifespan is extended when the RNAi against rpc-1 is restricted to the gut by using the VP303 strain at 25 °C in the presence of FUDR (log-rank test, P = 9 × 10−3; control, n = 84; rpc-1 RNAi n = 103). In a, b and d, a representative of two trials is shown. Source data

Extended Data Figure 3 Genes corresponding to unique Pol III subunits in Drosophila.

The genes encoding the unique Pol III subunits were identified in fruitflies based on their homology to the yeast genes (BLAST, followed by reverse BLAST), or to the human orthologue.

Extended Data Figure 4 Inhibition of Pol III extends lifespan of flies.

a, Summary of fly lifespan experiments, including the representative trials presented in the figures, but excluding those with rapamycin (see Extended Data Fig. 8a). Experiments were performed on females unless otherwise noted. The total number of animals in the trial = dead + censored. log-rank test P value is reported. b, c, Feeding with RU486 does not have an effect on the lifespans of: UAS-dC160RNAi-only controls (b; log-rank test, P = 0.28; control, n = 142; RU486, n = 146); or TIGS-only controls (c, log-rank test, P = 0.41, control, n = 141 RU486, n = 145). d, Inducing dC53RNAi in the gut by feeding RU486 to female TIGS > dC53RNAi flies extends their lifespan (log-rank test, P = 3 × 10−6; control, n = 143; RU486-treated, n = 139). e, Inducing dC160RNAi predominantly in the fat body by feeding RU486 to S1106 > dC160RNAi flies has no effect on their lifespan (log-rank test, P = 0.21; negative control, n = 158; RU486, n = 155). f, Inducing dC160RNAi in neurons by feeding RU486 to elavGS > dC160RNAi females by RU486 has a modest effect on their lifespan (P = 0.03, log-rank test; negative control, n = 148; RU486, n = 155). g, RU486 feeding does not have an effect on the lifespan of the GS5961-only controls (log-rank test, P = 0.88; negative controls, n = 89; RU486, n = 91). Experiments in bg were performed as single trials. Source data

Extended Data Figure 5 TIGS is active in ISCs.

a, b, Images from the posterior region of the mid-gut showing GFP expression driven by TIGS in the presence of RU486, and stained with antibodies against Prospero (a) and HRP (b). GFP is expressed in cells with small nuclei that are negative for Prospero in a, and those that are positive for HRP in b. Examples of both cell types are indicated with arrows on the merged images. GFP-positive cells can be observed whose morphology and staining pattern correspond closely to those of the ISCs (small nucleus, small cell size, Prospero-negative, HRP-positive; see ref. 48 regarding HRP). TIGS has a complex expression pattern, showing variation between neighbouring cells of the same type and between gut regions. TIGS appears to be active in at least some ISCs. Images are representative of two animals. Images were acquired at 40× (a) or 20× (b) magnification.

Extended Data Figure 6 Effects of dC160RNAi induction in Drosophila adult gut.

ac, Induction of dC160RNAi in the gut of TIGS > dC160RNAi females results in: decreased levels of 45S pre-rRNA (a; MANOVA, P = 4 × 10−3; left to right, c.i. = 0.95–1.1, 0.85–1.0, 0.95–1.1 and 0.81–0.92; EST, 5′ external transcribed spacer; ITS, internal transcribed spacer), indicating a reduction in Pol I activity as a result of Pol III–Pol I crosstalk; unaltered levels of mRNAs encoding ribosomal proteins (b; RNA-seq data, no significant differences at 10% false discovery rate; DESeq2, n = 3 biologically independent samples), indicating no crosstalk between Pol III and Pol II; decreased protein synthesis (c; two further biological repeats and quantification related to Fig. 2e; two-sided t-test, P = 4 × 10−3, n = 3 biologically independent samples; negative controls, c.i. = 0.65–1.4; RU486, c.i. = −0.033–0.68). df, Feeding RU486 to female TIGS-only control flies does not result in a significant decreases in: levels of pre-tRNAs (d; MANOVA, P = 1 × 10−4; left to right, c.i. = 0.96–1.0, 1.1–1.2, 0.93–1.1, 1.1–1.2, 0.91–1.1 and 1.2–1.3); levels of 45S pre-rRNA (e; MANOVA, P = 2 × 10−4; left to right, c.i. = 0.94–1.1, 1.1–1.3, 0.94–1.1 and 1.1–1.2); protein synthesis (f; two-sided t-test, P = 0.74, n = 3 biologically independent samples). gi, Induction of dC160RNAi in the guts of TIGS > dC160RNAi females does not result in significant changes to: total gut protein content (g; two-sided t-test, P = 0.43); female fecundity (h; two-sided t-test, P = 0.51); whole-fly body weight, triacylglycerol or protein content (i; two-sided t-test, P = 0.58, 0.40 or 0.16, respectively). j, Feeding RU486 to TIGS-only control females does not result in increased resistance to tunicamycin (log-rank test, P = 0.89; negative control, n = 149; RU486, n = 153; single trial). Bar charts show mean ± s.e.m.; n, number of biologically independent samples; overlay, individual data points. Gel source data is shown in Supplementary Fig. 1. Source data

Extended Data Figure 7 Regulation of Pol III activity by TORC1 in Drosophila.

a, The antibody raised against a recombinant fragment of Drosophila TOR protein21 and used for ChIP (Fig. 3a) recognizes a single band of the expected size on western blots of S2 cell extracts. b, The same antibody can immunoprecipitate (IP) TOR from S2 cells expressing endogenous and Flag-tagged TOR. c, It can also immunoprecipitate endogenous TOR, and the intensity of this band is reduced upon RNAi treatment against TOR in S2 cells with dsRNA. Single experiments were performed for ac; the ability of the TOR RNAi to reduce the intensity of the band was confirmed in an independent experiment. d, ChIP using a different antibody against Drosophila TOR (raised against a peptide22) shows that relative enrichment of Pol III-transcribed genes is higher than that of Pol II-transcribed genes (linear model with an a priori contrast, P = 2 × 10−4; n = 3 biologically independent samples; left to right, c.i. = 1.6–2.6, 0.81–2.3, 1.1–2.7, 0.77–2.8, −0.24–2.5, −0.065–2.0 and 0.13–1.7). e, No enrichment of Pol III-transcribed genes over Pol II-transcribed genes is observed after mock ChIP with no antibody (linear model with an a priori contrast, P = 0.09, n = 3 biologically independent samples). f, Rapamycin feeding results in a decrease in total RNA content of the adult gut (two-sided t-test, P < 10−4). g, Rapamycin feeding results in reduction of pre-tRNAs relative to total RNA in the fly gut (MANOVA, P = 10−4). h, i, Rapamycin feeding does not result in a reduction of pre-rRNA in the fly gut relative to U3 (h; MANOVA, P < 10−4) or total RNA (i; MANOVA, P = 0.57). j, HA–Maf1 induction specifically in the gut by feeding RU486 to female TIGS > HA–Maf1 flies extends their lifespan (log-rank test, P = 0.006; control, n = 153; RU486, n = 146; single trial). Bar charts show mean ± s.e.m.; n, number of biologically independent samples; overlay, individual data points. Gel source data is shown in Supplementary Fig. 1. Source data

Extended Data Figure 8 Relationship between TORC1 and Pol III.

a, Summary of fly lifespan experiments examining the epistasis between Pol III and TORC1 inhibition (top), including the results of CPH analyses (bottom). The summary (top) shows log-rank test P values, relative to the no-RU486, no-rapamycin control, and the total number of animals in the trial = dead + censored. b, Induction of dC53RNAi in the adult gut by feeding RU486 to female TIGS > dC53RNAi flies, and rapamycin feeding both extend lifespan and their effects are not additive (for statistical analysis see a; control, n = 135; RU486, n = 135; rapamycin, n = 120; rapamycin + RU486, n = 137; single trial). cf, Rapamycin, but not induction of dC160RNAi in the gut of female TIGS > dC160RNAi flies with RU486, reduces phosphorylation of S6K in the gut (linear model; rapamycin, P = 3 × 10−4; RU486, P = 0.77; interaction, P = 0.55; left to right, c.i. = 0.51–1.5, 1.0–1.2, 0.33–0.73 and 0.08–0.91) and whole flies (linear model; rapamycin, P < 10−4; RU486, P = 0.10; interaction, P = 0.16; left to right, c.i. = 0.77–1.2, 0.60–1.0, 0.016–0.19 and 0.019–0.15). Additional biological repeats related to Fig. 3g are presented for the gut (c) and the whole fly (d). These are quantified in e and f, respectively. cf show data from four biologically independent samples. Gel source data are shown in Supplementary Fig. 1. Source data

Extended Data Figure 9 Inhibition of Pol III in the gut preserves organ health.

a, Induction of dC160RNAi in the gut by feeding RU486 to female adult TIGS > dC160RNAi flies suppresses accumulation of pH3-positive cells in old flies (two-tailed t-test, P = 1 × 10−3; control, c.i. = 58–110; RU486, c.i. = 10–46). b, Induction of dC160RNAi in the gut by feeding RU486 to adult female TIGS > dC160RNAi flies suppresses loss of gut barrier function (number of Smurfs) in old flies (χ2-test, P = 5 × 10−4; control, c.i. = 16–26%; RU486, c.i. = 8.7–16%, percentage of Smurfs). c, rpc-1 RNAi suppresses the severity of the age-related loss of gut barrier function in worms (OLR; effect of age, P < 10−4; rpc-1 RNAi, P = 0.51; interaction, P = 0.01; left to right, c.i. = 5.0–31%, 16–50%, 24–48%, 25–51%, 53–78% and 34–66%, percentage of Smurf grades 3 and 4). Age-related loss of gut barrier function in worms has been described previously32. d, Induction of dC160RNAi in the gut by feeding RU486 to adult male TIGS > dC160RNAi flies results in a small but significant extension of lifespan (log-rank test, P = 0.03; no-RU486, n = 141; RU486, n = 139; single trial). Bar charts show mean ± s.e.m.; n, number of animals; overlay, individual data points. Source data

Supplementary information

Life Sciences Reporting Summary (PDF 74 kb)

Supplementary Fig. 1

This file contains source data for gels. (PDF 2114 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Filer, D., Thompson, M., Takhaveev, V. et al. RNA polymerase III limits longevity downstream of TORC1. Nature 552, 263–267 (2017). https://doi.org/10.1038/nature25007

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.