Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. 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 longevity. 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.
Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. 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 longevity. 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.
The labour of transcription in the eukaryotic nucleus is divided amongst Pol I,
II and III1,4. This specialisation is evident in the biogenesis of the translation
machinery, a task for which all three Pols are co-ordinately required: Pol I generates
the 45S pre-rRNA that is subsequently processed into mature rRNAs, Pol
II transcribes various RNAs including messenger RNAs (mRNAs) encoding ribosomal proteins
(RPs), while Pol III provides the tRNAs and 5S rRNA. To match the
extrinsic conditions and the intrinsic need for protein synthesis, this costly process
of generating protein synthetic capacity is tightly regulated by the key driver of
cellular anabolism: TORC15,6. The central position of TORC1 in the control of fundamental
cellular processes is mirrored by the striking impact of its activity on organismal
physiology: following the initial discovery in worms7, the inhibition of TORC1 has been demonstrated to extend lifespan in all
organisms tested, from yeast to mice8,9, with beneficial effects on a range of age-related
diseases and dysfunctions3,10. TORC1 strongly activates Pol III transcription5,6 and this
relationship suggests the possibility that inhibition of Pol III promotes longevity.
Here, we test this hypothesis.In S. cerevisiae, each of the 17 Pol III subunits is encoded by
an essential gene. We generated a yeast strain in which its largest subunit (C160,
encoded by RPC160/RPO314) is
fused to the auxin-inducible degron (AID). The fusion protein can be targeted for
degradation by the ectopically expressed E3 ubiquitin ligase (OsTir) in the presence of
indole-3-acetic acid (IAA)11 to achieve
conditional inhibition of Pol III (Extended Data Fig.
1a). We confirmed that IAA treatment triggered the degradation of the fusion
protein (Fig. 1a) and observed IAA improve the
survival of the RPC160-AID strain upon prolonged culture (Fig. 1b). In addition, IAA treatment of the control
strain lacking the AID fusion reduced its survival, relative to same strain in absence
of IAA and the RPC160-AID strain in presence of IAA (Extended Data Fig. 1b). Hence, Pol III depletion
appears to extend yeast chronological lifespan. Note that IAA had no substantial effect
on the survival of a strain carrying the AID domain fused to the largest subunit of Pol
II (RPB220-AID), which appeared to survive better than the control
strain in the presence of IAA (Extended Data Fig. 1a and
b), indicating inhibition of Pol II may also extend chronological lifespan.
Yeast chronological lifespan is a measure of survival in a nutritionally-limited,
quiescent population. Replicative lifespan, on the other hand, measures the number of
daughters produced by a single mother cell in its lifetime. We found no evidence that
inhibition of Pol III causes an increase in yeast replicative lifespan (Extended Data Fig. 1c).
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 any 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
RPC160-AID 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, Yeast replicative lifespan (top panels)
is not altered by 1-naphthaleneacetic acid (NAA; analogue of IAA) while cell
cycle duration (bottom panels) is. Both were assessed in the
pADH-OsTir RPC160-AID strain on a microfluidics
dissection platform. The concentrations of NAA span the dynamic range where
the degree of protein depletion can be efficiently modulated in this
set-up30. The same control data
are shown in each panel for comparison. For each NAA concentration one
experiment was performed. For replicative lifespans, 95% CIs are indicated
by shading, or in brackets for median lifespan, together with
log-rank p value. 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.
Figure 1
Inhibition of Pol III extends lifespan.
Treatment of the RPC160-AID-myc pADH-OsTir-myc budding yeast
strain with 0, 0.125 and 0.25 mM IAA: a, triggers degradation of
C160-AID-myc and b, extends its chronological lifespan, measured as
colony formation after normalisation for optical density and 10-fold serial
dilution ([a] and [b] show a representative of two experimental trials). Feeding
N2 worms with E. coli expressing the rpc-1
RNAi construct from L4 stage: c, reduces the levels of
rpc-1 mRNA (p<10-4, two-tailed
t-test) and d, extends their lifespan relative to
vector alone at 20°C in presence of FUDR (p=0.03, log-rank
test, n= 86 control, 94 rpc-1 RNAi animals;
representative of three trials). Female flies heterozygous for the
dC53 allele display: e, a
reduction in dC53 transcript (p=10-4,
two-tailed t-test, 95% confidence intervals [CI] = 0.89-1.1
wt, 0.64-0.71 dC53) and f, extended
lifespan (p=6x10-13, log-rank test, n= 152 control,
144 dC53 animals; single trial). Bar charts show
mean ± Standard Error of the Mean (SEM), with n= number of biologically
independent samples indicated and overlay showing individual data points. For
more detailed demography and summary of worm and fly lifespan trials see Extended Data Fig. 2c and 4a. For gel source
data, see Sup. Fig.
1.
The observed increase in yeast chronological lifespan may simply be indicative of
increased stress resistance and hence bear limited relevance to organismal ageing. To
examine the role of Pol III in organismal ageing directly, we turned to animal models.
We treated C. elegans from the L4 stage with RNAi against
rpc-1, the worm orthologue of RPC160, achieving a
partial knockdown of its mRNA (Fig. 1c). This
consistently extended worm lifespan at both 20°C and 25°C (Fig. 1d; Extended Data
Fig. 2a and b, see c for summary of worm lifespans). To reduce
Pol III activity in the fruit fly, we backcrossed a P-element insertion deleting the
transcriptional start site of the gene encoding the Pol III-specific subunit C53
(CG5147, henceforth called
dC53, Extended Data
Fig. 3) into a healthy, outbred D. melanogaster population.
Homozygous dC53 mutants were not viable but
heterozygous females had a partial reduction in dC53 mRNA and lived
longer than controls (Fig. 1e and f; see Extended Data Fig. 4a for summary of fly lifespans).
Taken together, our data strongly indicate that Pol III limits lifespan in multiple
model organisms and, conversely, that partial inhibition of its activity is an
evolutionarily conserved longevity intervention.
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 absence of FUDR
(p<10-3
log-rank test, n= 100 control and rpc-1
RNAi treated animals). b, It is also extended at 25°C in
presence of FUDR (p=9x10-3, log-rank test, n= 60
control, 77 rpc-1 RNAi animals). c, Summary of
each worm lifespan experiment performed including the representative trials
presented in the figures. Log-rank test p value is
reported. The total number of animals in the trial = dead + censored. In
general, fewer worms were censored in control vs rpc-1 RNAi
conditions (average of N2 at 25°C 25% vs 38%; average of N2 at
20°C 53% vs 73%; average of VP303 at 25°C 3% vs 4% and average
of VP303 at 20°C 37% vs 54%) which is likely to be due to an
increased number of gut explosions in the rpc-1 RNAi
treated worms. On average 84.9% of control and 85.6% of
rpc-1 RNAi-treated censored events 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 using VP303 strain, at
25°C in presence of FUDR (p=9x10-3, log-rank
test, n= 84 control, 103 rpc-1 RNAi treated
animals). In (a), (b) and (d), a representative of two trials is shown.
Extended Data Figure 3
Genes corresponding to unique Pol III subunits in
Drosophila.
The genes encoding the unique Pol III subunits were identified in
fruit flies 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 fly lifespan.
a, Summary of each fly lifespan experiment performed
including the representative trials presented in the figures but excluding
the ones 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. RU486 feeding does
not have an effect on the lifespans of: b,
UAS-dC160 alone (p=0.28,
log-rank test, n= 142 -RU486, 146 +RU486 animals); or
c, TIGS alone controls (p=0.41,
log-rank test, n= 141 -RU486, 145 +RU486 animals).
d, Inducing dC53 in the gut
of TIGS>dC53 females by RU486 extends
their lifespan (p=3x10-6, log-rank test, n= 143
-RU486, 139 +RU486 animals). e, Inducing
dC160 predominantly in the fat body
of S females by
RU486 has no effect on their lifespan (p=0.21, log-rank
test, n= 158 -RU486, 155 +RU486 animals). f,
Inducing dC160 in neurons of
elavGS>dC160 females by RU486
has a modest effect on their lifespan (p=0.03, log-rank
test, n= 148 -RU486, 155 +RU486 animals). g, RU486
feeding does not have an effect on the lifespan of the
GS5961 alone control (p=0.88, log-rank
test, n= 89 -RU486, 91 +RU486 animals). In (b) to (g) the
single trial performed is shown.
The longevity of an animal can be governed from a single organ. In the worm, this
role is often played by the gut12,13. To restrict the rpc-1
knock-down to the gut, we used rde-1 null worms whose RNAi machinery
deficiency is restored solely in the gut by gut-specific rde-1
rescue14. rpc-1 RNAi
extended the lifespan of this strain, both at 20°C and 25°C (Fig. 2a, Extended Data
Fig. 2d). Similarly, in the adult fly, driving an RNAi construct targeting
the RPC160 orthologue (CG17209, henceforth called
dC160,
Extended Data Fig. 3) with the mid-gut-specific,
RU486-inducible driver (TIGS) extended female lifespan (Fig. 2b), while the presence of the inducer (RU486)
did not affect survival of the control lines (Extended
Data Fig. 4b and c). The longevity phenotype could also be recapitulated with
RNAi against another Pol III subunit (dC53, Extended Data Fig. 4d), indicating it is not due to off-target
effects, or subunit-specific. The longevity phenotype appeared specific to the gut,
since no significant lifespan extension was observed upon induction of
dC160 in the adult fly fat-body and only a
modest, albeit significant extension resulted from neuronal induction (Extended Data Fig. 4e and f); fat-body and neurons
being other two sites often associated with longevity13.
Figure 2
Gut-specific inhibition of Pol III extends lifespan, reduces protein
synthesis and increases tolerance to proteostatic stress.
a, Activating RNAi against rpc-1 specifically in
the worm gut, using the VP303 strain, extends worm lifespan at 20°C in
presence of FUDR (p=0.02, log-rank test, n= 90 control, 67
rpc-1 RNAi animals; representative of two trials).
b, Feeding RU486 to
TIGS>dC160 female fruit flies to
induce dC160 in the gut alone extends their
lifespan (p=6x10-16, log-rank test, n= 150 -RU486,
157 +RU486 animals; representative of three trials). c, Feeding
RU486 to GS5961>dC160 female fruit flies
to induce dC160 in the ISCs alone extends their
lifespan (p=2x10-4, log-rank test, n= 139 -RU486,
142 +RU486 animals; representative of three trials). Inducing
dC160 in the gut with RU486 feeding of
TIGS>dC160 females leads to:
d, reduction in pre-tRNAs (mean ± SEM, p=0.04,
Multivariate Analysis of Variance
[MANOVA], n= 10 biologically independent samples per -/+RU486
condition, CI= 0.91-1.1, 0.76-1.0, 0.90-1.1, 0.75-0.92, 0.88-1.1, 0.68-1.0 left
to right); e, reduction in gut protein synthesis, as quantified by
ex-vivo puromycin incorporation and western blotting
(representative of three biologically independent repeats; see Extended Data Fig. 6c); f,
improved survival in response to tunicomycin challenge (p=3x10-15,
log-rank test, n=185 animals per condition; representative
of two trials). For more detailed demography and summary of lifespan trials see
Extended Data Fig. 2c and 4a. For gel
source data, see Sup. Fig.
1.
Worm gut is composed of only post-mitotic cells. In flies, like in mammals, the
adult gut epithelium contains the mitotically active ISCs15. ISC homeostasis is important for longevity16 and the TIGS gut driver appears active in at
least some ISCs (Extended Data Fig. 5), prompting
us to further restrict dC160 induction to solely this
cell type. ISC-specific dC160, achieved with the
GS5961 driver, was sufficient to promote longevity (Fig. 2c, see Extended
Data Fig. 4b and g for controls). In summary, Pol III activity in the gut
limits survival in worms and flies, and in the fly, Pol III can drive ageing
specifically from the gut stem cell compartment.
Extended Data Figure 5
TIGS is active in ISCs.
Images from the posterior region of the midgut showing GFP
expression driven by TIGS in the presence of RU486 and
stained with: a, anti-Prospero; b, anti-HRP. GFP
expression can be observed in cells with small nuclei that are
Prospero-negative in (a) and those that stain with anti-HRP in (b). Examples
of both types are indicated with a white
“>” on the merged images. GFP-positive
cells can be observed whose morphology and staining pattern correspond
closely to that of the ISCs (small nucleus, small cell size,
Prospero-negative, anti-HRP-positive [see ref. 48 regarding anti-HRP]). TIGS has a
complex expression pattern, showing variation between neighbouring cells of
the same type and between gut regions. TIGS appears active
in at least some ISCs. Images are representative of two animals.
We assessed the consequences of Pol III inhibition in the fly gut. Pol III acts
to generate precursor-tRNAs (pre-tRNAs) that are rapidly processed to mature tRNAs. Due
to their short half-lives, pre-tRNA are suitable readouts of in-vivo
Pol III activity. Profiling the levels of pre-tRNA,
pre-tRNA and
pre-tRNA, relative to the levels of
U3, a small nucleolar RNA transcribed by Pol II17, revealed a moderate but significant reduction
in Pol III activity upon gut-specific induction of dC160
(Fig. 2d). The three Pols can be directly
coordinated for the generation of translation machinery18. Indeed, Pol III inhibition had knock-on effects on Pol I but not Pol
II-generated transcripts, revealing a partial cross-talk (Extended Data Fig. 6a and b). Consistent with reduced Pol III activity,
dC160 reduced protein synthesis in the gut
(Fig. 2e, Extended Data Fig. 6c). These effects (reduction in pre-tRNAs or protein
synthesis) were not observed after feeding RU486 to the driver-alone control (Extended Data Fig. 6d - f). The reduction in protein
synthesis was not pathological: total protein content of the gut was unaltered;
fecundity, a sensitive readout of a female’s nutritional status, was unaffected;
and the flies’ weight, triacylglycerol and protein levels also remained unchanged
(Extended Data Fig. 6g - i). Reduced protein
synthesis can liberate protein-folding machinery from protein production and increase
homeostatic capacity19. Indeed, inducing
dC160 in the gut increased the resistance of
adult flies to a proteostatic challenge with tunicamycin (Fig. 2f, and Extended Data Fig. 6j for
TIGS-alone control). Hence, Pol III can fine-tune the rate of
protein synthesis in the adult fly gut, without obvious detrimental outcomes, while
increasing resistance to proteotoxic stress.
Extended Data Figure 6
Effects of dC160 induction in
Drosophila adult gut.
Induction of dC160 in the guts of
TIGS>dC160 females results
in: a, decreased levels of 45S pre-rRNA
(p=4x10-3, MANOVA, CI= 0.95-1.1, 0.85-1.0,
0.95-1.1, 0.81-0.92 left to right; EST = 5’ external transcribed
spacer, IST = internal transcribed spacer), indicating a reduction in Pol I
activity as a result of Pol III – Pol I crosstalk; b,
unaltered levels of mRNAs encoding ribosomal proteins (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; c, decreased protein
synthesis (two further biological repeats and quantification related to
Fig. 2e; p=4x10-3,
two-sided t-test, n=3 biologically independent samples,
CI= 0.65-1.4 -RU486, -0.033-0.68 +RU486). RU486 feeding of
TIGS-alone control females does not result in a
significant decrease in: d, levels of pre-tRNAs
(p=1x10-4, MANOVA, CI= 0.96-1.0, 1.1-1.2,
0.93-1.1, 1.1-1.2, 0.91-1.1, 1.2-1.3 left to right); e, levels
of 45S pre-rRNA (p=2x10-4,
MANOVA, CI= 0.94-1.1, 1.1-1.3, 0.94-1.1, 1.1-1.2 left
to right); f, protein synthesis (p=0.74, two-sided
t-test, n=3 biologically independent samples).
Induction of dC160 in the guts of
TIGS>dC160 females does not
result in significant changes to: g, total gut protein content
(p=0.43, two-sided t-test); h, female
fecundity (p=0.51, two-sided t-test); i,
whole-fly body weight, triacylglycerol or protein content (p=0.58, 0.40,
0.16 respectively, two-sided t-test). j, RU486
feeding of TIGS-alone control females does not result in
increased resistance to tunicamycin (p=0.89, log-rank test,
n= 149 -RU486, 153 +RU486 animals; single trial). Bar charts show mean
± SEM, with n= number of biologically independent samples indicated
and overlay showing individual data points. For gel source data see Sup. Fig. 1.
Having demonstrated the relevance of Pol III for ageing, we examined whether it
acts downstream of TORC1 for lifespan using the fruit fly. Numerous observations in
several organisms support the model where TORC1 localises on Pol III-transcribed loci
and promotes the phosphorylation of the components of Pol III transcriptional machinery
to activate transcription, in part by inhibition of the Pol III repressor, Maf15. Using chromatin immunoprecipitation (ChIP) with
two independently generated antibodies against Drosophila TOR20,21, we
observed the kinase enriched on Pol III-target genes in the adult fly, relative to Pol
II targets (Fig. 3a; Extended Data Fig. 7a to d, and e for mock ChIP). Inhibition of TORC1 by
feeding flies rapamycin reduced the levels of pre-tRNAs in whole flies (Fig. 3b). Rapamycin also reduced pre-tRNA levels
specifically in the gut relative to U3 (Fig 3c). Since rapamycin results in re-scaling of the gut, evidenced by the
reduction in the organ’s total RNA content (Extended Data Fig. 7f), we also confirmed that the drug reduced pre-tRNA
levels relative to total RNA (Extended Data Fig.
7g). Interestingly, rapamycin did not cause a decrease in
45S pre-rRNA in the gut (Extended
Data Fig. 7h and i), suggesting a lack of sustained Pol I inhibition.
Additionally, gut-specific over-expression of Maf1 reduced the levels
of pre-tRNAs and extended lifespan (Fig 3d, Extended Data Fig. 7j), confirming this Pol III
repressor acts on Pol III in the adult gut. Our data are consistent with TORC1 driving
systemic and gut-specific Pol III activity in the adult fruit fly.
Figure 3
Pol III acts downstream of TORC1 for lifespan.
a, Relative enrichment of Pol III-transcribed genes is higher than
that of Pol II-transcribed genes after ChIP against TOR
(p<10-4, Linear Model
[LM] with an a priori contrast, n= 3
biologically independent samples). Rapamycin feeding causes a decrease in
pre-tRNAs relative to U3 in: b, whole female flies
(p=0.01, MANOVA, CI= 0.76-1.2, 0.58-0.79, 0.75-1.3, 0.49-0.79,
0.70-1.3, 0.48-0.78 left to right); c, their guts (p=0.01,
MANOVA, CI= 0.98-1.1, 0.90-1.0, 0.87-1.1, 0.74-0.99,
0.90-1.1, 0.77-0.97 left to right). d, Induction of
Maf1 in the guts of TIGS>HA-Maf1
females by RU486 feeding reduces the levels of pre-tRNAs relative to U3
(p=4x10-3, MANOVA, CI= 0.87-1.1, 0.74-1.0,
0.90-1.1, 0.82-1.0, 0.76-1.2, 0.53-1.0 left to right). e, Induction
of dC160 in the adult guts by RU486 feeding of
TIGS>dC160 females and rapamycin
feeding both extend lifespan and are not additive (effect of rapamycin
p=2x10-14, effect of RU486 p<2x10-16,
interaction p=7x10-9, Cox Proportional Hazards
[CPH], n= 135 control, 144 +RU486, 141 +rapamycin and 146
+RU486 and rapamycin animals; single trial). f, Induction of
dC160 in the ISCs by RU486 in
GS5961>dC160 females and
rapamycin both extend lifespan and are not additive (effect of rapamycin
p=8x10-14, effect of RU486 p=2x10-5, interaction
p=3x10-7, CPH, n= 113 control, 130 +RU486, 145
+rapamycin and 144 +RU486 and rapamycin animals; single trial). Rapamycin but
not dC160 induction in the gut by RU468 feeding
of TIGS>dC160 females leads to:
g, reduction in S6K phosphorylation in the gut or the whole fly
(representative of four biologically independent repeats; see Extended Data Fig. 8c-f); h,
reduction in egg laying (effect of rapamycin p<10-4, RU486
p=0.87 and interaction p=0.96, LM). i, Model of
the relationship between TORC1, Pol III and lifespan. Bar charts show mean
± SEM, with n= number of biologically independent samples indicated and
overlay showing individual data points. For more detailed demography, statistics
and summary of lifespan trials see Extended Data
Fig. 8a. For gel source data see Sup. Fig.1.
Extended Data Figure 7
Regulation of Pol III activity by TORC1 in
Drosophila.
a, The antibody raised against a recombinant fragment
of Drosophila TOR protein 20 and used for ChIP (Fig.
3a) recognises a single band of the expected size on western
blots of S2 cell extracts. b, The same antibody can
immunoprecipitate (IP) dTOR from S2 cells expressing the endogenous and
FLAG-tagged dTOR. c, It can also IP endogenous dTOR and the
intensity of this band is reduced upon treatment of S2 cells with dsRNA
against dTOR. For (a) to (c), a single experiment was
performed; the ability of the dTOR RNAi to reduce the
intensity of the band was confirmed in an independent experiment.
d, Relative enrichment of Pol III-transcribed genes is
higher than that of Pol II-transcribed genes after ChIP using a second
antibody against Drosophila TOR (raised against a
peptide21, p=2x10-4;
LM with an a priori contrast, n= 3
biologically independent samples, CI= 1.6-2.6, 0.81-2.3, 1.1-2.7, 0.77-2.8,
-0.24-2.5, -0.065-2.0, 0.13-1.7 left to right). e, No
enrichment for Pol III-transcribed genes over Pol II-transcribed genes is
observed after mock ChIP with no antibody (p=0.09, LM with
an a priori contrast, n=3 biologically independent
samples). f, Rapamycin feeding results in a decrease in total
RNA content of the adult gut (p<10-4, two-sided
t-test). g, Rapamycin feeding results in
reduction of pre-tRNAs relative to total RNA in the fly gut
(p=10-4, MANOVA). Rapamycin feeding does not
result in a reduction of pre-rRNA in the fly gut relative to:h,
U3 (p<10-4, MANOVA);
i, total RNA (p=0.57, MANOVA).
j, Feeding RU486 to TIGS>HA-Maf1
female fruit flies to induce HA-Maf1 in the gut alone
extends their lifespan (p=0.006, log-rank test, n= 153
-RU486, 146 +RU486 animals; single trial). Bar charts show mean ±
SEM, with n= number of biologically independent samples indicated and
overlay showing individual data points. For gel source data see Sup. Fig. 1.
To examine whether Pol III is downstream of TORC1 for lifespan, we combined
adult-onset Pol III inhibition with rapamycin treatment. Rapamycin feeding or
gut-specific dC160 resulted in the same magnitude of
lifespan extension (Fig. 3e). The two treatments
were not additive (see Extended Data Fig. 8a for
summary and statistical analyses), consistent with their acting in the same longevity
pathway. The same was observed with RNAi against dC53 in the gut (Extended Data Fig. 8b), as well as when
dC160 expression was restricted to the ISCs
(Fig. 3f). Importantly, rapamycin feeding also
inhibited the phosphorylation of TORC1 substrate, S6 kinase3 (S6K), in both the gut and the whole fly, and decreased fecundity,
whilst gut-specific induction of C160 did not (Fig. 3g and h, Extended Data Fig. 8c - f). This confirms that Pol III inhibition does not
impact TORC1 activity, neither locally nor systemically, and hence, Pol III acts
down-stream of TORC1 in ageing (Fig. 3i).
Extended Data Figure 8
Relationship between TORC1 and Pol III.
a, Summary of fly lifespans examining the epistasis
between Pol III and TORC1 inhibition (top), including the CPH analyses
results (bottom). In the summary (top), log-rank test p
value, relative to -RU486 -rapamycin control, is reported and the total
number of animals in the trial = dead + censored. b, Induction
of dC53 in the adult guts by RU486 feeding
of TIGS>dC53 females and rapamycin
feeding both extend lifespan and are not additive (for statistical analysis
see [a]; n= 135 control, 135 +RU486, 120 +rapamycin and 137 +RU486 and
rapamycin animals; single trial). c - f, Rapamycin
but not induction of dC160 in the guts of
TIGS>dC160 females by RU486
reduces the phosphorylation of S6K in the gut (effect of rapamycin
p=3x10-4, RU486 p=0.77 and interaction p=0.55,
LM, CI= 0.51-1.5, 1.0-1.2, 0.33-0.73, 0.08-0.91 left to
right) and whole flies (effect of rapamycin p<10-4, RU486
p=0.10 and interaction p=0.16, LM, CI=0.77-1.2, 0.60-1.0,
0.016-0.19, 0.019-0.15 left to right). Further biological repeats related to
Fig. 3g are presented in (c) for
the gut and in (d) for the whole fly. These are quantified in (e) and (f)
respectively. In (c) to (f), data from four biologically independent samples
are shown. For gel source data see Sup. Fig. 1.
TORC1 inhibition is known to ameliorate age-related pathology and functional
decline of the gut22. We examined whether
inhibition of Pol III was sufficient to block the dysplasia resulting from ISC
hyperproliferation and mis-differentiation by assessing the characteristic,
age-dependent increase in dividing, phospho-histone H3 positive (pH3+) cells16. Inducing dC160
in the fly gut or solely in the ISCs ameliorated this pathology (Extended Data Fig. 9a, Fig. 4a and
b). The treatment was also sufficient to counteract the age-related loss of
gut barrier function, decreasing the number of flies displaying extra-intestinal
accumulation of a blue food dye (“smurf” phenotype23, Extended Data Fig. 9b,
Fig. 4c). Interestingly, we also found that
rpc-1 RNAi feeding reduced the severity of age-related loss of
gut-barrier function in worms (Extended Data Fig.
9c). In Drosophila, gut health24 and TORC1 inhibition25
are specifically linked to female survival. Indeed, induction of
dC160 in the gut had a sexually dimorphic effect
on lifespan, as the effect on males, albeit significant, was reduced in magnitude
relative to females (Extended Data Fig. 9d).
Overall, our data show that gut/ISC-restricted inhibition of Pol III, which extends
lifespan, is sufficient to ameliorate age-related impairments in gut health, which may
be causative or correlate with this longevity.
Extended Data Figure 9
Inhibition of Pol III in the gut preserves organ health.
a, dC160 induction in
the gut of adult TIGS>dC160 females
by RU486 feeding supresses accumulation of pH3 positive cells in old flies
(p=1x10-3, 2-tailed t-test, CI= 58-110
-RU486, 10-46 +RU486). b, dC160
induction in the gut of adult
TIGS>dC160 females by RU486
feeding supresses loss of gut barrier function (number of
“smurfs”) in old flies (p=5x10-4,
χ, CI=16-26 -RU486, 8.7-16
+RU486 % smurf). c, rpc-1 RNAi suppresses the
severity of the age-related loss of gut barrier function in worms (effect of
age p<10-4, rpc-1 RNAi p=0.51,
interaction p=0.01, Ordinal Logistic Regression, CI=5.0-31,
16-50, 24-48, 25-51, 53-78, 34-66 % smurf grades 3 and 4). Age-related loss
of gut barrier function has been previously described for worms31. d,
dC160 induction in the gut of adult
TIGS>dC160 males by RU486
feeding results in a small but significant extension of lifespan (p=0.03,
log-rank-test, n= 141 –RU486, 139 +RU486
animals; single trial). Bar charts show mean ± SEM with n= number of
animals indicated and overlay showing individual data points.
Figure 4
Stem cell-restricted Pol III inhibition improves age-related dysplasia and
gut barrier function.
dC160 induction in the ISCs of adult
GS5961>dC160 females by RU486
feeding supresses age-related accumulation of pH3 positive cells:
a, images of pH3 staining in the posterior mid-gut at 70 d of age
(white bar = 100 μm, white “>” marks pH3+ cells,
representative of seven -RU486 and nine +RU486 animals); b, the
number of pH3+ cells per gut (effect of age p<10-4, RU486
p=2x10-3, interaction p=2x10-3, LM,
young: 7-9 d, old: 56-70 d, CI= 2.6-8.6, 1.8-9.4, 14-36, 6.0-14 left to right).
c, dC160 induction in the ISCs
of adult GS5961>dC160 females by RU486
feeding reduces the age-related increase in the number of flies with a leaky gut
(effect of age p<10-4, RU486 p=0.09, interaction p=0.01,
Ordinal Logistic Regression, young: 21 d, old: 58 d, CI=
0.19-0.28, 1.5-1.8, 18-19, 14-15 % smurf left to right). Bar charts show mean
± SEM, with n= number of biologically independent animals indicated and
overlay showing individual data points.
Our study demonstrates that adult-onset decrease in the growth-promoting,
anabolic function mediated by Pol III in the gut, and specifically in the stem cell
compartment, is sufficient to recapitulate the longevity benefits of rapamycin
treatment. Pol III activity is essential for growth6; its detrimental effects on ageing suggest an antagonistic pleiotropy26 where wild-type levels of Pol III activity are
optimised for growth and reproductive fitness in early life but prove detrimental for
later health. We reveal a fundamental role for Pol III in adult physiology, implicating
wild-type Pol III activity in age-related stem cell dysfunction, declining gut health
and organismal survival, downstream of nutrient signalling pathways. The longevity
resulting from partial Pol III inhibition in adulthood likely stems from the reduced
provision of protein synthetic machinery, however, differential regulation of tRNA genes
or Pol III-mediated changes to chromatin organisation may also be involved, as has been
suggested in other contexts2. The strong
structural and functional conservation of Pol III in eukaryotes suggests that studies of
its influence on mammalian ageing are warranted and may lead to important therapies.
Methods
Yeast stocks, chronological lifespans and microfluidics assessment
pMK43-based cassette was integrated into w303 MATa leu2-3,112
trp1-1 can1-100 ura3-1 pADH-OsTir-9Myc::ADE2::ade2-1 his3-11,15 to
produce RPC160 or RPB220 C-terminal AID
fusions as described11, confirmed by PCR
and absence of growth in presence of 2.5 mM IAA.Primers for strain construction:Primers for verification:For chronological lifespans, the strains were grown to exponential phase
(OD600~0.4) in Synthetic Complete medium (2% glucose, 0.5%
ammonium sulphate, 0.17% yeast nitrogen base, 0.001% adenine, uracil,
tryptophan, histidine, arginine, methionine, 0.0025% phenylalanine, 0.003%
tyrosine, lysine, 0.004% isoleucine, 0.005% glutamate, aspartate, 0.0075%
valine, 0.01% threonine, 0.02% serine and leucine [w/v]), the culture split and
treated with IAA in acetone or acetone alone (0.1%, day 0) and kept with
aeration and shaking at 30°C. Cell were harvested for protein extraction
after 30 min. Cultures essentially reached stationary phase after 24h. The
viability was measured on the indicated days by plating 5 μl of 10-fold
serial dilutions starting from initial concentration corresponding to
OD600=0.5 on YEPD plates and growth for 2 d at 30°C.For replicative lifespan, cells from single colonies were inoculated in
10 mL of minimal medium27 with 1%
glucose, 0.02% leucine and 0.001% tryptophan, arginine, histidine and uracil
(w/v) and pH 5 maintained with K-Phthalate-KOH buffer. The 10 mL cultures were
cultivated overnight in 100 mL shake flasks at 30°C and 300 rpm. Still
exponential, they were diluted next morning to OD600 of 0.005-0.01
and cultivated for several hours to OD600 of 0.045-0.09 when they
were loaded into the microfluidics device as described28,29. The growth
medium was aerated in advance by shaking for at least two hours. Trapped in the
device, the cells were constantly provided with fresh medium containing the
synthetic auxin hormone 1-naphthaleneacetic acid (NAA) at the concentrations of
0.0005, 0.001, 0.005 or 0.01 mM; the control did not contain NAA. These
concentrations of the hormone span through the dynamic range of the auxin-based
degron system where the degree of protein depletion can be efficiently modulated
in the set-up used30. Temperature of
30°C was maintained throughout the experiment.Microscopic imaging in the bright field channel was performed for up to
5 days with the time interval of 5 minutes, using a Nikon Ti-E inverted
microscope equipped with a 40x Nikon Super Fluor Apochromat objective, and
halogen lamp with additional UV-blocking filter. For each cell, the time points
of (1) the budding events, (2) the eventual cell losses due to wash away, or (3)
cell death were recorded by visual inspection of the movies with the help of
ImageJ and a custom written macro. For assessment of cell division times, the
first six cell cycles of each cell were used.
Worm husbandry, lifespans and gut integrity assay
Prior to experiments animals were maintained at 20°C and grown
for at least three generations with ample OP50 Escherichia coli
food to assure full viability. The rpc-1 RNAi clone, gene code
C42D4.8, was obtained from the Ahringer library. Lifespan assays were performed
on HT115 E. coli expressing either the rpc-1
RNAi plasmid or pL4440 empty vector control. Experiments were carried out at
both 20°C and 25°C. Worms were scored as dead or alive at
intervals and worms that crawled off the plate or died from explosion or bagging
phenotypes were censored. rpc-1 RNAi treatment from L4 stage
increased the incidence of a vulval explosion phenotype (noted in Extended Data Fig. 2c). However, we found
that at 25°C this phenotype was greatly reduced (Extended Data Fig. 2c). For gut-restricted RNAi, the VP303
strain was used14. The
“smurf” assay for gut integrity was carried out essentially as
described31. Worms were aged from the
L4 stage at 25°C and on the appropriate day soaked in blue dye for 3
hours. The dye was removed by allowing the worms to crawl around on a bacterial
lawn for 30 minutes prior to microscopy analysis. Individual worms were scored
from 0-4 for their degree of “smurfness” with 0 being no blue
beyond the gut barrier and 4 being completely blue.
Fly husbandry, lifespan, tunicamycin survival, smurf and fecundity
assays
The outbred, wild-type stock was collected in 1970 in Dahomey (now
Benin) and has been kept in population cages to maintain lifespan and fecundity
at levels similar to wild-caught flies. w
mutation was backcrossed into the stock and Wolbachia cleared
by tetracycline treatment. TIGS32 (a.k.a. TIGS-2), GS596133, S, elavGS,
UAS-HA-Maf136,
UAS-dC160 and
UAS-dC53 (v30512 and v103810 from Vienna
Drosophila Resource Centre), and dC53
(CG5147 from Bloomington Stock
Centre) were all backcrossed at least 6 times into the
w Dahomey background.Stocks were maintained and experiments conducted at 25°C on a
12L:12D cycle at 60% humidity, on SYA food37 containing 10% brewer’s yeast, 5% sucrose, and 1.5% agar
(all w/v; with propionic acid and Nipagin as preservatives). RU486 (Sigma) and
Rapamycin (LC Laboratories, both dissolved in ethanol) were added to
200μM final concentration as required. Tunicamycin (Cell Signaling, DMSO
stock) was used in food with only sugar and agar at concentration of 10mg/l. For
control treatments, equivalent volumes of the vehicle alone were added.Flies were reared at standardised larval density and adults collected
over 12 h, allowed to mate for 48 h and sorted into experimental vials at a
density of 15 flies per vial (10 flies per vial for RNA extractions). For
lifespan experiments, flies were transferred to fresh vials and their survival
scored three times a week. Flies were transferred onto food containing
tunicamycin on day 9 and survival scored once or twice daily. For smurf assays,
at the indicated age the flies were placed on SYA food containing 2.5% (w/v)
blue dye (FD&C blue dye no. 1, Fastcolors) for 48 h and scored as full
smurfs if completely blue or partial smurfs if the dye had leaked out of the gut
but not reached the head. Eggs layed over ~24h were counted on day 10.
Other phenotypic tests were performed essentially as described38.
RNA extraction, qPCR and RNA-Seq
Synchronised populations of worms were placed on control or
rpc-1 RNAi at the L4 stage, grown at 20°C and
harvested after 4 days. Ten whole adult flies, or ten dissected mid-guts, were
harvested on day 7-9. Total RNA was isolated using TRIZOL (Invitrogen). RNA
isolation was quantitative – the amount obtained was proportional to the
starting amount. RNA was converted to cDNA using random hexamers and Superscript
II (Invitrogen). Quantitative PCR was performed using Power SYBR Green PCR
Master Mix (ABI), with the relative standard curve method. For worms,
rpc-1 transcript levels were normalised to the geometric
mean of three stably expressed reference genes cdc-42,
pmp-3 and Y45F10D.4 as described39. For flies, primers specific for
pre-tRNAs were designed based on previous biochemical characterisation40 or predicted intronic sequences41.Primer sequences:Flies:Worms:For RNA-Seq, RNA was further cleaned up with Qiagen RNeasy (Qiagen),
ribo-depleted (Ribo-Zero Gold; Illumina) and sequenced on the Illumina platform
at Glasgow Polyomics (75 bp, pair-end reads). Transcript abundance was estimated
with Salmon42 (https://combine-lab.github.io/salmon/) in quazi-mapping mode
onto all Drosophila cDNA sequences (BDGP6), imported with
tximport (https://bioconductor.org/packages/release/bioc/html/tximport.html)
into R (https://www.r-project.org/) and differential expression
determined with DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html)
using dissection batch as covariate at 10% false discovery rate43.
Western blots, immunoprecipitation (IP), S2 dsRNA treatment, translation
assays and ChIP
Proteins were extracted from yeast (10 ml culture), S2 cells (0.1-2 ml
culture; S2 cells were obtained from L. Partridge), 10 flies or ten dissected
mid-guts with trichloroacetic acid, washed and re-suspended in SDS-PAGE loading
buffer, separated by SDS-PAGE and transferred to nitrocellulose. Western blots
were performed with anti-puromycin 12D10 antibody (Millipore), anti-Actin
(Abcam, ab1801 or ab8224), anti-Myc (Sigma), anti-FLAG (Sigma),
anti-phospho-T398-S6K (Cell Signaling, 9209), anti-S6K antibody44 or anti-TOR antibody20.IPs were performed on ~2mg of total protein extracted from 2-5 ml
of S2 cell culture (transfected with pAFW-dTOR, treated with dsRNA or untreated)
into 50mM HEPES-KOH pH 8, 100 mM KCl, 5 mM EDTA, 10% glycerol, 0.5% NP-40 and
protease inhibitors with 0.5 μl of anti-dTOR serum20, washed five times with the same buffer and eluted into
SDS-PAGE sample buffer. dsRNA against dTOR corresponds to
fragment 3694-4208 bp of the dTOR open reading frame (this is
DRSC02811 from DRSC/TRiP) and was generated with Megascript RNAi Kit (Thermo
Fisher Scientific).Relative translation rates were determined with the SUnSET assay45: 10 mid-guts of 7 day old flies per
sample were dissected in ice-cold PBS and kept in 200 μl of ice-cold
Schneider’s medium followed by incubation in 1 ml of Schneider’s
medium with 10 μg/ml puromycin for 30 min at 25°C. 333 μl
of 50 % trichloroacetic acid were added to stop the reaction. Level of puromycin
incorporation was determined by western blots.ChIP was performed as described46
on chromatin prepared from 7-day old, wild-type females using either the
anti-TOR antibody raised against a recombinant TOR protein fragment20, or anti-TOR raised against a
peptide21. The mock control included
no antibody. Enrichment after IP was measured relative to input with qPCR.
Primers for 5’ and 3’ end of aop and the P2
InR promoter have been described46,47.Further primers used:
pH3, Prospero and anti-HRP staining
Guts were dissected at indicated ages in ice-cold PBS and immediately
fixed in 4% formaldehyde for 30 minutes. The staining was performed essentially
as described38 with anti-phospho-H3
antibody (Cell Signaling, 9701), anti-Prospero (Developmental Studies Hybridoma
Bank) or anti-HRP48. Guts were mounted in
mounting medium with DAPI (Vectastain). pH3 positive cells per midgut were
counted on a fluorescence microscope. Representative images were acquired with
the Zeiss LSM700 confocal microscope.
Statistical Analysis
Fly and worm: Survival assays were analysed with
log-rank test in Excel (Microsoft) or JMP (SAS), or with
CPH in R using the survival package
(https://cran.r-project.org/web/packages/survival/index.html).
All other tests were performed in JMP. Data obtained from dissections, ChIP or
westerns were scaled to dissection/chromatin/replicate batch, except for pH3
counts, to account for batching effects. MANOVA was used to
test for overall effect of RU486 or rapamycin feeding. For ChIP analysis,
“gene” was used as covariate in a LM with an
a priory contrast comparing Pol III- to Pol II-transcribed
genes. All regression models had a fully factorial design.Yeast microfluidics platform: The data, including the number of buds
produced by each cell and its final event (death or washout), were used for
Kaplan-Meier estimation of survival curves with the Lifelines module
(Davidson-Pilon, C., Lifelines, (2016), Github repository, https://github.com/CamDavidsonPilon/lifelines) in Python.
Plotting and statistical analysis were done in Python.
Inhibition of Pol III in yeast.
a, The growth of strains carrying
pADH-OsTir with RPC160-AID,
RPB220-AID or the control lacking any 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
RPC160-AID 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, Yeast replicative lifespan (top panels)
is not altered by 1-naphthaleneacetic acid (NAA; analogue of IAA) while cell
cycle duration (bottom panels) is. Both were assessed in the
pADH-OsTir RPC160-AID strain on a microfluidics
dissection platform. The concentrations of NAA span the dynamic range where
the degree of protein depletion can be efficiently modulated in this
set-up30. The same control data
are shown in each panel for comparison. For each NAA concentration one
experiment was performed. For replicative lifespans, 95% CIs are indicated
by shading, or in brackets for median lifespan, together with
log-rank p value. 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.
Inhibition of Pol III extends worm lifespan.
a, Lifespan is extended by feeding N2 worms with
rpc-1 RNAi at 20°C in absence of FUDR
(p<10-3
log-rank test, n= 100 control and rpc-1
RNAi treated animals). b, It is also extended at 25°C in
presence of FUDR (p=9x10-3, log-rank test, n= 60
control, 77 rpc-1 RNAi animals). c, Summary of
each worm lifespan experiment performed including the representative trials
presented in the figures. Log-rank test p value is
reported. The total number of animals in the trial = dead + censored. In
general, fewer worms were censored in control vs rpc-1 RNAi
conditions (average of N2 at 25°C 25% vs 38%; average of N2 at
20°C 53% vs 73%; average of VP303 at 25°C 3% vs 4% and average
of VP303 at 20°C 37% vs 54%) which is likely to be due to an
increased number of gut explosions in the rpc-1 RNAi
treated worms. On average 84.9% of control and 85.6% of
rpc-1 RNAi-treated censored events 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 using VP303 strain, at
25°C in presence of FUDR (p=9x10-3, log-rank
test, n= 84 control, 103 rpc-1 RNAi treated
animals). In (a), (b) and (d), a representative of two trials is shown.
Genes corresponding to unique Pol III subunits in
Drosophila.
The genes encoding the unique Pol III subunits were identified in
fruit flies based on their homology to the yeast genes (BLAST, followed by
reverse BLAST), or to the human orthologue.
Inhibition of Pol III extends fly lifespan.
a, Summary of each fly lifespan experiment performed
including the representative trials presented in the figures but excluding
the ones 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. RU486 feeding does
not have an effect on the lifespans of: b,
UAS-dC160 alone (p=0.28,
log-rank test, n= 142 -RU486, 146 +RU486 animals); or
c, TIGS alone controls (p=0.41,
log-rank test, n= 141 -RU486, 145 +RU486 animals).
d, Inducing dC53 in the gut
of TIGS>dC53 females by RU486 extends
their lifespan (p=3x10-6, log-rank test, n= 143
-RU486, 139 +RU486 animals). e, Inducing
dC160 predominantly in the fat body
of S females by
RU486 has no effect on their lifespan (p=0.21, log-rank
test, n= 158 -RU486, 155 +RU486 animals). f,
Inducing dC160 in neurons of
elavGS>dC160 females by RU486
has a modest effect on their lifespan (p=0.03, log-rank
test, n= 148 -RU486, 155 +RU486 animals). g, RU486
feeding does not have an effect on the lifespan of the
GS5961 alone control (p=0.88, log-rank
test, n= 89 -RU486, 91 +RU486 animals). In (b) to (g) the
single trial performed is shown.
TIGS is active in ISCs.
Images from the posterior region of the midgut showing GFP
expression driven by TIGS in the presence of RU486 and
stained with: a, anti-Prospero; b, anti-HRP. GFP
expression can be observed in cells with small nuclei that are
Prospero-negative in (a) and those that stain with anti-HRP in (b). Examples
of both types are indicated with a white
“>” on the merged images. GFP-positive
cells can be observed whose morphology and staining pattern correspond
closely to that of the ISCs (small nucleus, small cell size,
Prospero-negative, anti-HRP-positive [see ref. 48 regarding anti-HRP]). TIGS has a
complex expression pattern, showing variation between neighbouring cells of
the same type and between gut regions. TIGS appears active
in at least some ISCs. Images are representative of two animals.
Effects of dC160 induction in
Drosophila adult gut.
Induction of dC160 in the guts of
TIGS>dC160 females results
in: a, decreased levels of 45S pre-rRNA
(p=4x10-3, MANOVA, CI= 0.95-1.1, 0.85-1.0,
0.95-1.1, 0.81-0.92 left to right; EST = 5’ external transcribed
spacer, IST = internal transcribed spacer), indicating a reduction in Pol I
activity as a result of Pol III – Pol I crosstalk; b,
unaltered levels of mRNAs encoding ribosomal proteins (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; c, decreased protein
synthesis (two further biological repeats and quantification related to
Fig. 2e; p=4x10-3,
two-sided t-test, n=3 biologically independent samples,
CI= 0.65-1.4 -RU486, -0.033-0.68 +RU486). RU486 feeding of
TIGS-alone control females does not result in a
significant decrease in: d, levels of pre-tRNAs
(p=1x10-4, MANOVA, CI= 0.96-1.0, 1.1-1.2,
0.93-1.1, 1.1-1.2, 0.91-1.1, 1.2-1.3 left to right); e, levels
of 45S pre-rRNA (p=2x10-4,
MANOVA, CI= 0.94-1.1, 1.1-1.3, 0.94-1.1, 1.1-1.2 left
to right); f, protein synthesis (p=0.74, two-sided
t-test, n=3 biologically independent samples).
Induction of dC160 in the guts of
TIGS>dC160 females does not
result in significant changes to: g, total gut protein content
(p=0.43, two-sided t-test); h, female
fecundity (p=0.51, two-sided t-test); i,
whole-fly body weight, triacylglycerol or protein content (p=0.58, 0.40,
0.16 respectively, two-sided t-test). j, RU486
feeding of TIGS-alone control females does not result in
increased resistance to tunicamycin (p=0.89, log-rank test,
n= 149 -RU486, 153 +RU486 animals; single trial). Bar charts show mean
± SEM, with n= number of biologically independent samples indicated
and overlay showing individual data points. For gel source data see Sup. Fig. 1.
Regulation of Pol III activity by TORC1 in
Drosophila.
a, The antibody raised against a recombinant fragment
of Drosophila TOR protein 20 and used for ChIP (Fig.
3a) recognises a single band of the expected size on western
blots of S2 cell extracts. b, The same antibody can
immunoprecipitate (IP) dTOR from S2 cells expressing the endogenous and
FLAG-tagged dTOR. c, It can also IP endogenous dTOR and the
intensity of this band is reduced upon treatment of S2 cells with dsRNA
against dTOR. For (a) to (c), a single experiment was
performed; the ability of the dTOR RNAi to reduce the
intensity of the band was confirmed in an independent experiment.
d, Relative enrichment of Pol III-transcribed genes is
higher than that of Pol II-transcribed genes after ChIP using a second
antibody against Drosophila TOR (raised against a
peptide21, p=2x10-4;
LM with an a priori contrast, n= 3
biologically independent samples, CI= 1.6-2.6, 0.81-2.3, 1.1-2.7, 0.77-2.8,
-0.24-2.5, -0.065-2.0, 0.13-1.7 left to right). e, No
enrichment for Pol III-transcribed genes over Pol II-transcribed genes is
observed after mock ChIP with no antibody (p=0.09, LM with
an a priori contrast, n=3 biologically independent
samples). f, Rapamycin feeding results in a decrease in total
RNA content of the adult gut (p<10-4, two-sided
t-test). g, Rapamycin feeding results in
reduction of pre-tRNAs relative to total RNA in the fly gut
(p=10-4, MANOVA). Rapamycin feeding does not
result in a reduction of pre-rRNA in the fly gut relative to:h,
U3 (p<10-4, MANOVA);
i, total RNA (p=0.57, MANOVA).
j, Feeding RU486 to TIGS>HA-Maf1
female fruit flies to induce HA-Maf1 in the gut alone
extends their lifespan (p=0.006, log-rank test, n= 153
-RU486, 146 +RU486 animals; single trial). Bar charts show mean ±
SEM, with n= number of biologically independent samples indicated and
overlay showing individual data points. For gel source data see Sup. Fig. 1.
Relationship between TORC1 and Pol III.
a, Summary of fly lifespans examining the epistasis
between Pol III and TORC1 inhibition (top), including the CPH analyses
results (bottom). In the summary (top), log-rank test p
value, relative to -RU486 -rapamycin control, is reported and the total
number of animals in the trial = dead + censored. b, Induction
of dC53 in the adult guts by RU486 feeding
of TIGS>dC53 females and rapamycin
feeding both extend lifespan and are not additive (for statistical analysis
see [a]; n= 135 control, 135 +RU486, 120 +rapamycin and 137 +RU486 and
rapamycin animals; single trial). c - f, Rapamycin
but not induction of dC160 in the guts of
TIGS>dC160 females by RU486
reduces the phosphorylation of S6K in the gut (effect of rapamycin
p=3x10-4, RU486 p=0.77 and interaction p=0.55,
LM, CI= 0.51-1.5, 1.0-1.2, 0.33-0.73, 0.08-0.91 left to
right) and whole flies (effect of rapamycin p<10-4, RU486
p=0.10 and interaction p=0.16, LM, CI=0.77-1.2, 0.60-1.0,
0.016-0.19, 0.019-0.15 left to right). Further biological repeats related to
Fig. 3g are presented in (c) for
the gut and in (d) for the whole fly. These are quantified in (e) and (f)
respectively. In (c) to (f), data from four biologically independent samples
are shown. For gel source data see Sup. Fig. 1.
Inhibition of Pol III in the gut preserves organ health.
a, dC160 induction in
the gut of adult TIGS>dC160 females
by RU486 feeding supresses accumulation of pH3 positive cells in old flies
(p=1x10-3, 2-tailed t-test, CI= 58-110
-RU486, 10-46 +RU486). b, dC160
induction in the gut of adult
TIGS>dC160 females by RU486
feeding supresses loss of gut barrier function (number of
“smurfs”) in old flies (p=5x10-4,
χ, CI=16-26 -RU486, 8.7-16
+RU486 % smurf). c, rpc-1 RNAi suppresses the
severity of the age-related loss of gut barrier function in worms (effect of
age p<10-4, rpc-1 RNAi p=0.51,
interaction p=0.01, Ordinal Logistic Regression, CI=5.0-31,
16-50, 24-48, 25-51, 53-78, 34-66 % smurf grades 3 and 4). Age-related loss
of gut barrier function has been previously described for worms31. d,
dC160 induction in the gut of adult
TIGS>dC160 males by RU486
feeding results in a small but significant extension of lifespan (p=0.03,
log-rank-test, n= 141 –RU486, 139 +RU486
animals; single trial). Bar charts show mean ± SEM with n= number of
animals indicated and overlay showing individual data points.
Authors: Yu-San Yang; Masato Kato; Xi Wu; Athanasios Litsios; Benjamin M Sutter; Yun Wang; Chien-Hsiang Hsu; N Ezgi Wood; Andrew Lemoff; Hamid Mirzaei; Matthias Heinemann; Benjamin P Tu Journal: Cell Date: 2019-04-11 Impact factor: 41.582
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