A mitochondrial unfolded protein response-independent role of DVE-1 in longevity regulation

Abstract

The special AT-rich sequence-binding (SATB) protein DVE-1 is widely recognized for its pivotal involvement in orchestrating the retrograde mitochondrial unfolded protein response (mitoUPR) in C. elegans. In our study of downstream factors contributing to lifespan extension in sensory ciliary mutants, we find that DVE-1 is crucial for this longevity effect independent of its canonical mitoUPR function. Additionally, DVE-1 also influences lifespan under conditions of dietary restriction and germline loss, again distinct from its role in mitoUPR. Mechanistically, while mitochondrial stress typically prompts nuclear accumulation of DVE-1 to initiate the transcriptional mitoUPR program, these long-lived mutants reduce DVE-1 nuclear accumulation, likely by enhancing its cytosolic translocation. This observation suggests a cytosolic role for DVE-1 in lifespan extension. Overall, our study implies that, in contrast to the more narrowly defined role of the mitoUPR-related transcription factor ATFS-1, DVE-1 may possess broader functions than previously recognized in modulating longevity and defending against stress.

Full text

A mitochondrial unfolded protein response-independent role of
DVE-1 in longevity regulation
Yi Sheng1, Adriana Abreu1, Zachary Markovich1, Pearl Ebea1, Leah Davis1, Eric Park1,
Peike Sheng2, Mingyi Xie2,6, Sung Min Han1, Rui Xiao1,3,4,5,6,7,*
1Department of Physiology and Aging, College of Medicine, University of Florida, Gainesville, FL
32610, USA
2Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida,
Gainesville, FL 32610, USA
3Institute on Aging, University of Florida, Gainesville, FL 32610, USA
4Center for Smell and Taste, University of Florida, Gainesville, FL 32610, USA
5Genetics Institute, University of Florida, Gainesville, FL 32610, USA
6UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA
7Lead contact
SUMMARY
The special AT-rich sequence-binding (SATB) protein DVE-1 is widely recognized for its pivotal
involvement in orchestrating the retrograde mitochondrial unfolded protein response (mitoUPR)
in
C. elegans
. In our study of downstream factors contributing to lifespan extension in sensory
ciliary mutants, we find that DVE-1 is crucial for this longevity effect independent of its
canonical mitoUPR function. Additionally, DVE-1 also influences lifespan under conditions of
dietary restriction and germline loss, again distinct from its role in mitoUPR. Mechanistically,
while mitochondrial stress typically prompts nuclear accumulation of DVE-1 to initiate the
transcriptional mitoUPR program, these long-lived mutants reduce DVE-1 nuclear accumulation,
likely by enhancing its cytosolic translocation. This observation suggests a cytosolic role for
DVE-1 in lifespan extension. Overall, our study implies that, in contrast to the more narrowly
defined role of the mitoUPR-related transcription factor ATFS-1, DVE-1 may possess broader
functions than previously recognized in modulating longevity and defending against stress.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
*Correspondence: rxiao@ufl.edu.
AUTHOR CONTRIBUTIONS
R.X. and Y.S. conceived and designed the study. Y.S. performed most experiments with the assistance of A.A., Z.M., P.E., L.D., E.P.,
P.S., M.X., and S.M.H. R.X. and Y.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS
During the preparation of this work the authors used GPT-4o in order to improve the readability of the text. After using this tool/
service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114889.
HHS Public Access
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. Author manuscript; available in PMC 2024 December 16.
Published in final edited form as:
Cell Rep
. 2024 November 26; 43(11): 114889. doi:10.1016/j.celrep.2024.114889.
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Graphical abstract
In brief
DVE-1 is well recognized as a key regulator of mitoUPR in
C. elegans
. However, Sheng et al.
show that DVE-1 can affect longevity through various pathways independent of mitoUPR. These
longevity pathways reduce DVE-1 nuclear accumulation and promote its cytosolic translocation,
indicating a role for cytosolic DVE-1 in lifespan regulation.
INTRODUCTION
The primary cilium, functioning as an immotile antenna-like sensory organelle, is widely
distributed across most vertebrate cells, where it serves diverse crucial roles spanning
embryonic development, tissue maintenance, and sensory signal transduction.1 In the
nematode
C. elegans
, 60 out of 302 neurons in the adult hermaphrodite are ciliated
sensory neurons.2–4 Analogous to their vertebrate counterparts, sensory cilia in
C. elegans
play pivotal roles in sensory perception, organismal development, and neuroendocrine
communication.5 Interestingly, studies involving
C. elegans
sensory ciliary mutants have
consistently shown that defects in sensory cilia correlate with extended lifespan.6 Similarly,
mutations in the
Or83b
gene, essential for recruiting odorant receptors to the ciliated
dendrites of
Drosophila
olfactory neurons, also significantly increase lifespan in flies.7,8
Furthermore, the acute loss of olfaction in adult mice has been demonstrated to protect
against diet-induced obesity and promote metabolic well-being.9 These findings suggest an
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intriguing possibility that sensory perception, particularly chemosensation, may inherently
contribute to aging processes. Downstream of the sensory ciliary signaling, the FOXO
family transcription factor DAF-16 has been shown to mediate the greatly extended lifespan
of various ciliary mutants.6 Nonetheless, direct evidence linking primary ciliary signaling to
DAF-16/FOXO activation is still limited, and it remains unknown whether other signaling
molecules may also mediate the prolongevity effect of ciliary mutants.
Mitochondrial dysfunction stands as a hallmark of aging across diverse species.10
Originating from a bacterial endosymbiont lineage with roots dating back at least
1.45 billion years,11,12 mitochondria intricately regulate longevity. According to the
free radical theory of aging,13 toxic reactive oxygen species (ROS), emanating from
mitochondria, inflict damage upon cellular components such as proteins, lipids, DNA,
and RNA. The accumulation of such damage over time is purported to underlie the
aging process at the organismal level. However, subsequent investigations have unveiled
a paradoxical phenomenon: low concentrations of ROS-generating compounds, such as
paraquat or juglone, demonstrate a remarkable ability to extend lifespan in
C. elegans
,14–
16 exemplifying the concept of hormesis. Intriguingly, mild suppression of mitochondrial
respiration emerges as a consistent promoter of longevity across multiple species, including
C. elegans
,17
Drosophila
,18 and mice.19–21 These observations suggest an evolutionarily
conserved role of mitochondrial respiration in modulating lifespan.
How does the mild inhibition of mitochondrial respiration lead to lifespan extension?
Retrograde signaling from mitochondria to the nucleus plays a key role. Among different
types of mitochondrial retrograde signaling, the mitochondrial unfolded protein response
(mitoUPR) has been shown to be a major contributor in mitochondrial stress-induced
longevity.22–24 Although the concept of mitoUPR initially surfaced in mammalian cells,25
its underlying mechanism is probably best studied in
C. elegans
.26 At least two transcription
factors are involved in mitoUPR activation in
C. elegans
, the bZIP Activating Transcription
Factor associated with Stress (ATFS-1) and the homeodomain transcriptional regulator
Defective proVEntriculus (DVE-1).26–28 While the regulatory mechanisms governing
ATFS-1 have been extensively investigated, revealing its dual nuclear localization sequence
(NLS) and mitochondrial-targeting sequence (MTS),29 insights into DVE-1’s role remain
less explored. Under normal mitochondrial conditions, the stronger mitochondrial-targeting
signal directs ATFS-1 into the mitochondrial matrix, where it undergoes degradation by the
resident protease LONP-1.29 However, during mitochondrial stress, compromised import
efficiency redirects ATFS-1 to the nucleus via its NLS, instigating the transcriptional
activation of various genes crucial for proteostasis, mitochondrial biogenesis, and
antioxidant defense.26,29 In a pathway parallel to that of ATFS-1, DVE-1 either genetically
or physically interacts with chromatin remodelers LIN-65 and MET-2 as well as a small
ubiquitin-like protein UBL-5 to relay various mitochondrial stress signals to the nucleus
and activate mitoUPR.23,27 Although the critical role of DVE-1 in mitoUPR induction was
identified earlier than ATFS-1,27,28 questions persist regarding its mechanism for monitoring
mitochondrial health and potentially additional cellular functions beyond mitoUPR.
In this study, we conducted a targeted RNAi screen, which led to the identification of
DVE-1 as an important regulator influencing sensory cilia-mediated longevity. Surprisingly,
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DVE-1 seems to modulate cilia-mediated longevity in a mitoUPR-independent manner, as
many ciliary mutants do not trigger mitoUPR activation, and other key mitoUPR regulators
such as ATFS-1, LIN-65, MET-2, and UBL-5 do not have a significant impact on the
lifespan of sensory ciliary mutants. Furthermore, we established that DVE-1 also plays a
role in longevity modulation influenced by dietary restriction and germline signaling, again
without eliciting mitoUPR induction. Consequently, our present study unveils the mitoUPR-
independent function of DVE-1 in lifespan regulation, hinting at distinct downstream
effectors for the two pivotal transcription regulators, ATFS-1 and DVE-1, in modulating
mitoUPR and organismal aging.
RESULTS
Various elements of sensory cilia exert distinct effects on lifespan
Sensory cilia are evolutionarily conserved subcellular organelles that are essential for smell,
taste, vision, and hearing.30 Paradoxically,
C. elegans
mutants with a spectrum of sensory
cilia defects, ranging from complete absence to partial deletion or irregularities in ciliary
segments, tend to display extended lifespans.6 Given the complexity of ciliogenesis and
maintenance, involving numerous components (Figure 1A), we undertook a systematic
examination to elucidate the differential impacts of distinct sensory cilia components on
longevity. Specifically, we examined the lifespans of various mutants in intraflagellar
transport particle A (
daf-10
), intraflagellar transport particle B (
osm-1
), motor activator
(
dyf-1
), homodimeric kinesin motor (
osm-3
), dynein motor (
xbx-1
), heterodimeric kinesin
motor (
klp-11
), and BBS protein (
osm-12
).
In line with previous investigations,6 mutations in
daf-10
(Figure 1B) and
osm-1
(Figure
1C) significantly extend lifespan compared to the wild-type (WT) N2 control animals.
Furthermore, mutants such as
dyf-1
(
mn335
),
osm-3
(
p802
), and
xbx-1
(
ok279
) exhibited
prolonged lifespans, albeit to varying extents (Figures 1D–1F). Nonetheless, we also
observed that the kinesin-II mutant
klp-11
(
tm324
) exhibits a lifespan comparable to that
of the WT (Figure 1G), while the
osm-12
(
n1606
) mutant shows decreased longevity (Figure
1H). Notably, both KLP-11 and OSM-12 play pivotal roles in ciliogenesis and intraflagellar
transport.31,32 Since
klp-11
(
tm324
) and
osm-12
(
n1606
) mutants are known to have defective
sensory cilia,31,32 our findings underscore that not all sensory ciliary mutants display
enhanced lifespan, as previously suggested.6 Thus, disparate components of sensory cilia
may exert distinct influences on longevity.
The transcription regulator DVE-1 acts downstream of ciliary modulation of longevity
Virtually all pathways implicated in longevity converge on a handful of pivotal transcription
factors, regulators, and nuclear hormone receptors.33,34 While pioneering research has
established that the FOXO transcription factor DAF-16 operates downstream of ciliary
regulation of aging,6 the inability of the
daf-16
(
mu86
) null allele to fully revert the extended
lifespan of ciliary mutants to the
daf-16
(
mu86
) level suggests the involvement of additional
nuclear factors.6 Given that the
daf-10
(
p821
) mutant demonstrates the most pronounced
extension of lifespan among all ciliary mutants examined, we conducted a targeted RNAi
bacterial feeding screen against 512 distinct clones of transcription factors (including
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daf-16
), regulators, and nuclear hormone receptors available to us, using the
daf-10
(
p821
)
mutant as the reference. Among these candidates, we found that the lifespan-suppressive
effect of
dve-1
RNAi rivals that of
daf-16
RNAi (Figure 2A), suggesting a potential role for
DVE-1 downstream of ciliary modulation of longevity.
DVE-1, the
C. elegans
counterpart of mammalian special AT-rich sequence-binding
protein 1 and 2 (SATB1 and SATB2),27 primarily functions in modulating mitoUPR
by remodeling histone landscapes and the three-dimensional genome structure of
C.
elegans
.23,27 Consistent with prior findings,23,27
dve-1
RNAi markedly attenuated mitoUPR
activation triggered by
isp-1
RNAi, as evidenced by monitoring the well-characterized
zcls13
[
Phsp-6
::
gfp
] mitoUPR reporter line (Figures 2B and 2C).35 Since our current finding
suggested that DVE-1 acts downstream of DAF-10 in lifespan modulation, we reasoned
whether DVE-1 may also modulate the lifespans of other ciliary mutants. Importantly,
dve-1
null mutation is embryonically lethal.27 We obtained and tested two available
dve-1
mutants,
dve-1
(
tm4803
) and
dve-1
(
tm7599
), to assess their potential impact on lifespan.
Surprisingly, both mutants exhibited normal lifespans, similar to WT animals (data not
shown). The
dve-1
(
tm4803
) mutation causes a deletion in a segment of the predicted helix
III in the homeodomain and introduces a 65-bp insertion due to a splice-site alteration.36
By contrast, the
dve-1
(
tm7599
) mutation results in a 60-bp deletion in the fourth intron,
which probably does not affect DVE-1’s normal function. Since the
dve-1
(
tm4803
) and
dve-1
(
tm7599
) alleles are likely very mild mutations and neither allele affects lifespan, we
proceeded with
dve-1
RNAi to further investigate its role in ciliary modulation of longevity.
Compared to the empty-vector control,
dve-1
RNAi significantly and consistently reduced
lifespan in WT worms (Figure 2D). More importantly, it also substantially suppressed the
extended lifespan observed in the long-lived
osm-1
(
p808
),
dyf-1
(
mn335
),
osm-3
(
p802
), and
xbx-1
(
ok279
) mutant backgrounds (Figures 2E–2H). By contrast,
dve-1
RNAi had minimal
impact on the lifespans of the
klp-11
(
tm324
) and
osm-12
(
n1606
) ciliary mutants, which do
not exhibit longevity extension (Figures 2I and 2J). Therefore, DVE-1 emerges as a general
modulator of the prolonged longevity conferred by various ciliary mutations.
Previous studies have shown that DVE-1 is mainly expressed in head neurons
and the intestine.23,27,37,38 To pinpoint the tissue through which DVE-1
influences lifespan, we performed tissue-specific RNAi experiments. We used the
sid-1
(
qt9
);
alxIs9
[
vha-6p
::
sid-1
::
SL2
::
GFP
] line39 to knock down DVE-1 specifically in the
intestine and the
sid-1
(
qt9
);
sqIs71
[
rgef-1p
::
GFP
+
rgef-1p
::
sid-1
] line40 for neuron-specific
knockdown. Our results suggest that DVE-1 primarily affects longevity through its action in
the intestine (Figure S1A) rather than in neurons (Figure S1B).
mitoUPR is not required for ciliary modulation of longevity
How does DVE-1 regulate lifespan downstream of ciliary mutations? As the primary known
function of DVE-1 is to mediate mitoUPR in a pathway parallel to that of ATFS-1 (Figure
3A), we explored whether ATFS-1 might also participate in the downstream effects of ciliary
modulation on longevity. Employing a similar approach to our investigation of DVE-1, we
administered
atfs-1
RNAi to both WT and various ciliary mutant worms. Consistent with
prior studies,41,42 genetic disruption of
atfs-1
had no significant impact on the lifespan of
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WT worms (Figure 3B). Moreover, we observed that
atfs-1
RNAi did not alter the lifespans
of any of the ciliary mutants examined (Figures 3C–3I). Thus, unlike DVE-1, ATFS-1 does
not play a significant role in ciliary modulation of longevity.
Considering ATFS-1’s crucial role as a transcription factor in mitoUPR activation, the
absence of its effect on ciliary modulation of aging led us to question whether mitoUPR
is involved in this process. Indeed, we did not detect induction of
hsp-6
expression,
a well-established marker for mitoUPR activation,27,35 in the long-lived
daf-10
(
p821
),
normal-lifespan
klp-11
(
tm324
), or short-lived
osm-12
(
n1606
) mutant backgrounds (Figure
4A). Furthermore, we explored the potential impact of other key mitoUPR regulators known
to function alongside DVE-1, including LIN-65, MET-2, UBL-5, and CLPP-1 (Figure
3A).23,27 To our surprise, RNAi targeting these mitoUPR regulators did not suppress the
longevity conferred by ciliary mutations as effectively as
dve-1
RNAi (Figures 4C–4G).
In fact,
clpp-1
RNAi consistently extended the lifespan of the WT (Figure 4B) as well as
multiple ciliary mutant worms (Figures 4C, 4F, and 4G–4I), and knockdowns of all these
factors further promoted the longevity of
osm-3
(
p802
) mutant (Figure 4F). Collectively,
these results strongly suggest that DVE-1 acts in a mitoUPR-independent fashion in ciliary
modulation of lifespan.
Cytosolic translocation of DVE-1 in the daf-10 mutant background
DVE-1, along with its mammalian counterpart SATB, has been previously recognized as
a transcriptional regulator and orchestrator of three-dimensional genome organization.23,43
Upon mitochondrial stress, DVE-1 relocates from the cytosol to the nucleus, where it
triggers transcriptional activation of the mitoUPR.23,27 Given our findings suggesting that
DVE-1 modulates longevity conferred by ciliary mutations independently of mitoUPR, we
investigated whether nuclear translocation of DVE-1 is crucial for its role in longevity
regulation. As a positive control, we treated worms with
spg-7
(a mitochondrial protease)
RNAi, which is a well-established inducer of mitoUPR.44–46 As anticipated,
spg-7
RNAi
led to robust induction of
Phsp-6
::
gfp
expression in the
zcls13
[
Phsp-6
::
gfp
] reporter line
(Figures 5A and 5B), indicative of mitoUPR activation.35 Additionally, both
atfs-1
RNAi
and
dve-1
RNAi effectively suppressed the mitoUPR activation triggered by
spg-7
RNAi
(Figures 5A and 5B), confirming that both ATFS-1 and DVE-1 are essential regulators of
mitoUPR.
Previous research has demonstrated that mitochondrial stress leads to the nuclear
accumulation of DVE-1 during mitoUPR activation.23,27,37,38 In line with these findings,
we observed increased levels of nuclear DVE-1::GFP (Figures 5C and 5D) following
spg-7
RNAi treatment, as indicated by the
zcIs39
[
Pdve-1
::
dve-1
::
gfp
] translational reporter line.28
Although DVE-1::GFP fluorescence is readily detectable in various head neurons and
posterior intestinal cells (Figure S3A), the larger and more distinct cells of the posterior
intestine make it much easier to observe and quantify nuclear localization. In addition,
our tissue-specific RNAi experiment suggested that DVE-1 primarily regulates the lifespan
through its function in the intestine rather than neurons (Figure S1). Thus, we decided to
focus on the posterior intestine for further analysis of DVE-1 subcellular localization.
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Interestingly, while DVE-1::GFP was predominantly localized in the nucleus of WT worms,
its nuclear presence was significantly reduced in the
daf-10
(
p821
) mutant background
(Figures 5E and 5F). To further investigate, we performed additional imaging experiments
(multiple aligned worms at lower magnification) with DVE-1::GFP expressed in either
WT or
daf-10
(
p821
) mutant background. Again, we observed much-reduced nuclear
accumulation of DVE-1::GFP in the
daf-10
(
p821
) mutant (Figure 5G). Despite this, the total
fluorescence signal of DVE-1::GFP was actually higher in
daf-10
(
p821
) mutants (Figures
5G and S3A), suggesting that the
daf-10
(
p821
) mutant does not reduce the total expression
level of DVE-1.
To independently verify DVE-1’s cytosolic translocation in the
daf-10
(
p821
) mutant, we
created a transgenic line,
ruxEx437
[
Pdve-1
::
SL2
::
mCherry
::
H2B
;
Plin-44
::
gfp
];
zcIs39
[
Pdve-1
::
dve-1
::
GFP
];
daf-10
(
p8
21
), to label the nuclei of all DVE-1-expressing cells with mCherry-histone H2B fusion
proteins.47 Confocal microscopy with a 100× objective demonstrated that in WT worms,
most DVE-1::GFP signals were confined to the nuclei, whereas in
daf-10
(
p821
) mutants, a
substantial portion of DVE-1::GFP did not co-localize with mCherry H2B (Figures S3B and
S3C). This finding corroborates our lower-magnification imaging results, indicating that the
daf-10
(
p821
) mutation may promote the cytosolic translocation of DVE-1. Notably, a
previous study has shown that DVE-1 forms punctate structures in the nucleus.23 Although
green fluorescent puncta are also readily detectable in the cytoplasm of DVE-1 GFP worms,
their molecular nature was not confirmed in the same study.23 Intriguingly, we observed a
marked increase in cytosolic DVE-1 puncta in the
daf-10
(
p821
) mutant background (Figures
S3B and S3C). While the
C. elegans
intestine is well known to contain lysosome-related gut
granules that are autofluorescent,48 two considerations suggest that the green fluorescent
puncta observed in the cytoplasm of
daf-10
(
p821
) mutants are most likely not due to gut
granule autofluorescence. First, intestinal autofluorescence increases with age.48 As our
imaging experiment was performed with day-1 adult worms, the intestinal autofluorescence
was minimal compared to the DVE-1::GFP signal. Second, the cytosolic green fluorescent
puncta in WT worms were significantly less abundant than in
daf-10
(
p821
) mutants. If these
puncta were due to gut granule autofluorescence, we would expect a similar pattern in WT
worms.
Given that longevity in the
daf-10
(
p821
) mutant does not involve mitoUPR activation
and DVE-1 translocates from the nucleus to the cytosol in this mutant background, we
hypothesized that nuclear translocation of DVE-1 might not be essential for its role in
longevity regulation of ciliary mutants. To test this hypothesis, we generated transgenic
lines expressing DVE-1::YFP or DVE-1(ΔNLS)::YFP, where the predicted NLS sequence
(237FTQKRSSSRTL247, as predicted by the cNLS Mapper49) was deleted via site-directed
mutagenesis, in the WT background. Consistent with previous observations,23,27 WT DVE-1
was predominantly localized in the nucleus. In contrast, DVE-1(ΔNLS) lacking the NLS
sequence exhibited uniform distribution throughout the intestinal cell (Figures 5H and 5I).
Furthermore, we found that overexpression of both DVE-1 and DVE-1(ΔNLS) significantly
extended the lifespan to a comparable degree (Figure 5J). Given that WT DVE-1 is
predominantly nuclear, these findings suggest that both nuclear and cytosolic forms of
DVE-1 may contribute to longevity.
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DVE-1 acts in dietary-restriction-modulated and germline-signaling-modulated longevity
independently of mitoUPR
Besides mutations affecting sensory perception, many other longevity mutants and aging-
related genetic pathways have emerged over the past three decades.33,34 Our next objective
was to investigate whether DVE-1 also plays a role in these alternative longevity pathways.
DAF-2 serves as the primary receptor for insulin and insulin-like growth factors in
C.
elegans
,50,51 and the hypomorphic
daf-2
(
e1370
) mutant exhibits significantly prolonged
lifespan (Figure 6A). However, unlike the long-lived ciliary mutants,
dve-1
RNAi did
not significantly attenuate the lifespan extension observed in the
daf-2
(
e1370
) mutant
(Figure 6A). Thus, DVE-1 does not contribute to the longevity modulated by the insulin/
IGF-1 signaling (IIS) pathway. ISP-1 is a mitochondrial complex III component, and mild
inhibition of mitochondrial respiration by the
isp-1
(
qm150
) mutation is previously known to
extend the lifespan of
C. elegans
.52 Intriguingly,
dve-1
RNAi did not reduce the extended
lifespan of
isp-1
(
qm150
) mutant. In fact, it even further extended the lifespan of these
mutants (Figure 6B). Given that
isp-1
(
qm150
) is a strong inducer of mitoUPR42 whereas
dve-1
RNAi effectively suppressed mitoUPR triggered by
isp-1
RNAi (Figures 2B and 2C),
our findings suggested that DVE-1’s role in mitoUPR activation and longevity regulation
can be uncoupled.
A previous study showed that
atfs-1
RNAi did not affect
isp-1
mutants but shortened the
lifespan of
nuo-6
(a component of mitochondrial respiration complex I) mutants when
RNAi treatment started from the parental generation.41 Similar to
isp-1
RNAi,
nuo-6
RNAi strongly induced mitoUPR (Figure 6C). Very interestingly, while
atfs-1
RNAi greatly
suppressed mitoUPR induced by
nuo-6
RNAi,
dve-1
RNAi had little effect (Figures 6C and
6D). Conversely,
dve-1
RNAi was much more effective at reducing the lifespan extension
caused by
nuo-6
RNAi compared to
atfs-1
RNAi (Figure 6E). These results suggest
that DVE-1 is not required for mitoUPR activation but is crucial for lifespan extension
induced by
nuo-6
RNAi, further supporting the idea that DVE-1’s functions in mitoUPR
activation and lifespan modulation can be uncoupled. Moreover, this finding provides key
evidence that specific mitochondrial stress-induced mitoUPR can be differentially regulated
by ATFS-1 and DVE-1, highlighting their potentially distinct roles in mitoUPR activation
and longevity regulation.
Apart from IIS and mitochondrial signaling, dietary restriction and germline signaling
are recognized as significant contributors to longevity.33,34 The
eat-2
gene encodes an
acetylcholine-gated ion channel, and the
eat-2
(
ad465
) mutant, characterized by reduced
pharyngeal pumping rate, serves as a classic model of dietary restriction.53 Although
the dietary-restricted
eat-2
(
ad465
) mutant worms are long lived, their longevity is largely
abolished by
dve-1
RNAi (Figure 6F). On the other hand, the
glp-1
Notch receptor, a
crucial regulator of germ-cell fate, renders the germline-less
glp-1
(
e2141
) mutant long
lived at 25°C.54 Similar to the
eat-2
(
ad465
) mutant, the extended lifespan conferred by
the germline-lacking
glp-1
(
e2141
) mutant is greatly suppressed by
dve-1
RNAi (Figure
6G). Thus, DVE-1 is implicated in both dietary-restriction-mediated and germline-signaling-
mediated longevity.
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Given that DVE-1’s involvement in ciliary modulation of longevity does not rely
on mitoUPR, we investigated whether mitoUPR is induced in the
eat-2
(
ad465
) and
glp-1
(
e2141
) mutant backgrounds. Once again, we did not detect mitoUPR induction using
hsp-6
expression as a marker (Figures 6H and 6I). In fact, the mRNA expression level of
hsp-6
is even reduced in the
glp-1
(
e2141
) mutant (Figure 6I). Overall, our study challenges
the predominant role of DVE-1 in mitoUPR activation, supporting its capacity to influence
multiple longevity pathways independently of mitoUPR.
As DVE-1 acts in dietary-restriction-modulated and germline-signaling-modulated
longevity, we next examined the subcellular localization of DVE-1::GFP upon
eat-2
or
glp-1
RNAi treatment. Our high-magnification (Figures S4A and S4B) and low-magnification
(Figure S4C) imaging results demonstrated that both
eat-2
RNAi and
glp-1
RNAi
substantially decreased the nuclear accumulation of DVE-1, consistent with our observations
in the
daf-10
(
p821
) mutant. Given that DVE-1 regulates the lifespan downstream of sensory
cilia, dietary restriction, and germline signaling independently of mitoUPR activation, we
wondered whether these three genetic pathways might share some overlapping mechanisms.
To test this, we examined the lifespans of
daf-10
(
p821
) mutant worms treated with empty-
vector RNAi,
eat-2
RNAi, and
glp-1
RNAi, respectively. Interestingly, we did not observe
an additive effect of
eat-2
RNAi or
glp-1
RNAi on lifespan extension in
daf-10
(
p821
)
mutants (Figure 6J). Therefore, these longevity pathways may share a common underlying
mechanism by converging on DVE-1 to influence aging.
DVE-1 has broader roles beyond mitoUPR
Since our results suggest that DVE-1 can influence lifespan in a mitoUPR-independent
fashion whereas ATFS-1 plays a central role in mitoUPR, we hypothesize that the target
gene profiles might be different between DVE-1 and ATFS-1, two parallel nuclear factors
essential for mitoUPR activation in
C. elegans
.26 Through reanalysis of mRNA sequencing
(RNA-seq) data (GEO: GSE131611 and GSE141041) across three conditions (empty-vector
RNAi,
dve-1
RNAi, and
atfs-1
RNAi),55,56 we observed that
dve-1
RNAi resulted in a
substantially greater alteration in the abundance of differentially expressed target genes
compared to
atfs-1
RNAi (3,177 versus 156) (Figure 7A). Moreover, only a minimal
overlap of nine genes each was observed as shared targets between
dve-1
and
atfs-1
under
the non-stressed resting condition, both in the upregulated and downregulated gene lists
(Figure 7A). Given ATFS-1’s central role in mediating mitoUPR, the starkly distinct target
gene profiles between ATFS-1 and DVE-1 suggest that DVE-1 may possess multifaceted
functions beyond mitoUPR through direct or indirect regulation of gene expression.
Next, we performed gene enrichment analysis of DVE-1-dependent target genes using
WormCat.57 As expected, the predominant category of DVE-1 upregulated genes included
those involved in stress response and pathogen defense (Figure 7B), aligning with its
role in mitoUPR-related processes.26,58 However, DVE-1 also upregulated numerous
target genes associated with G protein-coupled receptors (GPCRs) and proteolysis (Figure
7B), indicating its involvement in mitoUPR-independent functions. Notably, a recent
study uncovered DVE-1’s ability to orchestrate a transcriptional program of the ubiquitin-
proteasome system independently of mitoUPR, facilitating the elimination of juvenile
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synapses in GABAergic neurons during circuit remodeling in
C. elegans
.36 This observation
corresponds with our bioinformatic analysis results. Another interesting observation is
that the total number of DVE-1-downregulated genes exceeded that of upregulated genes
(1,817 versus 1,360) (Figure 7A), implying an overall repressive role of DVE-1 in gene
regulation. Notably, among the major types of cellular components downregulated by
DVE-1, sperm proteins and cilia were over-represented (Figure 7C), which might explain
why DVE-1 is involved in sensory-cilia-modulated and germline-signaling-modulated
longevity. Interestingly,
daf-10, osm-1, xbx-1
, and
dyf-1
are among the list of 31 ciliary
genes (Table S1) that are significantly downregulated by DVE-1 according to our reanalysis
of mRNA sequencing data. Using a qPCR assay, we found that
dve-1
RNAi elevated the
expression levels of
daf-10, osm-1, xbx-1
, and
dyf-1
to varying extents in WT worms
(Figure S5A) as well as in
daf-10
(
p821
) (Figure S5B),
eat-2
(
ad465
) (Figure S5C), and
glp-1
(
e2141
) (Figure S5D) mutants. This confirms that DVE-1 generally suppresses the
expression of these ciliary genes.
In addition to the unstressed resting condition, we also examined the DVE-1-dependent
gene-expression profile under mitochondrial stress condition (
atp-2
RNAi, GEO:
GSE141041).55 Intriguingly, among the 218 upregulated genes induced by
atp-2
RNAi,
only a small subset (42) overlapped with the target genes regulated by DVE-1 under
resting conditions, with 39 upregulated and 3 downregulated by DVE-1 (Figure 7D). Since
atp-2
RNAi is a well-established inducer of mitoUPR,55,58 these results suggest that: (1)
DVE-1 can govern the expression of numerous additional genes under mitochondrial stress
conditions (e.g., 176 additional genes were induced by
atp-2
RNAi); and (2) the majority of
DVE-1 target genes do not coincide with its mitochondrial stress-related target genes. These
findings further bolster the notion that DVE-1 may harbor broader functional roles beyond
mitoUPR.
Intrigued by the results with WT worms, we next performed RNA-seq experiments (GEO:
GSE276887) to compare transcriptomic changes dependent on DVE-1 and ATFS-1 in
the
daf-10
(
p821
) and
eat-2
(
ad465
) mutant backgrounds, respectively. Unlike the patterns
observed in WT worms, both DVE-1 and ATFS-1 showed a significant increase in
upregulated genes compared to downregulated ones in these mutants. Specifically, for
DVE-1, there were 1,281 upregulated versus 65 downregulated genes in
daf-10
(
p821
)
mutants and 3,061 versus 856 in
eat-2
(
ad465
) mutants. Similarly, ATFS-1 showed 866
upregulated versus 90 downregulated genes in
daf-10
(
p821
) mutants and 2,372 versus 345 in
eat-2
(
ad465
) mutants (Figures S6A and S6D). This suggests that both DVE-1 and ATFS-1
enhance gene expression in these long-lived mutant backgrounds. Notably, a significant
overlap was observed between the genes upregulated by ATFS-1 and those upregulated
by DVE-1 in both
daf-10
(
p821
) (687 out of 866) and
eat-2
(
ad465
) (2,117 out of 2,372)
mutants. As mitoUPR is not activated in either
daf-10
(
p821
) or
eat-2
(
ad465
) mutants, these
overlapping genes likely do not participate in mitoUPR but are potentially crucial for the
extended lifespan observed in these mutants.
An additional notable finding is the shift in gene-category enrichment: in WT worms,
stress-response-related and proteolysis-related genes are prominently upregulated by DVE-1
(Figure 7B). In contrast, these categories are enriched among the DVE-1 downregulated
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genes in both
daf-10
(
p821
) (Figure S6C) and
eat-2
(
ad465
) (Figure S6F) mutants.
Conversely, major sperm-protein-related and extracellular-material-related genes, which are
downregulated by DVE-1 in WT worms, become upregulated in both
daf-10
(
p821
) (Figure
S6B) and
eat-2
(
ad465
) (Figure S6E) mutants. Collectively, these observations indicate that
DVE-1-dependent transcriptomic profiles undergo significant alterations in these longevity
mutants.
DISCUSSION
Although sensory perception is typically considered as a transient process, it can have a
long-lasting impact on organismal aging.59 Genetic studies in model organisms such as
C.
elegans
and
Drosophila
demonstrated that sensory perception, especially chemosensation,
could be intrinsically detrimental for longevity.6,7,60 By contrast, transient and early
exposure to adult female olfactory cues has been shown to extend the lifespan of offspring
female mice.61 Thus, the overall impact of chemosensation on longevity could be context
dependent. On the one hand, chemosensation might encode the environmental chemical
cues and regulate downstream satiety/hunger signals in individual animals. In line with
this view, smell itself can significantly suppress the extended lifespan of dietary-restricted
worms and flies.7,60 On the other hand, chemosensation also mediates many aspects of
social behaviors such as social feeding, caring for offspring, attracting mates, and avoiding
predators.62 How the downstream signaling pathways underlying these social behaviors
regulate longevity is unclear. A previous study with
C. elegans
ciliary mutants identified
the FOXO transcription factor DAF-16 as an essential downstream transcription factor
of sensory modulation of aging.6 Our current findings extended this work by showing
that different ciliary components could regulate lifespan in both directions. Moreover,
we identified DVE-1 as another key factor downstream of sensory modulation of aging.
Nevertheless, more studies are needed to dissect the genetic and biochemical pathways
linking various ciliary mutations to the activation of DAF-16 and DVE-1.
Most previous studies on DVE-1 have been focusing on its critical role in mediating
mitoUPR. However, unlike ATFS-1, a well-characterized downstream transcription factor
of mitoUPR, DVE-1 does not undergo translocation to the mitochondria. This raises the
intriguing question of how DVE-1 detects the impaired mitochondrial environment. One
possibility is that the altered level of certain metabolite(s) due to impaired mitochondrial
respiration may relay the information from mitochondria to DVE-1 and trigger its nuclear
translocation. For example, a decreased level of citrate and reduced production of acetyl-
coenzyme A upon mitochondrial stress have been shown to act as a signal for DVE-1
nuclear accumulation and mitoUPR activation.38 Although ATFS-1 and DVE-1 are generally
essential for inducing mitoUPR in
C. elegans
(except in cases such as the
nuo-6
RNAi-
induced mitoUPR as shown in Figures 6C and 6D), they may regulate distinct sets of
target genes and could play different roles in mitoUPR and other cellular processes. Our
current study demonstrated that while DVE-1 is required for ciliary modulation of longevity,
ATFS-1 is not involved in this process. Because DVE-1 regulates the global chromatin
structure and has much more target genes than ATFS-1, it is possible that DVE-1 may
act at a higher level than ATFS-1 to mount the transcriptional responses during mitoUPR
activation. Alternatively, DVE-1-mediated chromatin remodeling and genome reorganization
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might be a prerequisite for ATFS-1’s role in turning on the transcription of various
mitoUPR-related target genes.
Most target genes of DVE-1 do not overlap with that of ATFS-1 in the WT background,
indicating that DVE-1 may have mitoUPR-independent functions. Indeed, a key finding of
the current study is that DVE-1 can act in a mitoUPR-independent manner in multiple aging-
related signaling pathways (i.e., sensory perception, dietary restriction, germline signaling)
to modulate lifespan. Therefore, DVE-1 may have multiple parallel signaling outputs, among
which mitoUPR serves an important branch.
As a transcriptional regulator and genome organizer, DVE-1 and its mammalian homolog
SATB factors are known to shuttle between the cytoplasm and the nucleus to exert their
roles in chromatin organization and gene regulation.23,63,64 While most attention has been
drawn to the nuclear roles of DVE-1/SATB in previous studies, our results indicate that
nuclear accumulation of DVE-1 is reduced in the long-lived
daf-10
(
p821
),
eat-2
(
ad465
), and
glp-1
(
e2141
) mutant backgrounds. Two potential mechanisms may explain this reduction.
First, nuclear DVE-1 has an overall suppressive role in longevity, and the reduction of
nuclear DVE-1 is due to degradation. However, our findings that
dve-1
RNAi shortened
the lifespan while DVE-1::GFP overexpression extended the lifespan of WT animals refute
the idea that DVE-1 suppresses longevity. Second, these longevity mutants may promote
the cytosolic translocation of DVE-1, a hypothesis supported by our high-magnification
imaging of posterior intestinal cells in
daf-10
(
p821
) mutants. Additionally, lifespan assays
with an NLS-deleted DVE-1 overexpression line further support the prolongevity effect
of cytosolic DVE-1. Subcellular fractionation experiments in intestinal cells could provide
further validation of these findings.
SET-25 functions as the H3K9 trimethylation methyltransferase in
C. elegans
, and the
set-25
(
n5021
) mutation results in nuclear accumulation of DVE-1.23 Notably, this nuclear
accumulation of DVE-1 in
set-25
(
n5021
) mutant worms does not significantly extend
lifespan, nor does it impact the mild mitochondrial stress-extended longevity.23 Therefore,
nuclear accumulation of DVE-1 by itself could be insufficient to extend lifespan. Instead,
our findings point to a potential cytosolic role for DVE-1 in promoting longevity, possibly
through its interaction with other cytosolic binding partners. We therefore propose a dual-
action model for DVE-1, wherein it modulates mitoUPR, aging, and likely other cellular
processes in the nucleus while also integrating multiple aging-related signaling pathways
in the cytosol (Figure 7E). Taken together, our current study suggests that DVE-1 may
have broader functions beyond its known role in mitoUPR activation. In addition to its
established nuclear role in gene transcription related to mitoUPR and likely other processes,
its involvement in cytosolic longevity regulation warrants further exploration.
Limitations of the study
Our tissue-specific RNAi experiments showed that DVE-1 primarily influences longevity
through the intestine rather than neurons. Imaging data indicated that various longevity
mutants (i.e.,
daf-10
(
p821
),
eat-2
(
ad465
),
glp-1
(
e2141
)) reduce the nuclear accumulation
of DVE-1 in intestinal cells. However, it is unclear whether these mutations similarly
affect DVE-1 localization in neurons and, if so, what the physiological roles of neuronal
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cytosolic DVE-1 might be. In addition, although DVE-1 appears to form puncta in both
the nucleus and cytoplasm, the cytoplasmic puncta are notably larger and more numerous
in the
daf-10
(
p821
) mutant background. The physiological significance of these cytosolic
DVE-1 puncta and their role in integrating longevity pathways remains to be explored.
Lastly, our study demonstrated that DVE-1 and ATFS-1 can have distinct functions in the
nuo-6
RNAi-induced mitoUPR activation and lifespan extension. Further research is needed
to elucidate the differential roles of these two key regulators of mitoUPR.
RESOURCE AVAILABILITY
Lead contact—Further information and requests for resources and reagents should be
directed to and will be fulfilled by the lead contact, Rui Xiao (rxiao@ufl.edu).
Materials availability—All unique/stable reagents generated in this study are available
from the lead contact without restriction.
Data and code availability
•RNA-seq data comparing transcriptomic changes influenced by DVE-1 and
ATFS-1 in the
daf-10
(
p821
) and
eat-2
(
ad465
) mutant backgrounds have been
deposited in the Gene Expression Omnibus under accession number GEO:
GSE276887 and are publicly available as of the date of publication. Additionally,
this study analyzes existing publicly available datasets at GEO, specifically
accession numbers GEO: GSE131611 and GSE141041. All data reported in this
paper will be shared by the lead contact upon reasonable request.
•This paper does not report original code.
•Any additional information required to reanalyze the data reported in this paper
is available from the lead contact upon request.
STAR★METHODS
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
Animals—Unless otherwise noted, all
C. elegans
strains were maintained at 20°C
on standard Nematode Growth Medium (NGM) plates with OP50 bacteria. For RNAi
experiments, worms were fed HT115 bacteria harboring the specific RNAi plasmid. A
comprehensive list of the strains used in this study, along with their genotypes, is provided in
the key resources table.
Microbe strains—The
Escherichia coli
strain OP50 was used as the standard food source,
while the
Escherichia coli
strain HT115 was used for RNAi feeding in
C. elegans
.
METHOD DETAILS
RNAi feeding assay—Standard RNAi feeding experiments were conducted as described
before,65,66 and
Escherichia coli
HT115 (DE3) transformed with the empty vector L4440
was used as control for all RNAi feeding experiments. Except
isp-1
RNAi, all RNAi clones
used in this study were derived from the Ahringer RNAi library and sequence verified using
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Sanger sequencing. If not specified, all RNAi experiments were initiated from the larval
1 stage once the worms were hatched. To confirm the knockdown efficiency of some key
RNAi clones, we performed reporter imaging and qPCR assays, as detailed in the Figure
S2. Specifically, compared to the empty vector RNAi,
dve-1
RNAi significantly reduced the
expression of DVE-1::GFP fusion proteins (Figures S2A and S2B). Our qPCR results further
validated the knockdown efficiency of RNAi clones targeting
atfs-1
(Figure S2C),
lin-65
(Figure S2D),
met-2
(Figure S2E),
ubl-5
(Figure S2F), and
clpp-1
(Figure S2G).
Lifespan assay—Lifespan experiments were conducted primarily at 20°C, with the
exception of experiments involving the temperature-sensitive
glp-1
(
e2141
) mutant, which
were conducted at 25°C. In all trials, day 1 of adulthood was designated as the starting point.
Each lifespan assay comprised ~50 worms, which were transferred to fresh 60 mm NGM
plates every 2–3 days at a density of ~10 worms per plate. Worms were censored if they
exhibited behaviors such as crawling off the plate, exploding, bagging, or if they became
contaminated by non-specific bacteria or fungi, as previously described.65–67 Notably, many
sensory cilia mutants exhibit defects in chemosensation and other sensory functions, leading
to a higher likelihood of these worms crawling off the plates and sticking to the walls
of petri dishes. These intrinsic sensory defects contribute to elevated censor rates in some
of our lifespan analyses. The comprehensive statistical analysis summaries for all lifespan
experiments can be found in Table S2.
qPCR assay—For each genotype, we collected 100–150 age-synchronized worms for
total mRNA extraction. Total RNA was isolated using TRIzol (Invitrogen) following the
manufacturer’s protocol. cDNA synthesis was carried out using the High Capacity cDNA
Reverse Transcription Kit (ThermoFisher Scientific). All qPCR reactions were conducted in
10 μL volumes with a primer concentration of 1 μM mixed with PowerUp SYBR Green
Master mix (ThermoFisher Scientific) using a CFX96 Touch Real-Time PCR System (Bio-
Rad). The mRNA level of
act-1
was utilized for normalization in all qPCR experiments.
Fluorescence microscopy and GFP reporter assay—To monitor the mitoUPR,
age-synchronized
zcls13
[
hsp-6p
::
GFP
] worms were transferred onto RNAi plates seeded
with HT115 bacteria containing different RNAi plasmids. Following mitoUPR induction,
immobilized worms were imaged at room temperature using an Olympus BX51
fluorescence microscope equipped with a 10X UPlanFI objective and a QImaging
optiMOS camera. Image acquisition was performed using μManager software (https://
micro-manager.org/), and the fluorescence signals of entire worms were quantified using
ImageJ software (http://rsbweb.nih.gov/ij/). To enable comparison across different worms
and genotypes, fluorescence signals were normalized to the respective worm areas.
To assess the subcellular localization of DVE-1, we visualized the translational fusion
proteins DVE-1::GFP, DVE-1::YFP, and DVE-1(ΔNLS)::YFP expressed in their respective
transgenic worms. Imaging was conducted using either an Olympus BX51 fluorescence
microscope equipped with a 40X UPlanFI objective or an Andor DSD2 high-speed spinning
disk confocal system coupled with a Nikon TiE microscope under a 100X Plan Apo
objective.
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Bioinformatic analysis of the RNA sequencing results—The datasets containing
lists of ATFS-1 and DVE-1 target genes in the wild type background were retrieved
from previously published studies with accession numbers GSE131611 and GSE141041,
respectively.55,56 For the RNAseq experiments in the
daf-10
(
p821
) and
eat-2
(
ad465
) mutant
backgrounds, poly-A selected RNAs from age-synchronized day-one adult mutant worms
were sequenced by the University of Florida Interdisciplinary Center for Biotechnology
Research (ICBR). Gene expression profiles and identification of differentially expressed
genes were obtained following established methods.55,56 Subsequently, genes exhibiting
significantly upregulated expression (log2[Fold Change] > 1) and downregulated expression
(log2[Fold Change] < −1) were compared to generate the Venn diagram.
QUANTIFICATION AND STATISTICAL ANALYSIS
Data analysis was conducted using PRISM 10 (GraphPad Software) and SigmaPlot 15
(Systat Software). The log rank (Kaplan-Meier) test was applied for lifespan assays, while
one-way ANOVA or unpaired Student’s t-test were utilized for comparing distinct groups in
qPCR assays and imaging experiments. Throughout all experiments, statistical significance
was defined as a
p
value <0.05.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institute on Aging (R01AG063766 and P30AG028740
to R.X.), the National Institute on Deafness and Other Communication Disorders (T32DC015994 to Z.M.),
and the American Cancer Society (RSG-17-171-01-DMC to R.X.). We appreciate the
Caenorhabditis
Genetic
Center, which is supported by the National Institutes of Health Office of Research Infrastructure Programs (P40
OD010440), for providing strains.
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Highlights
•DVE-1 acts downstream of ciliary modulation of lifespan independent of
mitoUPR
•DVE-1 aids longevity from dietary restriction and germline signaling,
independent of mitoUPR
•Mutations in these longevity pathways reduce DVE-1 nuclear accumulation
•DVE-1 and ATFS-1 likely play separate roles in activating the mitoUPR
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Figure 1. Different ciliary mutants in lifespan regulation
(A) An illustration of
C. elegans
sensory cilium showing multiple different ciliary
components involved in ciliogenesis and maintenance. The impacts of these components
(highlighted in red) on longevity are studied in subsequent experiments (B–H).
(B) The
daf-10
(
p821
) mutant with defective intraflagellar transport particle A is significantly
longer lived than wild-type (WT) worms.
(C) The
osm-1
(
p808
) mutant with defective intraflagellar transport particle B is significantly
longer lived than WT worms.
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(D) The
dyf-1
(
mn335
) mutant with defective motor activator is significantly longer lived
than WT worms.
(E) The
osm-3
(
p802
) mutant with defective kinesin motor is significantly longer lived than
WT worms.
(F) The
xbx-1
(
ok279
) mutant with defective dynein motor is significantly longer lived than
WT worms.
(G) The
klp-11
(
tm324
) mutant with defective kinesin-II motor has a lifespan similar to that
of WT worms.
(H) The
osm-12
(
n1606
) mutant with defective BBSome is significantly shorter lived than
WT worms.
The comprehensive statistical analysis summaries for all lifespan experiments can be found
in Table S2. Of note, in the case of multiple ciliary mutants within the same batch of lifespan
experiments, the comparison may involve the use of a common WT control. For example,
(B), (C), (D), (F), and (G) are conducted with the same control, whereas (E) and (H) share
another control experiment.
Sheng et al. Page 22
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Figure 2. dve-1 RNAi suppresses ciliary mutations-conferred longevity
(A) In addition to
daf-16
RNAi,
dve-1
RNAi strongly suppresses the greatly extended
lifespan of
daf-10
(
p821
) mutant animals.
(B)
dve-1
RNAi strongly suppresses the
isp-1
RNAi-induced mitoUPR activation. The
zcls13
[
Phsp-6
::
gfp
] transcriptional reporter line for mitoUPR35 is used in this experiment.
Compared to the basal condition on the empty vector (EV) RNAi bacteria (top panels),
a 1:1 mixture of
isp-1
RNAi feeding bacteria with EV RNAi bacteria triggers robust
expression of Phsp-6::GFP (middle panels). However,
dve-1
RNAi strongly suppresses
isp-1
RNAi-induced Phsp-6::GFP expression (bottom panels). Scale bar, 100 μm.
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(C) Quantification of Phsp-6::GFP fluorescence intensity, as depicted in (B), was performed.
The sample sizes are 15 for EV(RNAi), 15 for EV(RNAi) +
isp-1
(RNAi), and 13 for
isp-1
(RNAi) +
dve-1
(RNAi). Data are presented as mean ± SEM. Statistical significance
was determined using one-way ANOVA. ****
p
< 0.0001. (D–H)
dve-1
RNAi significantly
shortens the lifespans of WT (D) as well as long-lived
osm-1
(
p808
) (E),
dyf-1
(
mn335
) (F),
osm-3
(
p802
) (G), and
xbx-1
(
ok279
) (H) mutant worms.
(I and J)
dve-1
RNAi has no significant impacts on the lifespans of
klp-11
(
tm324
) (I) and
osm-12
(
n1606
) (J) mutants.
Of note, (I), Figure 3H, and Figure 4H are derived from the same experiment and share a
common empty vector (RNAi) control.
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Figure 3. The mitoUPR transcription factor ATFS-1 is not involved in ciliary modulation of
longevity
(A) Illustration of signaling pathways required for mitoUPR activation in
C. elegans
.
Among them, two parallel pathways are centered around two transcription factors/regulators,
ATFS-1 and DVE-1, both of which are highlighted in blue.
(B–I)
atfs-1
RNAi has no significant impacts on the lifespans of WT (B) and
daf-10
(
p821
) (C),
osm-1
(
p808
) (D),
dyf-1
(
mn335
) (E),
osm-3
(
p802
) (F),
xbx-1
(
ok279
) (G),
klp-11
(
tm324
) (H), and
osm-12
(
n1606
) (I) mutant worms.
Of note, (C) and Figure 4C share the same empty vector (RNAi) control experiment,
whereas (I) and Figure 4I share another empty vector (RNAi) control experiment.
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Figure 4. Ciliary mutations-conferred longevity does not require mitoUPR
(A) No changes of the
hsp-6
mRNA level are observed in the long-lived
daf-10
(
p821
),
normal-lifespan
klp-11
(
tm324
) or short-lived
osm-12
(
n1606
) mutant backgrounds,
suggesting that mitoUPR is not induced in these ciliary mutants. Each genetic background
is analyzed using three biological replicates, and the data are presented as mean ± SEM. ns,
not significant (t test).
(B) LIN-65, MET-2, UBL-5, and CLPP-1 have all been reported to interact with DVE-1
to regulate mitoUPR in
C. elegans
. However, unlike the lifespan-shortening effect of
dve-1
Sheng et al. Page 26
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RNAi, disruption of
lin-65, met-2, ubl-5,
and
clpp-1
by RNAi does not shorten the lifespan
in WT worms.
clpp-1
RNAi significantly extends lifespan, which is a commonly seen
phenomenon, by mildly inhibiting the functions of mitochondrially localized proteins.
(C–G) Unlike the robust suppression of
dve-1
RNAi on the lifespans of long-lived ciliary
mutants, disruption of
lin-65, met-2, ubl-5,
and
clpp-1
by RNAi does not have a similarly
strong impact on the lifespans of
daf-10
(
p821
) (C),
osm-1
(
p808
) (D),
dyf-1
(
mn335
) (E),
osm-3
(
p802
) (F), and
xbx-1
(
ok279
) (G) mutant worms.
(H and I)
clpp-1
RNAi significantly extends the lifespan of
klp-11
(
tm324
) (H) and
osm-12
(
n1606
) (I) mutant worms, whereas
lin-65, met-2,
and
ubl-5
RNAi have no or modest
effects on their lifespans.
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Figure 5. Cytosolic DVE-1 mediates ciliary modulation of longevity
(A and B) Potent induction of mitoUPR by
spg-7
RNAi, which can be largely suppressed
by either
atfs-1
RNAi or
dve-1
RNAi. Shown are the representative images (A) and
quantification of Phsp-6::GFP fluorescence intensity (B). The sample sizes are 17 for
EV(RNAi), 20 for
spg-7
(RNAi) + EV(RNAi), 21 for
spg-7
(RNAi) +
atfs-1
(RNAi), and
27 for
spg-7
(RNAi) +
dve-1
(RNAi). Data are presented as mean ± SEM. ****
p
< 0.0001
(one-way ANOVA). Scale bar, 100 μm.
Sheng et al. Page 28
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(C and D)
spg-7
RNAi promotes nuclear accumulation of DVE-1, as monitored using
the
zcIs39
[
Pdve-1
::
dve-1
::
gfp
] translational reporter line. (C) Representative images of the
expression pattern of DVE-1::GFP in the posterior intestine. (D) Quantifications of the
DVE-1::GFP fluorescence intensity in the nucleus versus cytosol treated with EV RNAi
(sample size 68) or
spg-7
RNAi (sample size 129). Data are presented as mean ± SEM. **
p
< 0.01 (t test). Scale bar, 100 μm.
(E and F) Opposite to the impact of
spg-7
RNAi, the
daf-10
(
p821
) mutation reduces
nuclear accumulation of DVE-1. (E) Representative images of the expression pattern of
DVE-1::GFP in the posterior intestine. (F) Quantifications of the DVE-1::GFP fluorescence
intensity in the nucleus versus cytosol of WT (sample size 105) and
daf-10
(
p821
) mutant
worms (sample size 112). Data are presented as mean ± SEM. **
p
< 0.01 (t test). Scale bar,
100 μm.
(G) Representative images of a group of aligned worms demonstrate that nuclear
accumulation of DVE-1::GFP is greatly reduced in the posterior intestine of
daf-10
(
p821
)
mutants (lower panels) compared to WT (upper panels) worms. Scale bar, 100 μm.
(H and I) Two transgenic lines are created by overexpressing
Pdve-1
::
dve-1
::
yfp
::
SL2
::
mCherry
and
Pdve-1
::
dve-1
(
ΔNLS
)::
yfp
::
SL2
::
mCherry
in the N2
WT background, respectively. These lines utilize the endogenous
dve-1
promoter to drive
the expression of DVE-1::YFP and DVE-1(ΔNLS)::YFP fusion proteins. The inclusion of
the SL2::mCherry cassette enables bicistronic expression, allowing simultaneous expression
of DVE-1::YFP/DVE-1(ΔNLS)::YFP and mCherry. The NLS-deleted DVE-1 variant
translocates from the nucleus to cytosol. (H) Representative images of the expression
pattern of DVE-1::YFP and NLS-deleted DVE-1(ΔNLS)::YFP in the posterior intestine.
(I) Quantification of the DVE-1::YFP (sample size 40) and DVE-1(ΔNLS)::YFP (sample
size 30) fluorescence intensity in the nucleus versus cytosol. Data are presented as mean ±
SEM. ****
p
< 0.0001 (t test). Scale bar, 100 μm.
(J) The overexpression of either DVE-1 or NLS-deleted DVE-1(ΔNLS) variant can
significantly extend lifespan.
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Figure 6. DVE-1 acts in a mitoUPR-independent fashion downstream of dietary-restriction-
modulated and germline-signaling-modulated lifespan
(A and B)
dve-1
RNAi does not significantly suppress the extended lifespans of
daf-2
(
e1370
) (A) and
isp-1
(
qm150
) (B) mutant worms, indicating that DVE-1 is not
involved in the IIS-regulated and mitochondrial-signaling-regulated longevity. Of note, (A)
and (B) share the same empty vector (RNAi) and
dve-1
(RNAi) control experiments.
(C and D)
nuo-6
RNAi-induced mitoUPR is strongly suppressed by
atfs-1
RNAi, but not
dve-1
RNAi. Shown are the representative images (C) and quantification of Phsp-6::GFP
fluorescence intensity (D). The sample sizes are 14 for EV(RNAi), 18 for
nuo-6
(RNAi),
Sheng et al. Page 30
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35 for
nuo-6
(RNAi) + EV(RNAi), 43 for
nuo-6
(RNAi) +
dve-1
(RNAi), and 25 for
nuo-6
(RNAi) +
atfs-1
(RNAi). Data are presented as mean ± SEM. ns, not significant;
****
p
< 0.0001 (one-way ANOVA). Scale bar, 100 μm. (E) While
dve-1
RNAi does not
affect the mitoUPR induced by
nuo-6
RNAi, it significantly shortens the lifespan of
nuo-6
RNAi-treated worms. This lifespan reduction is even more pronounced than that caused by
atfs-1
RNAi.
(F and G) The extended lifespans conferred by the dietary-restricted
eat-2
(
ad465
) (F)
and germline-lacking
glp-1
(
e2141
) (G) mutants are greatly suppressed by
dve-1
RNAi,
supporting that DVE-1 is involved in dietary-restriction-modulated and germline-signaling-
modulated lifespan.
(H and I) The
eat-2
(
ad465
) (H) and
glp-1
(
e2141
) (I) mutants do not elevate the expression
level of
hsp-6
mRNA, suggesting that mitoUPR is not activated by dietary restriction or
germline ablation. Each genetic background is analyzed using three biological replicates,
and data are presented as mean ± SEM. ns, not significant; ***
p
< 0.001 (t test).
(J) Neither
eat-2
RNAi nor
glp-1
RNAi further extends the lifespan of
daf-10
(
p821
)
mutants, suggesting that these factors may share a common genetic mechanism in regulating
longevity.
Sheng et al. Page 31
Cell Rep
. Author manuscript; available in PMC 2024 December 16.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Figure 7. Bioinformatic analyses of DVE-1 target genes in the WT background
(A) Compared to the empty-vector RNAi-treated control worms,
dve-1
RNAi significantly
elevates (>2-fold increase) the expression levels of 1,817 genes while it represses (>2-
fold decrease) the expression levels of 1,360 genes. Thus, DVE-1 downregulates 1,817
genes and upregulates 1,360 genes under the non-stressed resting condition. In a similar
comparison, ATFS-1 downregulates 86 genes and upregulates 70 genes. Among all these
target genes, only nine each are shared targets of DVE-1 and ATFS-1 between upregulated
and downregulated categories.
Sheng et al. Page 32
Cell Rep
. Author manuscript; available in PMC 2024 December 16.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript

(B and C) Gene enrichment analysis of DVE-1 target genes under the non-stressed resting
condition. Among the upregulated genes, many are involved in stress response, GPCR
signaling, and proteolysis (B). By contrast, major sperm proteins, cilia, and extracellular
matrix components are over-represented in the DVE-1 downregulated genes (C).
(D) Under the mitochondrial stress condition (
atp-2
RNAi), 176 extra DVE-1-dependent
genes are induced compared to the non-stressed resting condition. However, the majority of
DVE-1 target genes are not induced by
atp-2
RNAi.
(E) A working model of DVE-1’s roles in longevity modulation and mitoUPR activation.
Sheng et al. Page 33
Cell Rep
. Author manuscript; available in PMC 2024 December 16.
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Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Sheng et al. Page 34
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Bacterial and virus strains
E. coli:
OP50 Caenorhabditis Genetics
Center (CGC) OP50
E. coli
: HT115 cells transformed with an empty pL4440 vector plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli:
HT115 cells transformed with a pL4440-
spg
-
7
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
nuo
-
6
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
atfs
-
1
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
dve
-
1
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
lin
-
65
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
met
-
2
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
isp
-
1
(RNAi) plasmid This study N/A
E. coli
: HT115 cells transformed with a pL4440-
ubl
-
5
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
clpp
-
1
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
eat
-
2
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
E. coli
: HT115 cells transformed with a pL4440-
glp-1
(RNAi) plasmid Ahringer RNAi library,
Horizon Discovery Cat# RCE1181
Chemicals, peptides, and recombinant proteins
Levermisole hydrochloride ThermoFisher Scientific Cat# AC187870500
PowerUp SYBR Green Master mix ThermoFisher Scientific Cat# A25776
5-fluro-2’-deoxyuridine (FUDR) Chem-Impex
International Cat# 00867
TRI Reagent Solution ThermoFisher Scientific Cat# AM9738
Critical commercial assays
High-Capacity cDNA Reverse Transcription Kit ThermoFisher Scientific Cat# 4368814
Deposited data
Raw and analyzed data This study GEO: GSE276887
Raw and analyzed data Shao et al.55 GEO: GSE141041
Raw and analyzed data Li et al.56 GEO: GSE131611
Experimental models: Organisms/strains
C. elegans
strain: N2 Bristol (Wild type) CGC N2
daf-10
(
p821
) CGC PR821, RRID:WB-
STRAIN:WBStrain00030799
osm-1
(
p808
) CGC PR808, RRID:WB-
STRAIN:WBStrain00030795
Cell Rep
. Author manuscript; available in PMC 2024 December 16.

Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Sheng et al. Page 35
REAGENT or RESOURCE SOURCE IDENTIFIER
ruxEx384
[
Pdve-1
::
dve-1
::
yfp
::
SL2
::
mCherry
+
Plin-44
::
gfp
] This study N/A
ruxEx388
[
Pdve-1
::
dve-1
(
ΔNLS
)::
yfp
::
SL2
::
mCherry
+
Plin-44
::
gfp
] This study N/A
dyf-1
(
mn335
) CGC SP1205, RRID:WB-
STRAIN:WBStrain00034356
osm-3
(
p802
) CGC PR802, RRID:WB-
STRAIN:WBStrain00030794
xbx-1
(
ok279
) CGC JT11069, RRID:WB-
STRAIN:WBStrain00022845
klp-11
(
tm324
) CGC VC1228, RRID:WB-
STRAIN:WBStrain00036431
osm-12
(
n1606
) CGC MT3645, RRID:WB-
STRAIN:WBStrain00027084
daf-2
(
e1370
) CGC CB1370, RRID:WB-
STRAIN:WBStrain00004309
isp-1
(
qm150
) CGC MQ887, RRID:WB-
STRAIN:WBStrain00026670
eat-2
(
ad465
) CGC DA465, RRID:WB-
STRAIN:WBStrain00005463
glp-1
(
e2141
) CGC CB4037, RRID:WB-
STRAIN:WBStrain00004531
zcls13
[
Phsp-6
::
gfp
] CGC SJ4100, RRID:WB-
STRAIN:WBStrain00034068
zcIs39
[
Pdve-1
::
dve-1
::
gfp
] CGC SJ4197, RRID:WB-
STRAIN:WBStrain00034074
zcIs39
[
Pdve-1
::
dve-1
::
gfp
];
daf-10
(
p821
) This study N/A
ruxEx434
[
Pdve-1
::
SL2
::
mCherry
::
H2B
+
Plin-44
::
gfp
];
zcIs39
[
Pdve-1
::
dve-1
::
gfp
]This study N/A
ruxEx437
[
Pdve-1
::
SL2
::
mCherry
::
H2B
+
Plin-44
::
gfp
];
zcIs39
[
Pdve-1
::
dve-1
::
gfp
];
daf-10
(
p821
)This study N/A
dve-1
(
tm4803
) NBRP TM4803
dve-1
(
tm7599
) NBRP TM7599
sid-1
(
qt9
);
sqIs71
[
rgef-1p
::
GFP
+
rgef-1p
::
sid-1
] CGC MAH677
sid-1
(
qt9
);
alxIs9
[
vha-6p
::
sid-1
::
SL2
::
GFP
] CGC MGH171, RRID:WB-
STRAIN:WBStrain00026494
Oligonucleotides
Refer to the list of oligonucleotides (Table S3)
Recombinant DNA
Pdve-1
::
SL2
::
mCherry
::
H2B
This study N/A
Pdve-1
::
dve-1
::
yfp
::
SL2
::
mCherry
This study N/A
Pdve-1
::
dve-1
(
ΔNLS
)::
yfp
::
SL2
::
mCherry
This study N/A
Software and algorithms
FIJI National Institutes of
Health RRID: SCR_002285
Graphpad PRISM version 10.3.0 GraphPad Software, Inc. RRID:SCR_000306
Sigmaplot 15 Grafiti LLC RRID:SCR_003210
Cell Rep
. Author manuscript; available in PMC 2024 December 16.