The microRNA miR-71 suppresses maladaptive UPR^mt signaling through both cell-autonomous and cell-non-autonomous mechanisms

Domain: biology
Published: 2025-12-14
Editorial outcome: major_revision

Abstract
Mitochondria play a central role in metabolism and biosynthesis, but function also as platforms that perceive and communicate environmental and physiological stressors to the nucleus and distal tissues. Systemic mitochondrial signaling is thought to synchronize and amplify stress responses throughout the whole body, but during severe or chronic damage, overactivation of mitochondrial stress pathways may be maladaptive and exacerbate aging and metabolic disorders. Here we uncover a protective micro(mi)RNA response to mtDNA damage in Caenorhabditis elegans that prolongs tissue health and function by interfering with mitochondrial stress signaling. Acting within muscle cells, we show that the miRNA miR-71 is induced during severe mitochondrial damage by the combined activities of DAF-16, HIF-1, and ATFS-1, where it restores sarcomere structure and animal locomotion by directly suppressing the inordinate activation of DVE-1, a key regulator of the mitochondrial unfolded protein response (UPRmt). Indirectly, miR-71 also reduces the levels of multiple neuro- and insulin-like peptides and their secretion machinery, resulting in decreased cell-non-autonomous signaling of mitochondrial stress from muscle to glia cells. miR-71 therefore beneficially coordinates the suppression of both local and systemic mitochondrial stress pathways during severe organelle dysfunction. These findings open the possibility that metabolic disorders could be ameliorated by limiting the overactivation of mitochondrial stress responses through targeted small RNAs.

Introduction
Mitochondria are compartmentalized hubs of cellular metabolism, biosynthesis, and signaling, and their dysregulation is closely associated with metabolic disorders and diseases of aging, including diabetes, neurodegeneration, and cancer1,2. Organisms frequently encounter both extrinsic and intrinsic stresses that perturb mitochondria and disrupt cellular homeostasis. An inevitable threat to correct mitochondrial function are mitochondrial genome (mtDNA) mutations and other molecular lesions, which accumulate in post-mitotic tissues through oxidative damage and duplication mistakes and can expand clonally in stem cell compartments3,4. mtDNA mutations can also be maternally inherited, culminating in congenital metabolic disorders. Both inherited and acquired mtDNA mutations can persist for the duration of an organism’s life, causing varying amounts of chronic mitochondrial stress that may contribute to disease5,6. As such, the capacity of cells to perceive, respond proportionately, and function under mtDNA stress underpins organismal health, especially during aging and mitochondrial disease conditions.

Metazoans have evolved mitochondrial-nuclear signaling pathways, such as the mitochondrial unfolded protein response (UPRmt)7–11, that can alleviate mitochondrial burden and promote recovery and overall organismal health. Genes that are upregulated as part of the UPRmt, which is activated by mtDNA lesions in both mammals and C. elegans12–16, encode protein functions that encompass a broad profile of processes that extend beyond direct mitochondrial activity, including promoting innate immunity and pathogen clearance, maintaining cytosolic proteostasis, activating anerobic metabolism and reactive oxygen species detoxification, and mediating signaling between tissues17–19. Although mitochondria-nuclear communication mechanisms differ between mammals and invertebrates, they are regulated by conceptually similar processes20. In C. elegans, where the regulatory mechanisms of the UPRmt are best characterized, multiple factors mediate UPRmt signaling. Nuclear localization of the homeobox protein DVE-1 mediates a branch of the transcriptional response to mitochondrial stress10 and its activity occurs downstream of chromatin remodeling driven by mitochondrial dysfunction. Upon mitochondrial stress, the histone methyltransferase MET-2 is required for nuclear translocation of the cytosolic protein LIN-65, a histone H3K9 methyltransferase that globally compacts chromatin while promoting the redistribution and transcriptional activity of DVE-1 in chromatin sites that remain open21. This suggests that DVE-1 may play a key role in the propagation of an epigenetic memory of mitochondrial stress experienced during early development that underpins changes in later life traits, such as adult lifespan21,22.

In addition to retrograde signaling, mitochondrial stress can be communicated systemically in multiple species. In C. elegans, mitochondrial damage restricted to neurons can activate UPRmt responses in peripheral tissues that are not experiencing stress themselves, such as the intestine or germline23,24, via cell-non-autonomous signaling mechanisms that involve the secretion of neurotransmitters such as glutamate, acetylcholine, tyramine, serotonin, as well as insulin-like peptides, neuropeptides, and Wnt/EGL-2025–28. This intercellular or inter-tissue communication of mitochondrial stress has been shown to occur in mammals and involves multiple circulating factors such as fibroblast growth factor 21 (FGF21), adrenomedullin 2 (ADM2), growth/differentiation factor 15 (GDF15), angiopoietin-like 6 (ANGPTL6), and various mitochondrial-derived peptides29. Intercellular communication of mitochondrial stress is thought to play a role in synchronizing and amplifying stress responses throughout the whole body, thereby coordinating homeostasis of the organism more effectively than if stress responses were restricted to within already affected cells.

However, under chronic mitochondrial stress, localized as well as systemic activation of mitochondrial stress responses could be detrimental to organismal fitness. Indeed, whereas acute UPRmt signaling is important for mitochondrial recovery following stress, chronic UPRmt activation is maladaptive as it can drive the propagation of mutant mitochondrial genomes14, induce or aggravate neuroinflammation and neurodegenerative disorders30,31, and contribute to the pathogenesis of diseases of aging, including cancers32. For instance, subpopulations of cancer cells that demonstrate persistent activation of the UPRmt display an adaptive metastatic advantage, with breast cancer patients with high UPRmt genetic signatures having significantly worse clinical outcomes32. Mechanisms that counteract both cell-autonomous and cell-non-autonomous mitochondrial stress signaling during chronic mitochondrial dysfunction are yet to be understood but could play an essential role in organismal homeostasis and be important for ameliorating a broad spectrum of disorders.

Here we reveal a miRNA-mediated protective response to chronic mtDNA damage within the muscle of C. elegans, which acts to simultaneously attenuate cell-autonomous as well as cell-non-autonomous UPRmt signaling by targeting DVE-1 and systemic mitochondrial stress signaling mechanisms. We find that during mitochondrial stress, miR-71 is strongly upregulated through a combination of HIF-1, ATFS-1, and DAF-16 nuclear localization, where it then recognizes and targets dve-1 transcripts for degradation. Overexpression of miR-71 restores muscle cell structure and function disrupted by chronic mtDNA damage and hyperactivated UPRmt. Remarkably, severe mitochondrial stress suppresses a broad range of cell-cell signaling molecules in a miR-71-dependent manner, suggesting that miR-71 silences systemic signaling during chronic mitochondrial stress possibly to prevent maladaptive inter-tissue responses. Indeed, muscle miR-71 limits distal DVE-1 nuclear accumulation in glial cells, upstream mediators of whole animal stress signaling, via the neuropeptide-like protein nlp-52. Together, our results reveal a small non-coding RNA regulatory network that antagonizes local and systemic mitochondrial quality control programs during chronic stress.

Results
miR-35 family and miR-71 are induced during mitochondrial stress
The broad abilities of small non-coding RNAs to regulate gene expression in a manner that promotes homeostasis during stress prompted us to investigate whether a miRNA response to mtDNA damage is activated in animals. miRNAs are a class of 21- to 23-nucleotide non-coding RNAs that post-transcriptionally downregulate genes by targeting mRNAs for translational inhibition or degradation33. We performed small RNA sequencing in genetically engineered C. elegans backgrounds that incur muscle-specific mitochondrial DNA double strand breaks (mtDSBs) via the expression of a mitochondria-targeted endonuclease (MTSPstI) under the myo-3 promoter (from hereon called myo-3p::mtDSB) (Fig. 1a, b)13. When compared to control animals expressing a catalytically inactive version of MTSPstI (myo-3p::mtDSB*), we identified 24 miRNAs that were significantly upregulated as a result of mtDNA lesion (FDR<0.05, log2 fold change >2, Supplementary Data Table 1). Interestingly, most of the members of the miR-35 family of miRNAs (miR-35/36/37/38/39/40/41-3p) were highly represented in this group (Fig. 1c). This miRNA family is widely conserved and its members share identical seed regions, and are therefore likely to target the same mRNAs and have similar biological functions34,35, which are as diverse as embryonic patterning36–38, sex determination39, germ cell apoptosis40, and hypoxia and starvation responses41,42. In addition to the miR-35 family, miR-71 was upregulated in response to mtDSBs (Fig. 1c). miR-71 has previously been shown to increase in abundance with age in C. elegans, and is indispensable for lifespan enhancements caused by starvation43–45 or germline removal46. It has also recently been demonstrated to mediate olfactory regulation of organismal proteostasis and longevity47. We confirmed that miR-35 family members and miR-71 were upregulated in animals during mtDSBs using quantitative (q)PCR (Fig. 1d, Extended Data Fig. 1a).

In addition to mtDSBs, which can be caused by replication fork arrest during mtDNA synthesis and various endogenous and exogenous genotoxins48, mtDNA mutations also pose a major risk to correct mitochondrial function when present at high enough heteroplasmy levels (the percentage of mitochondrial genomes carrying a mutation)49. We found that a large 3.1-kb mtDNA deletion (called uaDf5) that removes four protein-coding and seven tRNA genes (Fig. 1b)50,51 caused upregulation of both miR-71 and miR-35 at heteroplasmy levels of 60% (Fig. 1e, Extended Data Fig. 1b). Animals harboring high heteroplasmy levels (90%) of a smaller 100-bp mtDNA deletion (called mpt1, 12) within the ND1 gene (Fig. 1b) also induced miR-71 but not miR-35 (Fig. 1f, Extended Data Fig. 1d). Similarly, miR-71 but not miR-35 was induced in animals homozygous for a mutation in the proof-reading domain of the mitochondrial DNA polymerase γ (polg-1(srh1)) (Fig. 1g, Extended Data Fig. 1d), which causes increased levels of de novo mtDNA mutations while also reducing mtDNA copy number52. Finally, pharmacological inhibition of complex IV of the mitochondrial respiratory chain with sodium azide induced both miR-71 and miR-35 (Fig. 1h, Extended Data Fig. 1e). Because miR-71 was consistently induced by diverse forms of mtDNA damage, we focused on its role during mitochondrial dysfunction. Importantly, unlike mitochondrial stress, neither cytoplasmic nor endoplasmic reticulum stress induced miR-71 (Extended Data Fig. 1f-k), suggesting that it forms a small non-coding RNA response to stress originating from mitochondrial dysfunction.

miR-71 counteracts muscle cell dysfunction caused by mtDSBs
To determine whether an increase in the abundance of miR-71 is protective against cellular dysfunction caused by mtDNA damage, we tested whether miR-71 overexpression reduced the effects of mtDSBs on muscle cells. Animals expressing myo-3p::mtDSBs displayed disruptions in the alignment of actin filaments that form sarcomeres (Fig. 1i, j), the basic contractile units of muscle cells. The animals also exhibited severe decreases in their rates of muscle-driven body bends (Fig. 1k) after they were forced to initiate locomotion by being placed in liquid medium, a measure of maximal muscle functional capacity. Increasing miR-71 levels six-fold via expression of the transgene mir-71p::miR-71 (Extended Data Fig. 1l)46 mitigated the disruption to actin filaments (Fig. 1i, j) as well as the muscle dysfunction (Fig. 1k) caused by muscle-specific mtDSBs.

In animals carrying mir-71p::GFP53, we observed fluorescence in multiple tissues including the intestine, neurons, pharynx, vulval muscle, and body wall muscles (Extended Data Fig. 1m), suggesting, as previously described45,46, that miR-71 is expressed ubiquitously. Moreover, miR-71 is loaded into Argonaute silencing complexes in most major tissue types, including the body wall muscles, intestine, and neurons, suggesting widespread activity54. To determine whether miR-71 acts within muscle cells to protect against mtDSBs, we generated animals overexpressing miR-71 selectively in this tissue (myo-3p::miR-71) in backgrounds in which the endogenous mir-71 gene was deleted (mir-71(n4115)). Interestingly, deletion of mir-71 did not exacerbate muscle dysfunction during mtDSBs (Extended Data Fig. 1n), possibly due to redundancy in the system. However, we found that selective overexpression of miR-71 in muscle cells was sufficient to promote muscle recovery in the presence of muscle-specific mtDSBs (Fig. 1l). Selective overexpression of miR-71 in either the intestine (ges-1p::miR-71) or neurons (rgef-1p::miR-71) did not reduce the vulnerability of muscle cells towards mtDSBs (Fig. 1l). Collectively, these results suggest that upregulation of miR-71 in response to mtDNA damage is protective of tissue function in a cell-autonomous manner.

miR-71 targets the UPRmt regulator dve-1
Nearly all animal miRNAs regulate their target mRNAs by binding to short recognition sequences in their 3’ untranslated regions (UTRs) through complementarity, leading to mRNA degradation or translational repression55. 3’UTRs of target mRNAs often contain multiple recognition sites with either perfect or imperfect miRNA complementation. To identify targets of miR-71 (seed sequence: AGAAAG), the regulation of which could reduce cellular vulnerability toward mitochondrial damage, we used TargetScan56 to predict potential miR-71 3’UTR binding sites across the C. elegans transcriptome. We found 399 mRNAs with putative miR-71 recognition sites (UCUUUC) which we further sorted for mitochondrial-related functions. Interestingly, five potential miR-71 targets encoded key components of pathways involved in mitochondrial quality control and metabolic signaling, including dct-1 and atg-2 which mediate mitochondrial autophagy (mitophagy), dve-1 and hsp-6, which are involved in the UPRmt, and daf-2 which regulates insulin/IGF-1 signaling. To experimentally determine whether any of these genes were regulated by miR-71, we generated transgenic reporter strains where the 3’UTR of each putative target was incorporated into a fluorescent transgene and integrated as a single-copy genome insertion to ensure consistent expression (Extended Data Fig. 2a). Each C. elegans reporter strain, as well as a control line harboring the tbb-2 (tubulin) 3’UTR that does not contain a predicted miR-71 binding site, were crossed to animals overexpressing miR-71 (mir-71p::miR-71). Similarly to the tbb-2 3’UTR control reporter, the 3’UTR reporters for dct-1, atg-2, hsp-6 and daf-2 were not suppressed by miR-71 overexpression (Extended Data Fig. 2b). However, the reporter containing the 3’UTR of dve-1 was significantly suppressed (Fig. 2a, b), suggesting that miR-71 can negatively regulate a key transcription factor of the UPRmt.

To confirm this interaction, we mutated the two putative miR-71 binding sequences within the dve-1 3’UTR reporter (Fig. 2c) and remade the transgenic strains as above. Disrupting the putative miR-71 binding sites not only increased the overall fluorescent signal of the reporter but also abolished any suppression caused by miR-71 overexpression (Fig. 2d, e). We therefore conclude that dve-1 transcripts are true regulatory targets of miR-71, and that the predicted miR-71 binding sites within the dve-1 3’UTR are required for miR-71-mediated suppression. To determine whether miR-71 regulated the abundance of endogenous dve-1 transcripts, we quantified their mRNA levels using qPCR. In the absence of mtDNA damage, we found that dve-1 mRNA levels were not significantly affected by miR-71 overexpression (Fig. 2f). However, upon muscle-specific mtDSBs, dve-1 transcript levels increased six-fold. Overexpression of miR-71 in this background restored dve-1 transcript levels to basal levels (Fig. 2f), suggesting that miR-71 can post-transcriptionally attenuate dve-1 upregulation during severe and chronic mtDNA damage by promoting the degradation of dve-1 mRNAs. We did not identify putative miR-71 binding sites in genes encoding other UPRmt regulators, such as the transcription factor atfs-1 or the ubiquitin-like protein ubl-5, suggesting that dve-1 is the only major UPRmt regulator targeted by miR-71. Supporting this notion, unlike dve-1, we did not detect decreases in the transcript levels of atfs-1 or ubl-5 when miR-71 was overexpressed, while their abundance increased due to muscle-specific mtDSBs (Extended Data Fig. 2c, d).

miR-71 is cytoprotective against excessive UPRmt activation
As a transcriptional regulator of the UPRmt, DVE-1 activates a wide range of genes involved in mitochondrial repair, detoxification, and stress resistance10,57. It is therefore surprising that miR-71 acts to suppress dve-1 during mtDNA damage. To resolve this apparent paradox, we sought to understand how activation of the UPRmt affects cell function during mtDSBs. Using two fluorescent mitochondrial chaperone reporters, hsp-6p::GFP and hsp-60p::GFP9, which are commonly used to assess UPRmt activation, we observed increased GFP fluorescence in muscle cells in response to mtDSBs (Fig. 3a-d), suggesting that UPRmt activation is coincident with the upregulation of miR-71 during mtDNA damage stress. We hypothesized that miR-71 orchestrates negative feedback to reduce the activities of the UPRmt post-transcriptionally, which may be beneficial when UPRmt hyperactivation is detrimental to cellular homeostasis. Indeed, overexpression of a constitutively active version of the key UPRmt regulator ATFS-1 (ATFS-1nuc) selectively in muscle cells exacerbated muscle cell dysfunction in the presence of mtDSBs, indicating that chronic UPRmt activation is detrimental during severe mitochondrial stress (Fig. 3e). Supporting the hypothesis that miR-71 mitigates the detrimental effects of excessive UPRmt, overexpressing miR-71 significantly improved muscle function in animals expressing ATFS-1nuc (Fig. 3e).

Furthermore, several lines of evidence indicated that miR-71-mediated cytoprotection is caused by a reduction in the DVE-1-mediated UPRmt response to mtDSBs. First, deleting mir-71 exacerbated muscle dysfunction in animals overexpressing dve-1 in the presence of muscle-specific mtDSBs (Fig. 3f). Second, upregulation of dve-1 in response to mtDSBs was abolished by miR-71 overexpression (Fig. 2f). Third, hsp-6, a downstream effector of the UPRmt and a DVE-1 transcriptional target was upregulated in response to mtDSBs and suppressed when miR-71 was overexpressed under its native promoter or in a muscle-specific manner (Fig. 3g). Fourth, mutating the two miR-71 binding sites within the endogenous 3’UTR of dve-1 via genome editing significantly reduced miR-71-mediated improvements in muscle function (Fig. 3h) and muscle structure (Fig. 3i, j) in the presence of mtDSBs. And lastly, knocking down dve-1 by RNA interference (RNAi) emulated the effects of mir-71 overexpression, alleviating the mtDSB phenotype (Fig. 3k). Together, these results suggest that miR-71 functions to attenuate DVE-1-mediated UPRmt hyperactivity in response to chronic mtDNA damage.

miR-71 is regulated through the integration of ATFS-1, DAF-16 and HIF-1 activities
As our results implicated miR-71 as a negative feedback regulator of DVE-1-mediated UPRmt, we hypothesized that mir-71 itself must be regulated in proportion to mitochondrial stress levels in order to counteract UPRmt hyperactivation triggered by severe or chronic damage. Indeed miR-71 abundance correlated with the extent of mtDNA defects. For instance, miR-71 levels were not significantly increased in animals harboring uaDf5 at a heteroplasmy of 35% but increased more than nine-fold in animals with 60% uaDf5 heteroplasmy (Fig. 1e). miR-71 abundance also remained at normal levels in heterozygous polg-1(srh1) mutants but increased strongly in homozygous mutants (Fig. 1g). These results hinted at a mechanism whereby miR-71 upregulation is induced once mitochondrial stress levels surpass a certain threshold. Given that the levels of the pre-mir-71 precursor and mature miR-71 increased by the same amount in response to mtDSBs as well as sodium azide (Fig. 4a, Extended Data Fig. 3a), we reasoned that miR-71 was regulated through transcription, rather than through miRNA biogenesis.

To understand how mir-71 was transcriptionally upregulated in response to mitochondrial stress, we investigated the requirements of several conserved pathways which are known to act as sensors of mitochondrial and metabolic state, including ATFS-1 signaling. Our results revealed that mtDSBs promoted nuclear accumulation of ATFS-1::GFP in muscle cells (Fig. 4b, c), which alongside the induction of hsp-6p::GFP and hsp-60p::GFP (Fig. 3a–d), confirmed that mtDNA damage invokes ATFS-1-mediated UPRmt signaling. Furthermore, ATFS-1 was required for mir-71 upregulation in response to sodium azide, as miR-71 induction was suppressed in several atfs-1 loss-of-function mutants, including atfs-1(cmh15) null animals (Fig. 4d, Extended Data Fig. 3b). However, miR-71 levels were not increased in atfs-1(et15) mutants, which harbor a defective mitochondrial targeting signal in ATFS-1, rendering it constitutively nuclear localized and active (Extended Data Fig. 3c). This suggested that ATFS-1 is required but not sufficient to upregulate miR-71. As such, we next investigated whether other factors contribute to mir-71 upregulation during mitochondrial stress.

Analysis of the upstream promoter region of mir-71 revealed putative binding sites for ATFS-1 as well as several other transcription factors that are involved in cellular metabolic and oxidative stress signaling, including the FOXO transcription factor DAF-16 and the hypoxia-inducible factor 1 (HIF-1) (Extended Data Fig. 3d). DAF-16 and HIF-1 are regulated via shuttling from the cytoplasm to the nucleus, which can occur in response to starvation and hypoxic/oxidative cellular cues, respectively, but also upon mitochondrial stress58–60. We found that mtDSBs invoked nuclear translocation of both DAF-16::GFP and HIF-1::GFP in muscle cells (Fig. 4b, c). Furthermore, increases in miR-71 levels caused by sodium azide exposure were abolished in daf-16(mu86) mutants and significantly reduced in hif-1(ia07) mutants (Fig. 4e, f). These results suggest that ATFS-1, DAF-16, and HIF-1 activities are required but not sufficient to upregulate mir-71 expression in response to mitochondrial stress. Consistent with this notion, mir-71 upregulation was not further inhibited in daf-16(mu86);atfs-1(tm4525) or daf-16(mu86);atfs-1(tm4525);hif-1(RNAi) genetic backgrounds relative to individual genetic mutations in daf-16 or atfs-1 (Fig. 4g). Finally, constitutive DAF-16 activation caused by the daf-2(e1370) mutation61 was insufficient to increase miR-71 levels and instead reduced their abundance (Extended Data Fig. 3e). In addition, transgenic expression of an undegradable and therefore constitutively active HIF-1P621G variant62 was also unable to induce miR-71, similar to animals harboring an atfs-1(et15) constitutively active allele (Extended Data Fig. 3c and e). Moreover, in animals with double as well as triple mutant/transgene combinations that should activate two or all three pathways simultaneously, we still did not observe miR-71 upregulation (Extended Data Fig. 3e). These findings support the notion that ATFS-1, DAF-16, and HIF-1 activities are required but not sufficient to activate mir-71 transcription, and that other pathways, yet to be identified, also contribute to miR-71 induction during mitochondrial stress.

ATFS-1, DAF-16 and HIF-1 bind to the mir-71 promoter sequence
Mining of publicly available ChIP-sequencing data sets for ATFS-1 (GSE63803), DAF-16 (ENCSR946AUI), and HIF-1 (ENCSR991HIA) revealed that all three transcription factors occupied overlapping regions of the mir-71 promoter region during stress (Fig. 4h). We confirmed this physical interaction by simultaneously expressing ATFS-1, DAF-16, and HIF-1 in HEK293T cells, and demonstrating that they each could bind to a sequence corresponding to the mir-71 promoter that was also co-transfected into the cells (Fig. 4i, Extended Data Fig. 3f). However, coimmunoprecipitation experiments did not reveal any physical interaction between the three transcription factors (Extended Data Fig. 3g), suggesting that they regulated mir-71 independently. Together, these results suggest that during mtDNA damage, ATFS-1, DAF-16, and HIF-1 localize to the nucleus where they interact with the promoter of mir-71 to coordinate its induction. This tripartite mechanism of regulation could act to restrict mir-71 upregulation to scenarios in which mitochondrial damage is severe enough to activate all three stress signaling pathways simultaneously, such as when invoking mtDSBs.

miR-71 suppresses cell-non-autonomous signaling of mtDNA damage from the muscle to glia cells
We next asked whether miR-71 activity extended beyond localized suppression of dve-1. The coordinated regulation of mitochondrial functions across different tissues is believed to optimize the fitness of an organism. For example, the UPRmt and changes in mitochondrial dynamics can be non-autonomously regulated in the intestine by mitochondrial perturbation in neurons24–28,49. In animals expressing dve-1p::DVE-1::GFP, we found that muscle-specific mtDSBs induced nuclear accumulation of DVE-1::GFP in intestinal cells (Extended Data Fig. 4a) and other cells which resembled glia in the heads of animals (Fig. 5a). We therefore generated animals expressing a pan-glial fluorescent reporter (mir-228p::mKate2) and confirmed that glial DVE-1::GFP was activated by muscle-specific mtDSBs (Fig. 5b), suggesting that mtDNA damage within muscle cells can be communicated cell-non-autonomously to glia. Interestingly, deletion of mir-71 increased the basal signal of nuclear DVE-1::GFP in the glia (Fig. 5c, d), suggesting that miR-71 suppressed UPRmt activation. Furthermore, mir-71 deletion further enhanced the glial DVE-1::GFP signal induced cell-non-autonomously by muscle-specific mtDSBs (Fig. 5c, d), suggesting that miR-71 acts to suppress signaling that induces the UPRmt in distal cells not experiencing mitochondrial damage themselves. In support of this notion, overexpressing miR-71 (mir-71p::miR-71) produced the opposite effect and significantly reduced DVE-1::GFP signals in the glia following muscle-specific mtDSBs (Fig. 5e, f). Importantly, the DVE-1::GFP transgene harbors an unc-54 3’UTR that does not contain predicted miR-71 binding sites, indicating that miR-71 regulates glial UPRmt activation, rather than the expression of the reporter directly.

To determine whether miR-71 functions within muscle cells to attenuate cell-non-autonomous signaling of mitochondrial damage to glial cells, we crossed the UPRmt reporter DVE-1::GFP into animals overexpressing miR-71 specifically in muscle tissue (myo-3p::miR-71). Remarkably, this suppressed glial UPRmt induction invoked by muscle-specific mtDSBs (Fig. 5e, f), suggesting that miR-71 in the muscle cells can dampen cell-non-autonomous UPRmt signaling. Ruling out the possibility that miR-71 itself transported from muscle-to-glia to distally silence the UPRmt, the glial signal of a miR-71 activity reporter (his-72p::mCherryNLS::dve-1 3’UTR) was not reduced by the selective overexpression of miR-71 in muscle tissue (Extended Data Fig. 4b, c). Furthermore, we generated animals overexpressing miR-71 specifically in the glia (mir-228p::miR-71), which did not reduce cell-non-autonomous glial UPRmt induction (Fig. 5g, h), reinforcing the notion that miR-71 functions within muscle to suppress muscle-to-glia mitochondrial damage signaling. This activity required target recognition by miR-71 in the muscle, as overexpression of a scrambled seed-region variant (myo-3p::miR-71*, Extended Data Fig. 4d), which is incapable of binding target mRNAs, did not suppress glial DVE-1::GFP (Extended Data Fig. 4e, f). Interestingly, miR-71-mediated suppression of cell-non-autonomous UPRmt signaling appeared to be independent of its regulation of dve-1, as disruption of the miR-71 binding sites within the endogenous dve-1 3’UTR did not impact miR-71-mediated suppression of muscle-to-glia UPRmt activation (Extended Data Fig. 4g, h). Similarly, a dve-1(tm4803) mutation did not attenuate muscle-to-glia UPRmt activation (Extended Data Fig. 4i, j), further suggesting that dve-1 and its regulation by miR-71 has no influence on muscle-to-glia UPRmt signaling. These findings collectively indicate that miR-71 acts within muscle cells to limit the propagation of a cell-non-autonomous stress signal to glia, likely by repressing one or more target transcripts other than dve-1.

miR-71 suppresses peptides required for muscle-to-glia UPRmt signaling
To identify possible targets and a mechanism by which miR-71 suppressed cell-non-autonomous mitochondrial stress signaling, we performed RNA sequencing (RNA-seq) of mir-71 deletion mutants treated with the complex IV inhibitor sodium azide to identify transcripts regulated by miR-71 during mitochondrial stress. Under non-stressed conditions, we identified 608 transcripts with increased levels in mir-71 deletion animals (FDR<0.05; Fig. 6a, Supplementary Data Table 2), suggesting that these genes are either directly or indirectly regulated by miR-71. In animals additionally exposed to sodium azide, we identified 2,754 transcripts elevated in mir-71 mutants that did not overlap with basally regulated transcripts (FDR<0.05; Fig. 6a, Supplementary Data Table 2), representing genes that are normally suppressed by miR-71 exclusively during mitochondrial stress. In C. elegans, non-autonomous UPRmt signaling is mediated by multiple neurotransmitters, neuropeptides, and insulin-like peptides24–28. Remarkably, within the deregulated subset of transcripts, we identified a large number of mRNAs that encode neuropeptides and insulin-like peptides that are suppressed during mitochondrial stress in a miR-71-dependent manner (Fig. 6b and Extended Data Fig. 5a). This includes those previously reported to communicate mitochondrial stress systemically, including INS-27, INS-3528, FLP-163 and FLP-227 (Fig. 6b, Extended Data Fig. 5a, Supplementary Data Table 2), suggesting that miR-71 may act to dampen cell-non-autonomous mitochondrial stress signaling via regulation of signaling peptides.

To identify a specific peptide that could mediate muscle-to-glia UPRmt activation, we performed an RNAi screen against candidates identified by RNA-seq. Of the 70 genes tested (Supplementary Data Table 3), only suppression of nlp-52, which encodes a neuropeptide-like protein, significantly reduced glial UPRmt activation during muscle-specific mtDSBs (Fig. 6c, d), suggesting its involvement in muscle-to-glia mitochondrial stress signaling. We confirmed this result using the nlp-52(tm12973) mutant, which phenocopied nlp-52 RNAi (Fig. 6e, f). In further support, dense-core vesicle exocytosis, which is required for neuropeptide signaling and is mediated by UNC-31 (Ca2+-dependent activator protein for secretion, CAPS)25, is necessary for muscle-to-glia UPRmt signaling as RNAi of unc-31 reduced glial DVE-1::GFP activation in animals experiencing muscle-specific mtDSBs (Extended Data Fig. 5b, c).

Next, we generated animals in which nlp-52 was knocked-down exclusively in body wall muscle cells through the expression of complementary sense and antisense RNA sequences driven by the myo-3 promoter that target nlp-52 transcripts. Muscle-to-glia UPRmt signaling was almost completely abolished in these animals (Fig. 6g, h), suggesting that NLP-52 originating from the muscle is required to activate cell-non-autonomous UPRmt in the glia. Confirming this interpretation, we re-introduced a wild type copy of the nlp-52 gene under the control of a muscle-specific promoter (myo-3p) into nlp-52(tm12973) mutants, which restored muscle-to-glia UPRmt activation (Fig. 6e, f).

Methods
All resources used in this study are listed in Supplementary Data Table 4 to 6.

C. elegans strains and maintenance
A full list of strains made for this project are listed in Table S4. Animals were maintained according to established methods71 on Nematode Growth Medium (NGM) agar plates seeded with OP50 E. coli and cultured at 20°C unless otherwise indicated. Genetic crosses were performed with the aid of SoMarker72. Animals with Mos-mediated single-copy insertions of transgenes (MosSCI, foxSi alleles) were generated as described previously73. Briefly, gonads of 1-day-old adults of the MosSCI acceptor strains were injected with 50ng/μl of the corresponding plasmid (PureLink™ HiPure Plasmid Miniprep Kit, Invitrogen). After injection, animals were kept at 25°C to enable Mos-transposase-mediated insertion, which was confirmed by PCR approximately 2 weeks after injection. Animals carrying extrachromosomal arrays were obtained by injecting 10ng/μl of plasmid DNA (PureLink™ Quick Plasmid Miniprep Kit, Invitrogen) together with the co-injection marker odr-1p::dsRed or odr-1p::GFP (10ng/μl) into gonads of 1-day-old adults. Subsequent transmission was checked by following the co-injection marker.

Constructs
A full list of plasmid constructs made for this project are listed in Table S5. Most constructs were generated with overlap extension PCR (MegaWHOP), similar to previously performed74 using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific), T4 polynucleotide kinase (NEB), T4 DNA ligase (NEB), the restriction enzyme DpnI (cat. R0176, NEB), and DH5α competent bacteria. For genomic DNA (gDNA) template used for cloning, wild-type (N2) animals were lysed for 1h at 65 °C in Mitochondrial Lysis Buffer (MLB)75, supplemented with 0.1mg/ml proteinase K. gDNA was subsequently extracted by phenol:chloroform following standard procedure.

The mir-71 coding sequence was amplified from N2 gDNA using the oligos ik481 and ik474. Standard sequences of his-72, eft-3, myo-3, ges-1 and rgef-1 promoters, and the tbb-2 3’UTR, used in constructs can be found in the plasmids pSZ33, pSZ110, pSZ32, pSZ57, pSZ59, respectively75. To swap tbb-2 3’UTR in pSZ232 with alternative 3’UTR sequences, the dct-1 3’UTR was amplified with the oligos ik245 and ik246, atg-2 3’UTR with ik247 and ik248, hsp-6 3’UTR with ik249 and ik250, daf-2 3’UTR with ik251 and ik252, and dve-1 3’UTR with ik255 and ik256. The two mutations of the miR-71 binding motif in the dve-1 3’UTR were introduced by PCR using the oligos ik282/ik283 and ik293/ik294. The promoter region of mir-228 was amplified from N2 gDNA using the oligos ik473 and ik472. The mir-71 locus (mir-71p::mir-71::mir-71 3’UTR) was amplified from N2 gDNA using the oligos ik343 and ik463. All oligonucleotide sequences are listed in Table S6.

pSZ336 (CMVp::hif-1(P621G)::3xFLAG::P2A::atfs-1::2xMYC::T2A::daf-16::HA) was created by assembling gBlock cDNA fragments (IDT) of atfs-1 (NM_074114), daf-16 (NM_001381205) and hif-1 (NM_075607.8). C. elegans cDNA sequences were codon-optimized for expression in mammalian cells. HIF-1 degradation was prevented by using P621G mutation62. cDNA fragments were assembled using Phusion DNA polymerase (NEB), Taq DNA ligase (NEB), and T5 exonuclease (NEB) following the standard Gibson assembly protocol76,77.

Small RNA sequencing
Synchronized worm populations were obtained by egg pulse, which was performed by isolating 100 egg-laying adults for 2h in three biological replicates per strain, removing adults, and then allowing progeny to hatch and develop. When the progeny reached the L4 stage, larvae were collected in 1ml of M9 buffer with 0.01% Tween 20 and washed twice. 500μl of TRIzol was added to the pelleted animals and the samples were freeze-cracked three times in liquid nitrogen to break open the cuticle. The standard TRIzol extraction protocol was followed, and the final RNA pellet was diluted in 20μl of nuclease-free water. Small RNA sequencing libraries were prepared using the Illumina small RNA sequencing kit with 1μg of total RNA input. Libraries were checked for size on an Agilent 2100 Bioanalyzer system, and their concentration was measured using the Invitrogen™ Qubit™ Fluorometer. Libraries were pooled and sequenced on an Illumina NextSeq sequencer. The obtained reads were mapped to the C. elegans genome version ce10 and annotated to miRBase release 21. Data was thresholded to adjusted P≤0.05 and a fold change of 2 for downstream analysis. Fold changes were calculated based on the 2-ΔΔCt method.

Locomotion assay
For all locomotion assays, animals were synchronized by egg pulse by allowing 30 egg-laying adults to produce eggs for 2h before removing them from the NGM plate and raising the progeny to the L4 stage. The L4 animals were collected in 500μl of M9 buffer with 0.01% Tween 20 and transferred to 35mm NGM plates without food using 1ml of M9 buffer. After one minute of free swimming (adaptation time) the plates were placed under a Nikon SMZ745T stereomicroscope with a True-Chrome IIS camera (Tucsen Photonics) at 1.5x magnification and swimming behavior was recorded for 1min. Body bends per minute were quantified using WormLab software (MBF Bioscience). 720 frames of video were analyzed by the software and at least 250 continuously trackable frames from individual animals were used for quantification.

Microscopy and image analysis
For imaging, animals were immobilized in 10µl of 25mM Tetramisole (Sigma) on a 2% agarose (dissolved in H2O) pad on a glass slide and covered with a coverslip. Microphotographs were taken on a Zeiss Z2 imager microscope equipped with a Zeiss Axiocam 506 mono camera, using the ZEN 2 software (Zeiss, version 2.0.0.0). Image processing and analysis were performed with FIJI software78.

Real-time qRT-PCR analysis of mRNAs
Total RNA was extracted using TRIZol according to the manufacturer’s instructions and dissolved in 20–50µl of ultrapure water (Invitrogen), depending on RNA pellet size. 1µl was reverse-transcribed in a volume of 20µl containing 4µl of 5X ProtoScript II reaction buffer (NEB), 25µM dNTPs, 50µM oligo dT, 50µM random hexamers, 2µl DTT, 20 units of ProtoScript II reverse transcriptase (NEB), and 0.25µl of murine RNase inhibitor (NEB). The reaction was incubated at 42°C for 1h followed by enzyme inactivation at 65°C for 15min. cDNA was diluted 1:20 and real-time quantitative reverse transcriptase PCR (RT qPCR) was run on a LightCycler 480II (Roche) in a total volume of 10µl containing 5µl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1µl of cDNA and 500nM of each primer at the cycling conditions of 95°C for 3min, followed by 45 cycles of denaturation at 95°C for 10s, annealing at 60°C for 10s, and elongation at 72°C for 20s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. Samples were normalized to actin, a gene which did not change its expression level under the experimental conditions. Fold changes were calculated based on the 2-ΔΔCt method.

Real-time qRT-PCR analysis of miRNAs
Total RNA was Poly(A) tailed and reverse-transcribed as described previously79. Briefly, a total volume of 10µl containing 200ng of total RNA, 2µl 5X ProtoScript II reaction buffer (NEB), 25µM ATP, 25µM dNTPs, 50µM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42°C for 1h, followed by enzyme inactivation at 95°C for 5min. MiRNA-specific primers were designed as previously described79. cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480II (Roche) in a total volume of 10µl containing 5µl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1µl of cDNA and 250nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95°C for 3min, followed by 45 cycles of denaturation at 95°C for 10s, annealing at 60°C for 10s, and elongation at 72°C for 20s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. Samples were normalized to miR-59, a miRNA which did not change its expression level under the experimental conditions. Fold changes were calculated based on the 2-ΔΔCt method.

RNA sequencing
As above, synchronized worm populations were obtained by egg pulse, which was performed by isolating 100 egg-laying adults for 2h in three biological replicates per strain, removing adults, and then allowing the progeny to hatch and develop. When the progeny reached the L4 stage, larvae were collected in 1ml of M9 buffer with 0.01% Tween 20 and washed twice. 500μl of TRIzol was added to the pelleted animals and the samples were freeze-cracked three times in liquid nitrogen. The standard TRIzol extraction protocol was followed, and the final RNA pellet was diluted in 20μl of nuclease-free water. Sequencing library preparation, sequencing and data analysis were carried out by Azenta (https://www.azenta.com/). 1μg total RNA was used for library preparation with poly(A) mRNA isolated using Oligo(dT) beads. mRNA fragmentation was performed using divalent cations and high temperature, and cDNA was synthesized using random sequence primers. The purified double-stranded cDNA was treated to repair both ends and add a dA-tail in one reaction, followed by a T-A ligation to add adapters to both ends. Size selection of Adapter-ligated DNA was then performed using DNA Clean Beads and each sample was amplified by PCR using P5 and P7 primers and the PCR products were validated. Libraries with different indexes were then multiplexed and sequenced using a 2×150 paired-end (PE) configuration according to the manufacturer’s instructions. In order to remove technical sequences, including adapters, PCR primers, or fragments thereof, and quality of bases lower than 20, pass filter data in FASTQ format were processed by Cutadapt (V1.9.1, phred cutoff: 20, error rate: 0.1, adapter overlap: 1bp, min. length: 75, proportion of N: 0.1) to filter reads. The C. elegans reference genome sequence and gene model annotation files were downloaded from https://www.ncbi.nlm.nih.gov/assembly/GCF_000002985.6/. Hisat2 (v2.0.1) was used to index the reference genome sequence and to align filtered reads to the reference genome. FASTA format transcripts were converted from the known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end filtered data. Differential gene expression analysis was completed using the DESeq2 Bioconductor package, with an adjusted P value cutoff of P<0.05 for differentially expressed genes.

Pharmacological treatments
Sodium azide (Sigma) was dissolved in MilliQ H2O and diluted in M9 to a concentration of either 10mM (miR-71 induction experiments) or 20mM (hsp-6p::GFP experiments) and 0.2ml of this solution was then spread evenly over 35mm (4ml) NGM plates seeded with E. coli OP50 and left to absorb into NGM to reach final concentrations of 0.5mM and 1mM, respectively. Larval stage L1 individuals were transferred onto these plates and were raised to L4 and then analyzed (miR-71 induction experiments). For hsp-6p::GFP experiments, L4 animals were placed on the sodium azide plates for 5h and then assessed. Tunicamycin (Sigma) was dissolved into M9 buffer to a concentration of 0.4µg/ml and 0.2ml of this solution spread evenly over 35mm NGM plates seeded with E. coli OP50 and allowed to absorb into the NGM to reach a final concentration of 0.02µg/ml. L4 individuals were transferred onto these plates and allowed to develop for 5h before analysis.

HEK293T cell culture, transfection, and co-immunoprecipitation
HEK293T cells were grown in 10cm culture dishes in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum at 37°C and 5% CO2. When the cells reached 60-70% confluency, 10μg of plasmid pSZ336 (and for co-transfection 5μg of pSZ314) were transfected with X-tremeGENE™ 9 (Roche) in Opti-MEM medium (Gibco) according to the manufacturer’s instructions. When cells reached full confluency, they were washed with cold PBS and lysed in 500μl of lysis buffer supplemented with proteinase inhibitor (Sigma) and 1mM phenylmethylsulfonyl fluoride (PMSF) on ice for 30min with occasional vortexing. Debris was removed by centrifugation at 12,000xg at 4°C for 15min. An input of 60μl was kept and 400μl of supernatant were diluted with additional 600μl of supplemented lysis buffer and subjected to immunoprecipitation with either 20μl anti-MYC-beads (Pierce, 88842), 50μl of anti-FLAG beads (Pierce, A36797), or 25μl of anti-HA beads (Pierce, 88857) and incubated at 4°C overnight on a rotator. The beads were collected with a magnetic rack and washed 3 times with ice-cold 1X PBS and 3 times with ice-cold RIPA buffer (25mM Tris-HCl pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 300mM NaCl, followed by two more washes with ice-cold RIPA buffer containing 1M NaCl. The washed beads were resuspended in 60μl of lysis buffer and 20μl of 4X western blot sample buffer. The samples were then denatured at 95°C for 5min, spun at 20,000xg for 10min at room temperature and ran on a standard 10% SDS PAGE western blot. Coimmunoprecipitation was judged by detecting the individual protein tags.

Western blot
Samples were boiled in 1X western blot sample buffer at 95°C for 5min and centrifuged at 20,000xg for 10min at room temperature. The samples were then run on a 10% SDS polyacrylamide gel in running buffer (25mM Tris, 250mM glycine, 0.1% SDS) and blotted onto a PVDF membrane in ice-cold transfer buffer (25mM Tris, 250mM glycine, 20% ethanol). Membranes were blocked in TBST buffer (20mM Tris-HCl (pH 7.5), 137mM NaCl, 0.1% Tween 20) containing 5% bovine serum albumin (BSA, Moregate Biotech) and incubated with primary antibodies in TBST with 5% BSA overnight at 4°C on a shaker. After 3 washes with TBST, blots were incubated with the respective secondary antibody in TBST at room temperature for one hour and imaged on an Odyssey CLx near-infrared fluorescence imaging system (LI-COR). The antibodies and dilutions used in this study were: anti-HA (Cell Signaling, Cat. 3724), 1:5,000; anti-FLAG (Sigma-Aldrich, Cat. F3165), 1:5,000; anti-Myc (Invitrogen, Cat. PA1-981), 1:5,000; IRDye 680RD goat anti-mouse IgG (LI-COR, Cat. 68070), 1:20,000; and IRDye 800CW goat anti-rabbit IgG (LI-COR, Cat. 32211), 1:20,000.

Chromatin immunoprecipitation
HEK293T cells were transfected as described above, with each cell culture dish being treated as a biological replicate and three replicates being used in the study. After 24h, cells were washed 3 times with cold PBS and then fixed for 10min at room temperature in 1% PFA in PBS in a total volume of 10ml on an orbital shaker (20 rpm), after which, 1ml of 2.5M glycine was added to quench the fixation reaction and incubated for another 10min. Cells were then washed 3 times with PBS and scraped off the culture dish in 1ml of RIPA buffer (10mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM EDTA, 0.15M NaCl) supplemented with protease inhibitor (Sigma) and 1mM PMSF, and incubated on ice for 15min with occasional vortexing. Samples were then sonicated using a Covaris (M220 Focused-Ultrasonicator) for 450s at 25W, CBP 200, DF 20%. After centrifugation samples were spun at 10,000xg for 10min at 4°C. Supernatant was collected and 1% input were taken. The rest of the sample was split into three tubes and antibody-conjugated beads were added: 20μl of anti-MYC-beads (Pierce, 88842), 50μl of anti-FLAG beads (Pierce, A36797), or 25μl of anti-HA beads (Pierce, 88857). The samples were then incubated for 2h at 4°C, rotating (12 rpm). Magnetic beads were collected on a magnetic stand (BioRad) and washed 1X with 0.5ml TSE-150 (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl), 2X with 0.5ml TSE-500 (TSE with 500mM NaCl), 1X with 0.5 TSE-1M (TSE with 1M NaCl), 1X with 0.5ml LiCl (250mM LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1), and 2X with 0.5ml TE (10mM Tris pH 8.0, 1mM EDTA). Then, 2×0.2ml of elution buffer (1% SDS, 0.1M NaHCO3) was added to the beads with incubation for 15min on a rotator at room temperature. After adding 20μl of 5M NaCl, cross-links were reversed overnight at 65°C. The next day, 10μl of 0.5M EDTA, 20μl of 1M Tris-HCL pH 6.5, and 5μl of proteinase K (10mg/ml) were added and incubated for another hour at 65°C. DNA was then extracted by standard phenol-chloroform extraction and the pellet was diluted in 50μl of MilliQ H2O. The presence of amplicons of the mir-71 promoter was tested for by qPCR using the oligos ik393 and ik394 (see Table S6) as described above. Fold enrichment was obtained by comparing the amount of target sequence measured in the IP isolate to the amount measured in the nonspecific IgG control isolate.

RNA interference (RNAi)
RNAi was performed in a similar manner to that previously described80. Briefly, bacteria harboring sequenced-verified RNAi feeding vectors were obtained from the Ahringer library. IPTG plates were made by adding 238μl of Luria broth (LB) medium, 8μl of 1M IPTG (Promega), and 4μl of ampicillin (100mg/ml) to the surface of 35mm NGM plates and allowed to absorb into the NGM and dry. 200μl of the RNAi bacterial culture (OD 0.4-0.7) was seeded onto these plates, which were subsequently kept in the dark at room temperature to grow a lawn for 24h. Eggs were seeded onto the RNAi plates and animals raised to the L4 stage and analyzed. To generate strains expressing sense and antisense sequences targeting nlp-52 in muscle cells, the nlp-52 cDNA lacking start and stop codons was cloned into a MosSCI vector under the control of the muscle-specific myo-3 promoter and used to generate transgenic animals. The nlp-52 sequences were confirmed to have no off-target activity with DsCheck81.

RNAi by injection
RNAi by injection was performed as previously described82. Briefly, DNA from the hif-1 RNAi feeding clone was extracted with the Invitrogen High Pure kit. Five individual 50μl PCR reactions were set up with 100ng of plasmid using the T7 primer and Phusion polymerase. All reactions were run on separate lanes on a 1% agarose gel and the bands running at 1,200 bp were cut and DNA was extracted using the Monarch gel purification kit according to the manufacturer’s protocol. 4μg of the purified (pooled) DNA was transcribed into RNA with T7 RNA polymerase and rNTPs for 5h at 37°C. RNA was purified by standard TRIzol extraction (500μl per 100μl transcription reaction), and the pellet was resuspended in 50μl of nuclease-free water. 1-day-old adults were injected into the germline with the purified RNA and subsequent progeny were raised to L4 for analysis.

Phalloidin staining
Phalloidin staining was performed as previously described83. Briefly, 50 L4 animals were collected and washed in M9 buffer and snap-frozen in liquid nitrogen. After thawing, all remaining liquid was removed with a SpeedVac vacuum concentrator. Dried animals were then fixed with ice-cold acetone and dried under a fume hood. 5μl of Phalloidin–Atto 565 (Sigma) per tube was used for staining in a total volume 20μl for 30min in the dark. After 3 washes in M9 buffer with Tween, worms were mounted on agar pads and imaged as described above. The average number of lesions per cell was calculated for each animal. A health score was assigned to each individual, with a maximum score of 3 referring to all muscle cells showing perfect striation, 2.5 being similar but with small imperfections, 2 showing some lesions, and 1 referring to breaks in muscle fibers in all or most cells.

Statistical analysis
For experiments comparing two conditions, data were analyzed using unpaired two-way Student’s t-tests. For experiments with more than two conditions, data were analyzed using a one-way ANOVA with Tukey’s or Šídák’s multiple comparison tests. Statistical tests, n values, and replicate numbers are noted in the figure legends. Most statistical analyses were performed in GraphPad Prism (version 10.2.0).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study reveals that miR-71 protects against mtDNA damage in Caenorhabditis elegans by interfering with mitochondrial stress signaling, thereby extending tissue health and function. miR-71 is induced in muscle cells by DAF-16, HIF-1, and ATFS-1. During severe mitochondrial damage, miR-71 restores myofibril structure and animal motility by directly inhibiting the overactivation of DVE-1. Additionally, miR-71 reduces the transmission of mitochondrial stress signals from muscles to glial cells.\n\nThe mitochondrial unfolded protein response (UPRmt) is crucial for alleviating mitochondrial burden and promoting recovery, and the study highlights its potential adaptive failure in cases of severe or chronic damage. The research reveals a signaling axis between muscle and glial cells, emphasizing the importance of muscle cells in conveying mitochondrial health status information.\n\nThere are some questions that the authors need to address.\n1, In the case of mtDSB, miR-71 promotes the recovery of muscle cell function by reducing DVE-1. Can directly knocking down DVE-1 restore muscle function? Additionally, could other UPRmt inhibitors also restore muscle function?\n2, The regulation of miR-71 expression by the genes ATFS-1, DAF-16, and HIF-1 cannot be understood through knockdown experiments alone; overexpression studies are also required. For example, in ATFS-1 loss-of-function C. elegans, does the overexpression of DAF-16 and HIF-1 affect miR-71 expression?\n3, After mitochondrial stress is generated in muscle cells, it is transmitted to glial cells. Can the stress generated in glial cells regulate muscle cells in reverse and reduce muscle cell damage? In the case of mtDSB, if UPRmt is reduced only in glial cells without reducing it in muscle cells, will this alleviate the functional damage of muscle cells, or will it increase the damage to muscle cells?\nMinor issue:\nThe abbreviation mtDSB requires the full name.", "reviewer_2": "In this manuscript, the authors performed RNA sequencing in genetically engineered muscle-specific mtDSBs C. elegans backgrounds and identified 24 miRNAs that were significantly upregulated, including the miR-35 family of miRNAs and miR-71. Overexpression of miR-71 in muscular tissue is protective against cellular dysfunction caused by mtDSBs cell-autonomously. However, the deletion of mir-71 did not exacerbate muscle dysfunction during mtDSBs. The authors used TargetScan to predict the targets of miR-71 and found that dve-1 can be targeted for degradation. Meanwhile, they found that ATFS-1, DAF-16, and HIF-1 can bind the upstream promoter region of mir-71. Beyond the cell-autonomous effect, the authors also found that miR-71 can suppress cell-non-autonomous signaling of mtDNA damage from the muscle to glia cells through the regulation of unc-31 to suppress ins-11 and nlp-52, alleviating the activation of UPRmt. The authors concluded the cell-autonomous and cell-non-autonomous functions of miR-71 in regulating maladaptive UPRmt signaling. While the manuscript presents interesting findings on the role of miR-71 in regulating UPRmt signaling, several critical aspects diminish its overall significance and depth of mechanistic insight:\n\nMain points:\nThe study suggests that miR-71 targets DVE-1 and regulates UPRmt activation, but it lacks rigorous evidence to substantiate this claim. The authors primarily rely on miR-71 overexpression data without providing comprehensive analyses using miR-71 mutants or dve-1 3'UTR mutants under muscle-specific mtDSBs stress. This omission limits the depth of mechanistic understanding and raises questions about the robustness of the proposed pathway.\n\nAdditionally, the finding that mutation in mir-71 does not exacerbate muscle dysfunction during mtDSBs is contradictory to the proposed protective role of miR-71. Further experiments to examine muscle dysfunction in mir-71 mutants without mitochondrial stress would clarify the baseline effects of miR-71 and provide a more complete mechanistic insight.\n\nThe role of miR-71 in UPRmt regulation needs to be contextualized within the broader framework of known UPRmt pathways. The mainstream UPRmt regulation primarily involves ATFS-1, DVE-1, and UBL-5, which orchestrate a well-characterized response to mitochondrial stress. The authors should compare the impact of miR-71-mediated regulation to these established pathways, demonstrating whether miR-71 plays a redundant, synergistic, or distinct role.\n\nThe authors' finding that ATFS-1, DAF-16, and HIF-1 regulate miR-71 expression under mtDSBs stress is intriguing, but the functional relevance of this regulation remains unclear. How do these factors integrate with the canonical UPRmt pathway? Is miR-71 expression a downstream consequence or a parallel regulatory mechanism? Addressing these questions is crucial to establish the significance of miR-71 in the broader context of UPRmt regulation.\n\nThe manuscript proposes that miR-71 exerts protective effects without binding to its endogenous 3'UTR of dve-1p::DVE-1::GFP (The reporter was constructed in a vector containing unc-54 3’UTR). This paradoxical observation needs to be resolved through additional experiments. The authors should explore alternative mechanisms by which miR-71 might influence DVE-1 or other components of the UPRmt pathway.\n\nThe proposed cell-non-autonomous effects of miR-71 in suppressing signaling from muscle to glial cells are intriguing but underexplored. The roles of ins-11 and nlp-52 in this process should be validated through RNAi, tissue-specific knockout or knockdown, and tissue-specific rescue assays to understand their function in muscle and UPRmt signaling comprehensively.\n\nIn summary, the study in its current form lacks the significance and depth required to advance our understanding of UPRmt regulation. Addressing these critical points would enhance the mechanistic insight and position of miR-71 within the established landscape of UPRmt regulatory pathways.", "reviewer_3": "The study by Kirmes et al. describes the identification of miR-71 in muscles in response to severe mitochondrial stress and activation of DAF-16, HIF-1 and ATFS-1 and leads to degradation of DVE-1 to alleviate overactivation of the UPRmt.\nAdditionally, to the impact of miR-71 in the muscle, they propose that miR-71 leads to reduced secretion of neuro and insulin-like peptides which act to suppress muscle to glia axis of mitochondrial stress. Therefore, they suggest that miR-71 is a novel target of the UPRmt, that act to prevent its overactivation and leads to local and systemic beneficial effects.\nOverall, the introduction is well written and provide appropriate background and relevant literature. The inclusion of a nuclease dead control in the original screen is a strong control, data supporting the impact of miR71 following mitochondrial damage in muscle is strong, the results linking miR71 to the regulation of DVE1 in figure 2 is convincing and involves proper controls, same applies to all figures notably the control in figure 5 for regulation of UPRmt rather than reporter directly, that miR71 does not spread itself across\ntissues. Therefore, the study present compelling results and is very well designed.\nWhile the non cell-autonomous effect of mitochondrial stress in one tissue affecting another is already known, the novelty here is to add miR71 and the fact that the strength of signal is important.\n\nCritiques:\n1- miR-35 is activated significantly more than miR-71, miR246 seems activated at similar level to miR71, several stressors also activates mir35 but polG represses it , which is interested. Is there a specificity within the miR35 family? the focus on miR-71 or lack of analysis of miR-35 and miR-246 needs to be justified in a stronger manner, especially that the group have published in miR71 previously and therefore the choice of focusing on this particular miR come across as purely biased. The same applies to the focus on the peptides already known to regulate non-cell autonomous communication between tissues following mitochondrial stress in the screen in figure 6.\n2- Overexpression of miR71 increases daf2 significantly the receptor of insulin in muscle but this is not discussed, how it relates to the finding of insulin-like peptide in figure 6 (Extended data Fig.2).\n3- Choice of sodium azide for RNAseq is unfortunate and detract from main finding using a mtDNA specific DNA damage. While some validation is provided in fig. 6d and e, a more comparison of the gene expression in panel b and c following muscle and mitochondrial specific DNA damage should be provided."}, "author_response": "Point-by-point responses\nmiR-71 promotes muscle func6on during mitochondrial dysfunc6on by suppressing\nmaladap6ve UPRmt signaling.\nNew figures are referred to in the text below and textual changes to the manuscript are\ncoloured in red in the revised manuscript file.\nReviewer #1 (Remarks to the Author):\nThis study reveals that miR-71 protects against mtDNA damage in Caenorhabdi6s elegans\nby interfering with mitochondrial stress signaling, thereby extending 6ssue health and\nfunc6on. miR-71 is induced in muscle cells by DAF-16, HIF-1, and ATFS-1. During severe\nmitochondrial damage, miR-71 restores myofibril structure and animal mo6lity by directly\ninhibi6ng the overac6va6on of DVE-1. Addi6onally, miR-71 reduces the transmission of\nmitochondrial stress signals from muscles to glial cells.\nThe mitochondrial unfolded protein response (UPRmt) is crucial for allevia6ng\nmitochondrial burden and promo6ng recovery, and the study highlights its poten6al\nadap6ve failure in cases of severe or chronic damage. The research reveals a signaling axis\nbetween muscle and glial cells, emphasizing the importance of muscle cells in conveying\nmitochondrial health status informa6on.\nWe thank Reviewer #1 for their positive comments and insightful suggestions, which we\nhave addressed below and in the revised manuscript.\nThere are some ques6ons that the authors need to address:\n1, In the case of mtDSB, miR-71 promotes the recovery of muscle cell func6on by reducing\nDVE-1. Can directly knocking down DVE-1 restore muscle func6on? Addi6onally, could\nother UPRmt inhibitors also restore muscle func6on?\nWe have now performed experiments in which we knocked down dve-1 by targe=ng it with\nRNA interference (RNAi) and show that this helps to restore muscle func=on in animals with\nmuscle-specific mtDSBs (see Fig. 3k and addi=ons to the text in the revised manuscript). The\nlevel of muscle func=on improvement is similar to that obtained by miR-71 overexpression,\nsuppor=ng the conclusion that repression of dve-1 improves muscle func=on in the context\nof mtDSBs. We thank the reviewer for their sugges=on, which we believe further\nstrengthens the manuscript.\nAlthough we are not aware of any pharmacological inhibitors of the UPRmt, we have\npreviously demonstrated that loss-of-func=on muta=ons in a(s-1 (alleles cmh15 and\ntm4919), which also regulates the UPRmt, have no beneficial effect on muscle func=on in\nthe context of mtDSBs1. Importantly, restric=ng ATFS-1 to the mitochondria through muta=on of\nits nuclear localisa=on sequence does have a beneficial effect, which appears to be\nindependent of its UPRmt role1.\n1\n2, The regula6on of miR-71 expression by the genes ATFS-1, DAF-16, and HIF-1 cannot be\nunderstood through knockdown experiments alone; overexpression studies are also\nrequired. For example, in ATFS-1 loss-of-func6on C. elegans, does the overexpression of\nDAF-16 and HIF-1 affect miR-71 expression?\nWe thank the reviewer for their sugges=on. Importantly, the ac=vi=es of each of these\ntranscrip=on factors is not regulated by their levels of expression, but rather through\nregula=on of their transloca=on into the nucleus via changes in either mitochondrial import\nefficiency (ATFS-12), post-transla=onal modifica=on (DAF-163), or ac=ve degrada=on (HIF-14).\nTherefore, instead of overexpressing each gene, we used a cons=tu=vely ac=ve mutant for\na(s-1 (et15 allele), which results in exclusive nuclear localisa=on of ATFS-15 and a loss-of-\nfunc=on mutant of daf-2(e1370), which results in cons=tu=ve ac=va=on and nuclear\nlocalisa=on of DAF-166. In addi=on, we used an overexpression transgene of hif-1p::hif-1P621G\nthat carries a muta=on which renders HIF-1 undegradable by VHL proteins and is therefore\nac=ve and localises to the nucleus, even in the presence of oxygen4. We found that enhancing\nthe ac=vity or expression of each factor individually (even in wild type backgrounds where\neach of the other two factors remain present) was insufficient to increase mir-71 expression\nlevels and instead reduced miR-71 transcript levels in the case of a(s-1(et15) and daf-\n2(e1370) (Extended Data Fig. 3e).\nWe next crossed these mutants/transgenic strains together to create double and triple mutant\nstrains and measured mir-71 expression levels. Again, we did not observe increases in mir-71\nexpression in any mutant combina=on and instead observed a reduc=on in miR-71 transcripts\nin daf-2(e1370);hif-1p::hif-1P621G, a(s-1(et15);hif-1p::hif-1P621G, a(s-1(et15);daf-2(e1370), and\ntriple mutant/transgenic animals (Extended Data Fig. 3e).\nTogether, this new data supports the proposed mechanism that ac=va=on of ATFS-1, DAF-16,\nand HIF-1 individually is insufficient to induce mir-71 expression. It further suggests that\nalthough each factor is required (Fig. 4d-f), ac=va=on of all three factors together is also\ninsufficient and point to addi=onal requirements for mir-71 transcrip=onal regula=on. It also\nremains possible that daf-2(e1370) mutants ac=vate parallel pathways that could suppress\nmir-71. Therefore, these new results support the overall conclusions that mir-71 transcrip=on\nis regulated by the combined ac=vi=es of ATFS-1, DAF-16, and HIF-1 as well as possibly by\nother, yet to be iden=fied factors, during mitochondrial stress. The new results are included\nin the revised manuscript (Extended Data Fig. 3e) and reflected in the revised model (Fig 6i).\n3, A[er mitochondrial stress is generated in muscle cells, it is transmi\\ed to glial cells. Can\nthe stress generated in glial cells regulate muscle cells in reverse and reduce muscle cell\ndamage? In the case of mtDSB, if UPRmt is reduced only in glial cells without reducing it in\nmuscle cells, will this alleviate the func6onal damage of muscle cells, or will it increase the\ndamage to muscle cells?\nWe understand that Reviewer #1 wishes to know the impact of the cell-non-autonomous\nUPRmt response in the glial cells on muscle func=on in the context of mtDSBs. This is an\ninteres=ng ques=on and to address it, we generated new strains in which we overexpressed\nmiR-71 exclusively in the glia (via the glial-specific mir-228 promoter) to supress dve-1 ac=vity\nin only these cells. We crossed these transgenic animals with strains incurring muscle-specific\n2\nmtDSBs and found no change in muscle func=on (see Fig. 1 below). This result suggests that\nglial UPRmt ac=va=on does not influence muscle func=on affected by mtDSBs. In other words,\nglial stress responses do not appear to affect muscle func=on during stress.\nFigure 1. Quan=fica=on of body wave ini=a=on rate (using WormLab automated so]ware\nanalysis) of L4 animals placed in liquid. Bars represent mean ± SEM; n >3; one-way ANOVA\nwith Tukey’s post hoc test.\n4, The abbrevia6on mtDSB requires the full name.\nWe have now corrected this oversight in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors performed RNA sequencing in gene6cally engineered\nmuscle-specific mtDSBs C. elegans backgrounds and iden6fied 24 miRNAs that were\nsignificantly upregulated, including the miR-35 family of miRNAs and miR-71.\nOverexpression of miR-71 in muscular 6ssue is protec6ve against cellular dysfunc6on\ncaused by mtDSBs cell-autonomously. However, the dele6on of mir-71 did not exacerbate\nmuscle dysfunc6on during mtDSBs. The authors used TargetScan to predict the targets of\nmiR-71 and found that dve-1 can be targeted for degrada6on. Meanwhile, they found that\nATFS-1, DAF-16, and HIF-1 can bind the upstream promoter region of mir-71. Beyond the\ncell-autonomous effect, the authors also found that miR-71 can suppress cell-non-\nautonomous signaling of mtDNA damage from the muscle to glia cells through the\nregula6on of unc-31 to suppress ins-11 and nlp-52, allevia6ng the ac6va6on of UPRmt.\nThe authors concluded the cell-autonomous and cell-non-autonomous func6ons of miR-71\nin regula6ng maladap6ve UPRmt signaling. While the manuscript presents interes6ng\nfindings on the role of miR-71 in regula6ng UPRmt signaling, several cri6cal aspects\ndiminish its overall significance and depth of mechanis6c insight:\nWe thank Reviewer #2 for their insights and have addressed their concerns below.\n1, The study suggests that miR-71 targets DVE-1 and regulates UPRmt ac6va6on, but it\nlacks rigorous evidence to substan6ate this claim. The authors primarily rely on miR-71\noverexpression data without providing comprehensive analyses using miR-71 mutants or\ndve-1 3'UTR mutants under muscle-specific mtDSBs stress. This omission limits the depth\n3\nof mechanis6c understanding and raises ques6ons about the robustness of the proposed\npathway.\nWe thank the reviewer for their comment and would like to emphasise that mul=ple\northogonal lines of evidence provide evidence that miR-71 targets the 3’UTR of dve-1 during\nmitochondrial stress, which mi=gates the maladap=ve UPRmt. As outlined below, we have\nanalysed miR-71 dele=on mutants as well as dve-1 3’UTR mutants (in both transgene\nreporters and through CRISPR-Cas9 genome edi=ng of the endogenous gene).\n1) We generated transgenic reporter strains wherein the 3’UTR sequence of dve-1 was\nincorporated into a fluorescent transgene and integrated as a single-copy genome inser=on.\nWe also generated an iden=cal reporter strain (iden=cal genome inser=on site) with a control\ntbb-2 3’UTR which lacks any puta=ve miR-71 binding sites. Increasing the abundance of miR-\n71 by six-fold (Extended Data Fig.1l), which is similar to induc=on levels observed during\nmitochondrial stress (e.g. Fig. 1d, e, h), strongly suppressed the fluorescence signal of the dve-\n1 3’UTR reporter by half (P<0.0001; Fig. 2a and b) but had no effect on the signal of the tbb-2\n3’UTR reporter (P=0.2847; Extended Data Fig. 2b).\n2) To test whether the puta=ve miR-71 binding sites within the dve-1 3’UTR were required for\nmiR-71-mediated silencing of the dve-1 3’UTR reporter used above, we mutated these\nsequences (Fig. 2c) and reintroduced the transgene into the same genome inser=on site as\nthe original reporter. Scrambling the two puta=ve miR-71 binding sites completely abolished\nany suppression of the reporter by increased levels miR-71 (Fig. 2d and e). This result confirms\nthat miR-71 specifically targets and suppresses dve-1 via recogni=on of two binding sites in\nthe dve-1 3’UTR, valida=ng the precise predic=on of TargetScan and further strengthening the\nconclusion that dve-1 is a bona fide target of miR-71.\n3) Demonstra=ng the suppressive effect of miR-71 on endogenous dve-1 transcripts, we used\nqPCR to show that enhancing miR-71 levels prevented increases in dve-1 mRNA levels caused\nby mtDSB stress (Fig. 2f). Not only does this further support the ac=on of miR-71 on dve-1 but\nit also demonstrates that miR-71 ajenuates increases in dve-1 transcript abundance in the\ncontext of mtDSBs.\n4) We performed CRISPR-Cas9 genome editing of the endogenous dve-1 3’UTR to mutate\nthe experimentally validated miR-71 binding sites from point 2 above (also see Fig. 2c-e).\nThis abolished the ability of miR-71 to suppress muscle dysfunction (Fig. 3h) and actin\nfilament disorganisation (Fig. 3i and j) caused by mtDSBs. This experiment is what the\nreviewer has recommended, and not only does it further confirm that miR-71 targets dve-\n1 via recognition of its 3’UTR binding sites, but also demonstrates that miR-71-mediated\ntargeting of dve-1 is beneficial during muscle-specific mtDSBs.\n5) Downstream targets of DVE-1, such as the UPRmt eVector and mitochondrial\nchaperone hsp-6, are up-regulated in response to mtDSBs, but suppressed when miR-71\nlevels are increased (Fig. 3g). This supports a model of miR-71-mediated suppression of\nDVE-1, which ameliorates the up-regulation of its direct transcriptional targets during\nmitochondrial stress. Importantly, increasing miR-71 levels selectively in muscle cells\n4\nrecapitulates this result (Fig. 3g), indicating that miR-71 acts cell-autonomously to\nattenuate UPRmt activation during mtDSB stress.\n6) In animals with enhanced dve-1 levels, muscle dysfunc=on caused by mtDSBs is further\nexacerbated by the dele=on of mir-71 (Fig. 3f). This indicates that animals lacking miR-71 are\nsensi=ve to mtDSB, when the UPRmt is overac=vated through dve-1 overexpression.\n7) Finally, deletion of mir-71 increased the fluorescence signal of the UPRmt reporter (dve-\n1p::DVE-1::GFP), which was further increased in backgrounds with muscle-specific\nmtDSBs (Fig. 5c and d). This suggests that miR-71 suppresses UPRmt signalling.\nWe believe that the experimental evidence outlined above is compelling and strongly supports\nthe model that miR-71 targets dve-1, thereby regula=ng UPRmt ac=va=on. We further\nexperimentally validate the specific sequences in the dve-1 3’UTR that are required for this\nregula=on and show the phenotypic effects of this under the context of mtDSBs. This is\nsupported by Reviewer #3’s comments to the authors that “the results linking miR71 to the\nregula6on of DVE1 in figure 2 is convincing and involves proper controls, same applies to all\nfigures notably the control in figure 5 for regula6on of UPRmt rather than reporter directly,\nthat miR71 does not spread itself across 6ssues. Therefore, the study present compelling\nresults and is very well designed.”.\nWhile miR-71 is overexpressed in many of these experiments, so too is the fluorescent\nreporter used to assess miRNA suppression, which is the standard approach for defining bona\nfide miRNA targets in vivo. Importantly, the six-fold increase in abundance of miR-71 in the\noverexpression strain (Extended data Fig.1l) is within the range of the increase observed for\nthe endogenous miR-71 upon exposure to various mitochondrial stressors and is therefore a\nphysiologically relevant level of expression that matches stress-induced scenarios.\nSpecifically, muscle-specific mtDSBs induce miR-71 by ~4-fold (Fig. 1d), and animals\nharbouring the uaDf5/+ mtDNA deletion (60% heteroplasmy) or are treated with NaN\n3\nincrease miR-71 levels by ~9-fold (Fig. 1e) and ~6-fold (Fig. 1h), respectively.\nAs pointed out in comment 2 by the reviewer (below), loss of mir-71 does not exacerbate\nmuscle dysfunc=on caused by mtDSB. In the manuscript, we hypothesised that this may be\ndue to miR-71 func=onal redundancy, which is revealed if we enhance dve-1 expression levels\n(point 6 above). As such, we capitalised on the effects of increases in miR-71 levels rather than\nmir-71 dele=on, as overexpression recapitulates the induc=on of miR-71 during mitochondrial\nstress.\n2, Addi6onally, the finding that muta6on in mir-71 does not exacerbate muscle dysfunc6on\nduring mtDSBs is contradictory to the proposed protec6ve role of miR-71. Further\nexperiments to examine muscle dysfunc6on in mir-71 mutants without mitochondrial stress\nwould clarify the baseline effects of miR-71 and provide a more complete mechanis6c\ninsight.\nWe thank the reviewer for their comment, which we have addressed above. In addition,\nmiRNA redundancy is a significant factor in genetic studies, as the absence of a single\nmiRNA may not lead to an observable phenotype due to compensatory mechanisms by\n5\nother miRNAs. Therefore, the implica=on that miR-71 is func=onally redundant, in principle,\ndoes not contradict our model that miR-71 is protec=ve against the effect of mtDSBs. It does\nhowever, support the widely reported func=onal redundancy of miRNAs, whereby mul=ple\nmiRNAs can target individual transcripts and individual miRNAs can target mul=ple, dis=nct\ntranscripts.\n3, The role of miR-71 in UPRmt regula6on needs to be contextualized within the broader\nframework of known UPRmt pathways. The mainstream UPRmt regula6on primarily\ninvolves ATFS-1, DVE-1, and UBL-5, which orchestrate a well-characterized response to\nmitochondrial stress. The authors should compare the impact of miR-71-mediated\nregula6on to these established pathways, demonstra6ng whether miR-71 plays a\nredundant, synergis6c, or dis6nct role.\nWe thank the reviewer for their sugges=on. Considering that the manuscript establishes that\nmiR-71 directly targets dve-1, we have focussed our response on possible interac=ons\nbetween miR-71 and the other two components of the UPRmt, a(s-1, and ubl-5.\nUnlike dve-1, both a(s-1 and ubl-5 do not have puta=ve binding sequences for miR-71 and\nare therefore unlikely to be direct targets of miR-71. To confirm that this is true, we performed\nqPCR to determine whether the mRNA levels of each gene are reduced in animals with\nincreased miR-71 levels. Unlike dve-1 transcripts (Fig. 2f), we found increasing miR-71\nabundance did not decrease the levels of a(s-1 or ubl-5 transcripts (Extended Data Fig. 2c and\nd), sugges=ng that miR-71 does not target these genes. Therefore, we conclude that miR-71\nplays a dis=nct role by targe=ng only the DVE-1 branch of the UPRmt. These new results are\nincluded in the manuscript.\n4, The authors' finding that ATFS-1, DAF-16, and HIF-1 regulate miR-71 expression under\nmtDSBs stress is intriguing, but the func6onal relevance of this regula6on remains unclear.\nHow do these factors integrate with the canonical UPRmt pathway? Is miR-71 expression a\ndownstream consequence or a parallel regulatory mechanism? Addressing these ques6ons\nis crucial to establish the significance of miR-71 in the broader context of UPRmt regula6on.\nWe thank the reviewer for their comment and important ques=ons.\nATFS-1 is a key UPRmt regulator that can translocate to the nucleus and induce the\ntranscrip=on of ~300-400 genes that contribute to mitochondrial homeostasis and immunity7.\nHere, we find that it is also required for mir-71 induc=on during severe mitochondrial stress\n(Fig. 4d and Extended Data Fig. 3b). Although DAF-16 and HIF-1 are not integrated within the\ncanonical UPRmt pathway, we find that they are also required for the full transcrip=onal up-\nregula=on of mir-71 during mitochondrial stress (Fig. 4e-g). Because we have demonstrated\nthat miR-71 targets dve-1 (Fig. 2, 3h-j), both DAF-16 and HIF-1 appear to play a nega=ve\nfeedback role over the DVE-1 branch of the UPRmt. We propose that under severe and chronic\nmitochondrial stress condi=ons, all three transcrip=on factors are simultaneously ac=vated\nand localise to the nucleus (Fig. 4b and c), bind to the promoter of mir-71 (Fig. 4h and i), and\nup-regulate mir-71 expression, which subsequently exerts nega=ve feedback on DVE-1, thus\nlimi=ng maladap=ve UPRmt ac=va=on. In other words, our model suggests that miR-71 forms\na parallel regulatory mechanism that counteracts the DVE-1-mediated UPRmt pathway.\nlocalise to nuclei in response to mitochondrial stress8-10. We did not observe any gene=c\ninterac=on (Fig. 4d-g) or detect any physical associa=on between ATFS-1, DAF-16, and HIF-1\n(Extended Data Fig. 3f and g) that would suggest that they integrate on the same pathway and\ninstead have proposed a mechanism in which each factor is required to regulate mir-71\nexpression during mitochondrial stress in an independent manner.\nFurthermore, a cons=tu=vely ac=ve a(s-1(et15) mutant that localises to the nucleus and\nac=vates the UPRmt and various UPRmt reporters (e.g. hsp-6p::GFP) under normal condi=ons5\nis not sufficient to drive mir-71 upregula=on (Extended Data Fig. 3c). This suggests that mir-\n71 itself does not fall under the canonical UPRmt pathway mediated by ATFS-1. It also suggests\nthat ATFS-1 is not downstream of DAF-16 or HIF-1 signalling in this context. Instead, our results\nsuggest that the integra=on of several stress pathways ac=vated during mitochondrial stress\nis required for mir-71 regula=on. Moreover, these results suggest that mir-71 induc=on is not\nsimply a downstream consequence of UPRmt ac=va=on, but a nega=ve feedback mechanism\nthat is ac=vated in parallel during mitochondrial stress.\n5, The manuscript proposes that miR-71 exerts protec6ve effects without binding to its\nendogenous 3'UTR of dve-1p::DVE-1::GFP (The reporter was constructed in a vector\ncontaining unc-54 3’UTR). This paradoxical observa6on needs to be resolved through\naddi6onal experiments. The authors should explore alterna6ve mechanisms by which miR-\n71 might influence DVE-1 or other components of the UPRmt pathway.\nWe thank the reviewer for their comment and apologise for any confusion regarding the miR-\n71 mechanism.\nWe have proposed two dis=nct mechanisms (illustrated in Fig. 6i) by which miR-71 acts to\ndampen the UPRmt response during mitochondrial stress; 1) a cell-autonomous mechanism\nwhereby miR-71 directly binds to the 3’UTR of dve-1 transcripts, reducing its abundance, and\n2) a cell-non-autonomous mechanism in which miR-71 nega=vely regulates signalling pep=des\nthat are required to ac=vate the UPRmt in distal glia cells.\nThe dve-1p::DVE-1::GFP::unc-54 3’UTR transgene is strictly a reporter of UPRmt ac=va=on and\nnot a reporter of miR-71 ac=vity. We used this reporter to demonstrate that miR-71 reduces\nthe ac=va=on of the cell-non-autonomous UPRmt in distal glia cells (e.g. Fig. 5). In other words,\nmiR-71 does not directly regulate the dve-1p::DVE-1::GFP::unc-54 3’UTR transgene, but\ninstead regulates factors required for UPRmt ac=va=on in glial cells. The fact that the reporter\ntransgene does not have a dve-1 3’UTR has allowed us to ensure that this is the case. Reviewer\n#3 has noted that this is a good control in their comments to the authors.\n6, The proposed cell-non-autonomous effects of miR-71 in suppressing signaling from\nmuscle to glial cells are intriguing but underexplored. The roles of ins-11 and nlp-52 in this\nprocess should be validated through RNAi, 6ssue-specific knockout or knockdown, and\n6ssue-specific rescue assays to understand their func6on in muscle and UPRmt signaling\ncomprehensively.\nWe did already perform RNAi experiments for ins-11 and nlp-52 in the submijed manuscript.\nTo build on and strengthen these results, we analysed the induc=on of glial DVE-1::GFP in\ndele=on mutants of both factors but only observed a significant decrease in the glial signal for\nnlp-52(tm12973) and not for ins-11(tm1053) mutants. As such, we decided to focus on NLP-\n52. To understand the =ssue requirements of nlp-52, we performed several new experiments.\nFirst, we performed =ssue-specific rescue by expressing wild type nlp-52 under a muscle\nspecific promoter (myo-3p) in the nlp-52(tm12973) dele=on background. Muscle-specific\nexpression of nlp-52 restored glial DVE-1::GFP signals to control levels (Fig. 6e and f),\nsugges=ng that nlp-52 is sufficient in the BWM to signal muscle-specific mtDSB to the glia.\nSecond, we performed =ssue-specific knockdown of nlp-52 to determine whether it is\nrequired in the muscle to communicate muscle-specific mtDSBs to the glia. We made strains\nwhere nlp-52 was suppressed by RNAi only in the BWM through the expression of nlp-52\nsense and an=sense sequences under the myo-3 promoter. Muscle-specific RNAi of nlp-52\nsuppressed DVE-1::GFP ac=va=on in the glia (Fig. 6g and h), sugges=ng that NLP-52 is required\nin muscle cells to communicate mitochondrial stress localised to the muscle cells to the glia.\nTogether these new results further strengthen and support our conclusions that miR-71\nregulates muscle-to-glia cell-non-autonomous UPRmt ac=va=on via the regula=on of nlp-52 in\nthe muscle cells. In light of the new data and lack of support for a role for ins-11, we have\ndecided to only include nlp-52 in the manuscript.\nIn summary, the study in its current form lacks the significance and depth required to\nadvance our understanding of UPRmt regula6on. Addressing these cri6cal points would\nenhance the mechanis6c insight and posi6on of miR-71 within the established landscape of\nUPRmt regulatory pathways.\nWe thank the Reviewer for all of their important comments and sugges=ons. We believe we\nhave now addressed these and as a result enhanced the manuscript by improving the\nmechanis=c understanding of a new miRNA-mediated pathway that regulates the UPRmt.\nReviewer #3 (Remarks to the Author):\nThe study by Kirmes et al. describes the iden6fica6on of miR-71 in muscles in response to\nsevere mitochondrial stress and ac6va6on of DAF-16, HIF-1 and ATFS-1 and leads to\ndegrada6on of DVE-1 to alleviate overac6va6on of the UPRmt. Addi6onally, to the impact\nof miR-71 in the muscle, they propose that miR-71 leads to reduced secre6on of neuro and\ninsulin-like pep6des which act to suppress muscle to glia axis of mitochondrial stress.\nTherefore, they suggest that miR-71 is a novel target of the UPRmt, that act to prevent its\noverac6va6on and leads to local and systemic beneficial effects.\nOverall, the introduc6on is well wri\\en and provide appropriate background and relevant\nliterature. The inclusion of a nuclease dead control in the original screen is a strong control,\ndata suppor6ng the impact of miR71 following mitochondrial damage in muscle is strong,\n8\nthe results linking miR71 to the regula6on of DVE1 in figure 2 is convincing and involves\nproper controls, same applies to all figures notably the control in figure 5 for regula6on of\nUPRmt rather than reporter directly, that miR71 does not spread itself across 6ssues.\nTherefore, the study present compelling results and is very well designed.\nWhile the non cell-autonomous effect of mitochondrial stress in one 6ssue affec6ng another\nis already known, the novelty here is to add miR71 and the fact that the strength of signal\nis important.\nWe thank Reviewer #3 for their comments and enthusiasm for the manuscript.\nCritiques:\n1, miR-35 is activated significantly more than miR-71, miR-246 seems activated at similar\nlevel to miR-71, several stressors also activates mir35 but polG represses it , which is\ninterested. Is there a specificity within the miR35 family? the focus on miR-71 or lack of\nanalysis of miR-35 and miR-246 needs to be justified in a stronger manner, especially that\nthe group have published in miR-71 previously and therefore the choice of focusing on this\nparticular miR come across as purely biased. The same applies to the focus on the peptides\nalready known to regulate non-cell autonomous communication between tissues following\nmitochondrial stress in the screen in figure 6.\nWe thank the reviewer for their comments and ques=ons.\nWe do not believe there is specificity within the miR-35 family, all members of the miR-35\nfamily in Caenorhabditis elegans are transcribed as a single polycistronic primary\ntranscript encoded within an approximately 800-nucleotide cluster, indicating they are\nregulated by the same promoter. This transcript is processed into single miRNAS (mir-35-\n41) which all share the same seed region11. However, it is an interes=ng ques=on that may\nwarrant future study.\nAs noted on page 6, only miR-71 was consistently induced by mul=ple forms of mtDNA stress,\nwhich is why we focused on this par=cular miRNA. Specifically, miR-71 was induced by the\npresence of the mtDNA muta=on mpt1 (Fig. 1f) as well as by a muta=on in the proof-reading\ndomain of polg-1 (Fig. 1g). miR-35 was not induced under either of these condi=ons (Extended\nData Fig. 1c and d). Interes=ngly, miR-71 was also much stronger induced than miR-35 by\nanother mtDNA muta=on, uaDf5 (~9-fold versus ~5-fold; Fig. 1e and Extended Data Fig. 1b).\nBecause the response to mtDNA stress was consistent for miR-71 and not miR-35, we\npostulated that miR-71 was the most biological significant miRNA in mtDNA damage\nprocesses and therefore chose to focus on its mechanism of ac=on.\nIn the case of miR-246, we did not observe a protec=ve effect against mtDSBs when we\noverexpressed it and therefore did not focus on its ac=on any further.\nWe would like to clarify that we have not worked on miR-71 prior to this study. In an unbiased\nsequencing screen for Argonaute-loaded miRNAs, miR-71 was detected among various other\nmiRNAs to be loaded onto Argonaute proteins isolated from muscle cells (Brosnan et al.,\n2021)12, but we did not study its func=on.\nknowledge, NLP-52 has not previously been reported to play a role in cell-non-autonomous\nmitochondrial stress signalling. We did note in the text that other, previously characterised\npep=des involved cell-non-autonomous mitochondrial stress signalling (specifically INS-27,\nINS-35, FLP-1 and FLP-2) were revealed by our RNAseq experiment being poten=ally regulated\nby miR-71 during mitochondrial stress. However, INS-27, INS-35, FLP-1 or FLP-2 were not\nrequired for muscle-to-glia mitochondrial stress signalling (Extended Data Table 3).\n2, Overexpression of miR-71 increases daf-2 significantly the receptor of insulin in muscle\nbut this is not discussed, how it relates to the finding of insulin-like peptide in figure 6\n(Extended data Fig.2).\nWe thank the reviewer for raising this interes=ng point. We do not have an explana=on as to\nwhy overexpression of miR-71 increases the fluorescence signal of the daf-2 3’UTR reporter.\nAdmijedly, this effect is the opposite of an=cipated miRNA ac=vi=es and may therefore be\ncaused indirectly. It is true, that in the light of our findings around the insulin/IGF-1 signalling\n(IIS) pathway, this observa=on might gain in importance; our data shows that mir-71 is\ntranscrip=onally regulated by the insulin/IGF-1 signalling (IIS) factor DAF-16 (see Fig. 4), which\nis downstream of DAF-2. Moreover, mitochondrial stress and miR-71 impacts the levels of\nseveral insulin signalling pep=des (Extended Data Fig. 5a).\n3, Choice of sodium azide for RNAseq is unfortunate and detract from main finding using a\nmtDNA specific DNA damage. While some validation is provided in fig. 6d and e, a more\ncomparison of the gene expression in panel b and c following muscle and mitochondrial\nspecific DNA damage should be provided.\nWe thank the reviewer for their sugges=on. It is important to note that RNAseq was\nperformed on whole animals, and we hypothesised that systemic mitochondrial stress\ninduced by sodium azide treatment would enable us to detect important downstream targets\nof miR-71 more easily than =ssue-specific mtDSBs, which may be masked by the absence of\nmitochondrial stress in all other =ssues. As the Reviewer suggests, the experiments performed\nin Figs. 6c and d validate our RNAseq data in the context of muscle-specific mtDSBs and glia\ncell-non-autonomous signalling. However, to determine if it is possible to detect changes in\nins-11 and nlp-52 transcripts levels on whole animals experiencing muscle-specific mtDSBs,\nwe performed qPCR. As the results below show we cannot detect an increase in ins-11 or nlp-\n52 in mir-71 dele=on mutants when the mitochondrial stress is restricted to body wall muscle.\n1.5\n1.0\n0.5\n0.0\nmir-71(\nBs;\nS\nD\nmt\np::\no-3\nmy\negnahc\ndlof\n11-sni\nni\nP = 0.8978\n1.5\n1.0\n0.5\n0.0\nmyo-3p::mtDSB - +\nmir-71(n4115) - +\nWT n4115)\nmir-71(\nBs;\nS\nD\nmt\np::\no-3\nmy\negnahc\ndlof\n25-pln\nni\nP = 0.5129\nmyo-3p::mtDSB - +\nmir-71(n4115) - +\nFigure 2. Quan=ta=ve (q)PCR of (a) ins-11 and (b) nlp-52 levels in animals that have mtDSBs\ninduced in muscle cells in a mir-71 dele=on background. Columns represent mean ± SEM; n ≥\n3; unpaired, two-tailed t-test.\nReferences\nrepair over transcription. Nature Cell Biology 25, 1111-1120 (2023).\nhttps://doi.org/10.1038/s41556-023-01192-y\nMitochondrial and nuclear accumulation of the transcription factor ATFS-1\npromotes OXPHOS recovery during the UPR(mt). 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The additional results strengthened the author’s conclusion.", "reviewer_3": "The authors have addressed previous concerns and provided appropriate clarifications."}, "author_response": ""}]

Decision letter