Gain of function in linc-133 compensates for daf-18/PTEN loss and rescues survival

DAF-18, homology of PTEN, possess tumor suppressor activity. Loss of daf-18 causes cell growth in L1 diapause C. elegans is well studied; however, the reason why survival is dramatically shortened is not well elaborated. We found that linc-133 gain of function can fully restore the shortened survival caused by daf-18 loss. When lipid phosphatase activity of DAF-18 is defective, the linc-133 gain of function interacts with 14-3-3 proteins to obstruct DAF-16 translocation from the nucleus to the cytoplasm. However, the dysfunction of protein phosphatase activity of DAF-18 caused high levels of aggregated proteins. The linc-133 gain of function can induce HSP chaperones to select and process the aggregated proteins for degradation through ubiquitination. Our work demonstrates that protein homeostasis controlled by the protein phosphatase activity of DAF-18 is the main factor affecting survival and identifies a new function of the linc-133 gene, which can compensate for the loss of daf-18.

PTEN has been identified as a tumor suppressor in humans 6 .The C. elegans PTEN homolog DAF-18 also possesses tumor suppressor-like properties.Wild-type N2 L1arrested worms can live for approximately 20 days in sterilized liquid medium with no cell division.Loss of daf-18 can break this diapause and shorten survival drastically 2,3,7,8 .A variety of genes were reported to be related to L1 survival and the postembryonic cell developmental events that occur in daf-18 mutants 2,4,5,9,10 ; however, we found that none of them could fully rescue the survival of daf-18 worms during L1 arrest.DAF-18/PTEN is a dual-specificity phosphatase that has lipid and protein phosphatase activities [11][12][13] .Its better-known lipid phosphatase activity, which controls the insulin/insulin-like pathway (IIS), was reported to play a pivotal role in regulating cell development during L1 arrest 2,4,8,14 .However, genetic analyses show that the effect of daf-18 loss on L1 arrest survival may not only occur through the regulation of PI3K-DAF-16/FOXO signaling in IIS 2,5,7,8,10 .How downstream regulatory events control L1 arrest survival when daf-18 is lost still needs to be explored.
In this work, we demonstrated that both the lipid and protein phosphatase activities of DAF-18 are needed to fully rescue the shortened survival caused by daf-18 loss in L1arrested worms.The high internal aggregated protein level caused by the dysfunction of protein phosphatase activity of DAF-18 may be the main reason why the worms have a shorter survival.By using a traditional forward genetic method 15 , we identified that a gain-of-function mutation in linc-133 can extend the survival of worms during L1 arrest and fully recover the survival of daf-18 worms to that of wildtype worms.This new linc-133 mutation is capable of compensating for the consequences of the functional defects in DAF-18 lipid and protein phosphatase activities.

Both lipid and protein phosphatase activities of DAF-18/PTEN are needed to maintain survival
DAF-18/PTEN is well known for its lipid phosphatase activity, which plays an opposite role to AGE-1/PI3K in dephosphorylating phosphatidylinositol 3,4,5trisphosphate (PIP3) to PIP2 [16][17][18] to negatively regulate the function of DAF-16/FOXO in IIS 19 .We found that disrupting age-1 can partially rescue daf-18 survival during L1 arrest (Fig. 1a) and that the loss of daf-16 also reduced the survival of L1-arrested worms relative to that of N2 worms (Fig. 1b); however, the survival of the daf-16; daf-18 double mutant was even shorter than that of the daf-16 single mutants (Fig. 1b), suggesting that daf-18 also acts through mechanisms other than PI3K-AKT-DAF-16/FOXO to regulate L1 arrest survival.To confirm this speculation, we tested whether activated DAF-16 can fully rescue the shortened survival of daf-18.
We found that DAF-16 activation, through inhibiting AKT, only partially rescued the survival of daf-18 worms (Fig. 1c).Moreover, blocking IIS signaling by daf-2 can rescue the short sruvival of ins-3 (oe), an agonist gene of IIS, worms, but failed to rescue the short survival of daf-18 worms (Fig. 1d).These results suggested that the DAF-16/FOXO is not the main target regulated by DAF-18 to control L1 arrest survival.DAF-18/PTEN also has protein phosphatase activity, which is independent of the PI3K-DAF-16/FOXO pathway 12,[20][21][22] .To identify which phosphatase activity of DAF-18 was required for supporting L1 arrest survival, three variants of DAF-18 that correspond to known human PTEN variants were constructed and tested for survival rescuing activity 13 (Fig. 1e).The results showed that the protein phosphatase-defective (DAF-18 with D137A) and lipid phosphatase-defective (DAF-18 with G174E) constructs both partially rescued the short survival of daf-18 worms (Fig. 1f-g).DAF-18 (C169S) abolishes both lipid and protein phosphatase activity and is unable to rescue daf-18 survival during L1 arrest (Fig. 1h).We also generated a daf-18(D137A) mutation strain by using CRISPR/Cas9.Interestingly, daf-18(D137A) worms lived only approximately 5 days during L1 arrest (Fig. 1i).These results confirm that survival of L1 arrest requires both the lipid and protein phosphatase activities of DAF-18 and suggest that DAF-18 protein phosphatase activity may play a pivotal role in supporting survival during L1 arrest.

linc-133 gain-of-function mutation rescues the shortened survival caused by daf-18 loss
To identify the gene mutations that can fully rescue the shortened survival of daf-18 L1-arrested worms, a forward genetic screen was performed by using genome-wide mutagenesis with ethyl methanesulfonate (EMS) 15,23 .Thousands of F3 EMSmutagenized animals were screened, and two F3 progeny of EMS-treated daf-18 worms were found to have normal L1 arrest survival (Fig. 2a).A single-nucleotide polymorphism in linc-133, which we termed aqz1, was identified in these mutants by using the Sibling Subtraction Method 24 .linc-133(aqz1) fully recovered the survival of daf-18 L1-arrested worms to that of the N2 worms (Fig. 2b).linc-133 is predicted to be a non-coding RNA with no obvious role described so far.To further address the function of this new allele of linc-133, we made three RNAi versions to silence the expression of wild-type linc-133 (Fig. 2c).We found that all three RNAi plasmids failed to rescue the survival of L1 arrest daf-18 worms (Fig. 2d).Then, we used CRISPR/Cas9 to delete linc-133 (termed aqz2) and to make the aqz1 mutation (termed aqz3) (Fig. 2e).We found that the deletion version of linc-133(aqz2) failed to rescue the survival of L1 arrest daf-18 worms (Fig. 2f).We also overexpressed wildtype linc-133 in daf-18 worms, but linc-133(oe) had no obvious effect on the survival of L1-arrested worms (Fig. 2g).However, linc-133(aqz3), the CRISPR/Cas9 version of linc-133(aqz1), fully rescued the survival of L1-arrested daf-18(ok480) worms (Fig. 2h).These results suggest that the aqz1/3 alleles of linc-133 may be gain-offunction mutations.
We next wanted to know the location of its potential function inside the cells.Fortyeight short DNA nucleotide primers were used for RNA in situ hybridization to localize the mutated linc-133 25,26 .linc-133 was reported to be enriched in AVK cells, and our results confirmed that AVKs have high levels of mutated linc-133 (Fig. 2i) and that overexpression of linc-133 with aqz3 mutation in AVK cells can significantly rescue the survival of L1 arrest daf-18 worms (Fig. 2j), suggesting that linc-133 gain of function may play a cell nonautonomous role.However, the overall signal (outside AVK cells) appears much higher in linc-133 (aqz3) mutants compared to linc-133 (aqz2) null mutants suggesting that linc-133 may also be expressed outside AVK cells.
In total, these results suggest that both lipid and protein phosphatase activities can be compensated by linc-133 gain-of -function mutation and that linc-133 is a common effector for both DAF-18 activities.
These results suggest that the linc-133 gain-of-function mutant physically interacts with 14-3-3 to block DAF-16 from exiting the nucleus in daf-18 worms.
Most of these genes are heat shock genes which regulated by the transcription factor HSF-1 30 , so next we test whether the function of linc-133(aqz3) is related to HSF-1.

linc-133 gain of function enhances protein degradation
According to the RNA seq results, most of the genes, enriched in the top four pathways (Fig. 3c), actually encode the ubiquitous molecular chaperones HSP70, HSP90 and nucleotide exchange factor (NEF), which participate broadly in preventing protein aggregation and promoting the refolding of misfolded denatured proteins, solubilizing aggregated proteins and cooperate with cellular degradation machineries to clear protein aggregates 31 .When aggregated proteins are produced, these chaperons promote the degradation of aberrant proteins by the ubiquitin proteasome system to help maintain protein homeostasis and ensure survival (Fig. 6a) 31,32 .We speculated that the pressure of daf-18 loss may cause the cells to produce large amounts of aggregated proteins, and linc-133(aqz3) induces HSP chaperones to degrade these protein aggregates.To test this speculation, we used the protein aggregation marker Pnmy-2NMY-2::GFP (aggregation-prone protein) to measure the level of protein aggregation 33 .Our results showed that loss of daf-18 or loss of the protein phosphatase activity of DAF-18 caused the worms to produce high level aggregated proteins during L1 arrest, and linc-133(aqz3) significantly decreased the levels of protein aggregations (Fig. 6b).The results also showed that a mutant daf-18 rescue plasmid lacking the protein phosphatase activity failed to reduce the level of aggregated proteins (Fig. 6c).These results suggest that the protein phosphatase activity of DAF-18 controls protein aggregation.As our results showed that linc-133(aqz3) can rescue the dysfunction of protein phosphatase activity of DAF-18 through regulating HSF-1, as a transcription factor, which may play a key role in controlling unfolded proteins degradation.Indeed, knocking down hsf-1 significantly increased the level of aggregated proteins in daf-18(ok480);linc-133(aqz3) (Fig. 6d) and daf-18(D137A);linc-133(aqz3) (Fig. 6e) L1-arrested worms.Next, we also knocked down hsp-1, the target gene of HSF-1, in these worms, we found that the levels of aggregated proteins in daf-18(ok480);linc-133(aqz3) (Fig. 6f) and daf-18(D137A);linc-133(aqz 3) (Fig. 6g) were also significantly increased.
The aggregated proteins in cytoplasm can be selected, ubiquitinated and degraded by the ubiquitin proteasome system [34][35][36][37] (Fig. 6a).To test whether the ability of protein ubiquitination is affecting the survival of linc-133(aqz3) worms, we knocked down all the genes related to ubiquitination in ERAD, and found that knocking down the P97 coding genes cdc-48.1 or cdc-48.2shortened the survival of daf-18(ok480);linc-133(aqz3) (Fig. 7a) and daf-18(D137A);linc-133(aqz3) (Fig. 7b) worms during L1 arrest.These results show that linc-133(aqz3) works through protein degradation to support survival during L1 arrest.To further confirm this model, we tested whether the protein ubiquitination levels are changed in daf-18 and linc-133 worms.Recent study has shown that short-lived worms exhibit higher protein aggregation and lower ubiquitin protein levels.In contrast, long-lived worms have elevated levels of ubiquitinated proteins, which are degraded by the proteasome to reduce the accumulation of aggregated and unfolded proteins 38,39 .Our results showed that daf-18(ok480) and daf-18(D137A) (Fig. 7c) mutants had lower levels of K48-linked ubiquitinated proteins, and linc-133(aqz3) induced high levels of K48-linked ubiquitinated proteins (Fig. 7d).When we knocked down the P97 ubiquitin-selective chaperone coding gene cdc-48.2,the K48-linked ubiquitinated protein levels were decreased (Fig. 7e).Together, these results suggest that the loss of daf-18 leads to higher levels of aggregated proteins and that the protein phosphatase activity of DAF-18 may play a pivotal role in this process.linc-133(aqz3) can induce HSF-1-and P97related protein ubiquitination degradation to maintain protein homeostasis.

Discussion
Loss of daf-18 leads to two prominent phenotypes in L1 arrested worms: cell proliferation and survival decrease.Previous research has extensively studied cell divisions, including P, V, Q, and germ cell divisions, in daf-18 worms during L1 arrest, and many genes and signaling pathways such as TOR, TGF-beta, IIS, and MAPK have been implicated 4,5,8,40 .However, none of these genes were capable of fully rescuing the short survival of daf-18 L1 arrested worms (Fig. S1), despite some studies showing that IIS, TGF-beta have some functions on the survival of L1 arrested worms 2,3,5,7,9,41,42 .We used a traditional forward genetic screening method to identify a new gene mutation that can compensate for the loss of daf-18/PTEN (Fig. 8).In addition to the lipid phosphorylation of DAF-18 and related PI3K signaling, the protein phosphatase activity of DAF-18 also plays important roles in controlling survival.These findings are consistent with recent reports that protein phosphatase activity of DAF-18/PTEN has roles in controlling oncogenic signaling independently of PI3K-AKT 21,43,44 .We demonstrated that the protein phosphatase activity of DAF-18 is critical for the regulation of aggregated protein and protein homeostasis.linc-133 aqz3 can compensate for this by regulating the gene expression of the ERAD pathway, specifically by evoking the HSF-1 pathway to regulate heat shock chaperones that can select and identify aggregated proteins in the cytoplasm.These aggregated proteins can then be ubiquitinated and degraded, thereby maintaining protein homeostasis and supporting the survival of daf-18 mutants during L1 arrest.This is consistent with previous reports that short-lived worms, such as daf-18 mutants, have higher levels of unfolded and aggregated proteins and lower levels of ubiquitinated proteins, indicating a weakened ubiquitin degradation system 38 .daf-18 worms are more susceptible to heat stress and exhibit reduced mobility, likely due to the accumulation of aggregated proteins 45 .Our results also showed that the linc-133(aqz3) increased resistance to heat stress and improved mobility of daf-18 worms (Fig. S2).This further supports the conclusion of the study.We speculate that the function of HSF-1 can be independently evoked by linc-133(aqz3), despite the fact that the IIS pathway can also regulate HSF-1.However, we cannot exclude the possibility of inter-regulation between HSF-1 and the IIS pathway in linc-133(aqz3) worms based on our results.It is possible that linc-133(aqz3) regulates HSF-1 independently of IIS, as previous studies have reported that HSF-1 is regulated by IIS 46 , but still has functions independent of DAF-16 [47][48][49][50] .
The targets and mechanisms of linc-133 gain-of-function mutations that we identified in the L1 arrest stage may also be applicable to other life stages in worms.
Considering that the human ortholog of daf-18 is PTEN, a critical tumor suppressor gene, our findings may have implications for the study of PTEN-related diseases.It is worth exploring whether there are additional potential orthologs of linc-133 or alternative genes that have this function in humans.

C. elegans RNAi
RNAi feeding was conducted according to standard protocols 51 .The RNAi bacteria were prepared and seeded onto NGM plates containing 1 mM isopropyl-B-Dthiogalactopyranoside (IPTG).Eggs were grown on NGM plates until the L4 stage and then transferred to RNAi plates.C. elegans strains were well fed before phenotypic analysis.RNAi bacteria containing the empty vector L4440 were used as controls.The RNAi constructs were obtained from the Horizon RNAi library (Horizon Discovery Ltd).To silence other designated target genes, the PCR products were cloned into the bacterial expression vector L4440 between opposing phage T7 polymerase promoter sites.The feeding vector was then transformed into E. coli HT115.The efficiency of RNAi was confirmed by using real time PCR.The primers used to construct these RNAi plasmids are presented in Table S2.All worms used to perform RNAi experiments contained the neuronal SID-1 receptor unless otherwise stated.

Real-time PCR
L1-arrested worms sufficient to yield 50-100 µL of a tightly packed worm pellet were collected, and 1-1.5 µg/µL total RNA was extracted.Total RNA was isolated using RNAiso Plus (Takara) according to the manufacturer's protocol, and cDNA was generated using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme).Real-time PCRs were run using Cham Q Universal SYBR qPCR Master Mix (Vazyme) on a Roche LightCycler 480Ⅱ.The relative expression levels of the genes were assessed using the 2 -ΔΔCt method and normalized to the expression of the act-1 gene.P values were calculated using a two-tailed t test 3,52 .The primers for real-time PCR are listed in Table S2.

L1 arrest survival assay
Mixed-stage worms were collected and blenched in Eppendorf tubes (volume: 2 mL) to collect embryos.The embryos were resuspended and hatched in sterile M9 and incubated at 20 °C with low-speed rocking to enter L1 arrest.Two days later, a 20-50 µL aliquot with more than 50 L1-arrested worms was plated onto a 6 cm unseeded NGM plate.The numbers of live and dead worms were counted daily until all the larvae died.The percentages of live worms were calculated every day.The survival rates between strains were compared as described previously 3,53 , and the mean survival rate was calculated by the Kaplan-Meier method.The P values of the difference in overall survival rate were determined using the log-rank test.

RNA sequencing
Wild-type N2 and linc-133(aqz 3) L1-arrested worms were collected.RNA-seq was completed by Novogene Corporation (http://www.novogene.com).RNA was prepared using Trizol reagent.Total RNA quality was measured using a NanoDrop ND-1000 (Agilent Technologies).Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 µg of total RNA for the construction.RNA libraries were prepared for sequencing using standard Illumina protocols.Paired-end sequencing was performed on an Illumina NovaSeq 6000.Transcriptome alignment and quantification were performed using Hisat2 software.C. elegans genome version WBcel235 was used as the reference.KEGG pathway enrichment was performed using clusterProfiler software and the most up-to-date version of the KEGG database.

Fluorescence in situ hybridization
For smFISH, we used custom Stellaris probes specific for linc-133 lncRNA (48 probes) labeled with Quasar570 (excitation 548 nm, emission 566 nm).The probes were designed by Biosearch Technologies.The sequences of the new linc-133 probes can be found in Table S2.Sample preparation and hybridization were performed in tubes using previously described protocols with some modifications 25 .Images were recorded with a Leica TCS SP8 STED camera.

GFP fluorescence assay
To measure GFP fluorescence in L1-arrested worms, the day one L1-arrested worms were transferred onto 2% agar pads.Images were taken under a fluorescence microscope (Nikon DS-RI2).For each strain, at least three independent replicate experiments were carried out with at least 30 worms of each strain captured in each experiment.The relative GFP intensity was analyzed and calculated by using ImageJ.
The P values were determined by using a two-tailed t test.

DAF-16::GFP localization assay
To test DAF-16 translocation, a previously described standard method was used 52 .
Worms were maintained at 20 °C, transferred onto 2% agar pads for observation of DAF-16::GFP nuclear localization status.To test DAF-16 translocation from the nucleus, day one L1-arrested worms were heat shocked at 34 °C for 1 h to allow DAF-16 translocation into the nucleus.Then, the worms were either kept at 20 °C or treated with an Akt activator (SC79, diluted in M9 buffer; working concentration: 10 mg/kg SC79) for another half hour before observation.At least 80 animals were observed per treatment, and each treatment included three independent repeats.The localization status of DAF-16::GFP was scored and assigned to three categories: predominantly nuclear, partially nuclear and cytoplasmic.The location of DAF-16::GFP was monitored using a fluorescence microscope system.
((Nikon DS-RI2)).Accumulation of fluorescent signal in nuclei was scored as described previously 52 .P values were calculated using two-way ANOVA.
For the linc-133 overexpression strains, the gene sequence was amplified from C. elegans genomic DNA by using Phusion High-Fidelity PCR (Phusion Master Mix, Thermo Fisher) and cloned into a plasmid (L2528) containing its own promoter.
For the plasmids used to construct the daf-18 rescue strains, please see our previous publication 2 .

EMS screening
The daf-18(ok480) worms were used to perform genome-wide EMS mutagenesis for forward genetic screening 15 .In short, more than 800 synchronized L4 stage worms were incubated in a total volume of 4 mL of 50 mM EMS (Sigma) in M9 for 4 h at 20 °C, and approximately 20,000 F2 generation embryos were placed onto NGM plates.The F3 progenies were cultured in M9 to induce L1 arrest and screened for mutants that could survive with normal survival.The selected mutants were backcrossed into daf-18(ok480) 3 times.The mutations of interest were identified by using the recently published Sibling Subtraction Method 24 .
Antisense probes are reverse complements of the above and labeled with biotin.The Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific, Cat# 20164) was used to perform an RNA pull down assay to identify the specific proteins interacting with linc-133.The procedure was performed according to the manufacturer guidelines.After pull down, equal amounts of protein were separated by electrophoresis on 7.5% polyacrylamide gels (Epizyme Biotechnology, Cat# PG211).
Then, the gels were stained using a Fast Silver Stain Kit (Beyotime Biotechnology, Cat# P0017S) according to the manufacturer's instructions.After silver staining, RNA-binding protein complex-specific bands were cut out and sent to BGI Tech Solutions Co., Limited (Shenzhen, China) for mass spectrometry analysis.

Western blotting
Protein extraction from L1-arrested larvae was performed by ultrasonic cracking.We measured the protein concentration by using a BCA Protein Assay Kit (Solarbio. Cat#PC0020), separated protein samples via electrophoresis using SDS-containing polyacrylamide gels, and then transferred the separated protein samples onto polyvinylidene fluoride (PVDF) membranes.After blocking with 5% milk in TBST buffer for 1 h, the membranes were incubated at 4 °C overnight with the previously indicated primary antibodies.Afterward, the membrane was washed 3 times with TBST buffer for 10 min each.We incubated the membranes with the corresponding HRP-labeled secondary antibodies for 1 h at room temperature and then washed the membranes 3 times with TBST buffer.Finally, the proteins were visualized using enhanced chemiluminescence (Meilunbio), and the band intensity was determined with a FluorChem M Imaging System (ProteinSimple).The primary antibodies used in the study were anti-ubiquitin (K48 linkage-specific) (Abcam, Cat#ab140601), 14-3-3 polyclonal antibody (Proteintech, Cat#14503-1-AP), and beta tubulin antibody (Abways Technology, Cat#F084106).

RNA immunoprecipitation (RIP)
RIP was carried out according to previously described methods 55 .L1-arrested worms were harvested and rinsed twice with ice-cold PBS.The worms were suspended in 1 mL RIP buffer (nuclease-free) containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40, 100 units of RNase inhibitor, 2 mM ribonucleoside vanadyl complex (Sangon Biotech), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (MCE).Following brief sonication, worm lysate was centrifuged at 12,000 ×g for 15 min at 4 °C, and the supernatants were precleared with 10 μL of Protein Acoated agarose (Millipore).The precleared supernatants were then divided into two equal parts and incubated with 20 μL 14-3-3 polyclonal antibody and IgG overnight at of RIP buffer (nuclease-free) and resuspended in worm lysates containing antibody.
The samples were rotated for 1 h and then washed three times with RIP buffer.Then, the beads were spun down at 2000xg for 30 s, and the lysate was removed.The crosslinks were reversed by proteinase K buffer (50 mM Tris pH 7.4, 150 mM NaCl, 100 units RNase inhibitor, 0.5% SDS, and 200 μg/mL proteinase K) at room temperature for 30 min before being used for RNA extraction.RNA was subjected to real-time PCR analysis using primers to amplify linc-133.The primers used for realtime PCR are listed in Table S2.

Statistical analyses
All graphed data are presented as the mean ± SEM from at least 3 biological replicates performed in triplicate technical replicates.Statistical calculations were performed using GraphPad Prism 5 (GraphPad Software).The GFP mean intensity was quantified using ImageJ.The mean intensity of Western blotting bands was quantified using the Image J. The relative expression levels of genes were determined by using real-time PCR and the 2 -△△ CT method.The differences between two groups were analyzed using two-tailed Student's t test.For multiple comparisons, as in the DAF-16 nuclear localization assay, two-way ANOVA was used to determine the P values.For survival analysis, the mean survival rate was calculated by the Kaplan-Meier method.The P values of the difference in overall survival rate were determined using the log-rank test.A P value <0.05 was indicative of statistical significance.Statistical significance values were set as *p < 0.05, **p < 0.01, ***p < 0.001, n.s.= not significant.Further details are provided in the figure legends.was independently repeated at least three times.The mean survival rates were calculated using the Kaplan-Meier method, and P values were determined by using the log-rank test.All the survival data are summarized in Table S1.and the specific 14-3-3 antibody was used to do the blotting.(f) The 14-3-3 antibody was used to co-immunoprecipitated 14-3-3 proteins and linc-133 RNA.The RIP confirmed the linc-133 aqz3 can be immunoprecipitated together with 14-3-3 antibody.(g) The expression of linc-133 was not affected by daf-18 and linc-133 aqz3.(h) Knocking down 14-3-3 coding genes affected the survival of daf-18(ok480);linc-133(aqz3) worms.The mean survival rates were calculated using the Kaplan-Meier method, and P values were determined by using the log-rank test.All the survival data are summarized in Table S1.worms.Each set of survival experiments was independently repeated at least three times.The mean survival rates were calculated using the Kaplan-Meier method, and P values were determined by using the log-rank test.All the survival data are summarized in Table S1.

Figure 1 .
Figure 1.Both the lipid and protein phosphatase activities of DAF-18 affect L1 arrest survival.(a) Disruption of age-1 partially rescued the shortened survival of daf-18(ok480) L1arrested worms.(b) Disrupting daf-18 reduced the survival of daf-16 mutants even further.(c) The activation of daf-16 failed to fully rescue the shortened survival of daf-18(ok480) L1-arrested worms.(d) Unlike ins-3 worms, disruption of daf-2 failed to rescue the short survival of daf-18 worms.(e) Schematic of DAF-18 phosphatase and the mutations introduced in the experiments.Expression plasmids containing mutant sequences were used to generate the specific proteins.(f) The lipid phosphatase activity of DAF-18 partially rescued the survival of daf-18(ok480) L1arrested worms.(g) The protein phosphatase activity of DAF-18 partially rescued the survival of daf-18(ok480) L1-arrested worms.(h) Overexpression of daf-18 without

Figure. 5 .
Figure.5.linc-133 gain-of-function regulates HSF-1 to support the survival when the protein phosphatase activity of DAF-18 is dysfunctional.

Figure 6 .
Figure 6.linc-133 gain of function decreases the protein aggregation caused by loss of protein phosphatase activity of DAF-18.(a) Aggregated proteins selected and degraded in cytosol.(b) The protein aggregation affected by daf-18(D137A) and linc-133 gain of function were identified by using the

Figure. 8 .
Figure.8.The working model of linc-133 gain-of-function in daf-18-deficient L1arrested worms.Defects in lipid phosphatase activity can cause translocation of the transcription factor DAF-16 from the nucleus to the cytoplasm, and linc-133 gain-of-function may obstruct this translocation.The gain of function of linc-133 may also be involved in other AGE-1/PI3K-regulated biological processes through regulating HSF-1.Protein phosphatase activity plays a pivotal role in controlling L1 arrest survival.Loss of DAF-18 protein phosphatase activity causes the production of high levels of aggregated proteins in L1-arrested worms.The gain-of-function of linc-133 can induce HSP chaperones to select and process the aggregated proteins for degradation through ubiquitination modification.