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Endogenous siRNAs promote proteostasis and longevity in germline-less Caenorhabditis elegans

  1. Moran Cohen-Berkman
  2. Reut Dudkevich
  3. Shani Ben-Hamo
  4. Alla Fishman
  5. Yehuda Salzberg
  6. Hiba Waldman Ben-Asher
  7. Ayelet T Lamm
  8. Sivan Henis-Korenblit  Is a corresponding author
  1. The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Israel
  2. Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Israel
  3. Department of Neurobiology, Weizmann Institute of Science, Israel
Research Article
Cite this article as: eLife 2020;9:e50896 doi: 10.7554/eLife.50896
6 figures, 1 table and 9 additional files

Figures

Endo-siRNAs are required for the longevity of GSC(-) animals.

(A,C–E) Impairment of the endo-siRNA pathway by dcr-1(mg375), rrf-3(pk1426), ergo-1(gg98), or nrde-3(gg66) compromises the longevity of glp-1 mutants. (B) The longevity conferred by germline depletion via DGLA supplementation is perturbed by the mg375 mutation in the dcr-1 helicase domain. (F) Impairment of the dsRNA channel sid-1(pk3321) does not compromise the longevity of glp-1 mutants. Breslow (Generalized Wilcoxon) P-values between endo-siRNA mutants and corresponding animals with intact endo-siRNA are indicated between GSC(+) animals (in orange) or between GSC(-) animals (in red). See Supplementary file 1. Note that the dcr-1 and nrde-3 mutants have not been outcrossed. This may affect their lifespan phenotypes.

Figure 2 with 1 supplement
Endo-siRNAs are required for HSF-1 activation in GSC(-) animals.

(A) qRT-PCR of the indicated genes on day 1 of adulthood. Asterisks mark Student’s t-test values of p value<0.05 (N = 4). Note that the dcr-1 mutation reduced the transcript levels of all three chaperone genes in GSC(-) animals, consistent with the possibility that the activity of their upstream transcription factor HSF-1 has been compromised. Interestingly, the dcr-1 mutation also affected the levels of the f44e5.5 transcript in GSC(+) animals. Nevertheless, it did not significantly affect the transcript levels of the hsp-16.2 and hsp-12.6 chaperones in GSC(+) animals. Given that HSF-1 and some of its targets are expressed also in the germline (Ooi and Prahlad, 2017), to avoid biases due to the presence/absence of the germline tissue, comparisons should be made within GSC(-) animals or within GSC(+) animals (McCormick et al., 2012; Steinbaugh et al., 2015). See also Figure 2—figure supplement 1 and Supplementary file 2 and Supplementary file 3. (B) Representative western blot of endogenous HSF-1 in day one animals (upper panel) compared to loading control (lower panel). Boxplots represent the distribution of normalized HSF-1 levels per strain. Different shapes represent independent experiments (N = 7). P-values of One-Sample Test and One-Way ANOVA followed by Tukey's post hoc analysis across all seven experiments are indicated. See Supplementary file 8 for statistic details. (C) Bars represent mean of means of the number of HSF-1::GFP nuclear foci per hypodermal cell. Dots represent mean number of HSF-1 foci per cell with different shapes representing independent experiments. At least 140 cells per genotype were scored in a total of 3 independent experiments. P-values determined by One-Way ANOVA followed by Tukey's post hoc analysis are indicated. Data are presented as mean ± SEM. See Supplementary file 8 for statistic details. (D) Representative fluorescent micrographs of hypodermal cell nuclei in day three adults, harboring a single copy of the Phsf-1::hsf-1::gfp transgene upon heat shock stress. Exposures and contrast were adjusted for each picture independently to best emphasize foci amount. Nucleus boundaries are circled.

Figure 2—figure supplement 1
String analysis of 72 genes whose levels decreased by more than 1.5 fold in dcr-1 glp-1 double mutants compared to glp-1 mutant.
Endo-siRNAs are required for proteostasis maintenance in GSC(-) animals.

(A–B) Thermo-resistance was examined in age-synchronized animals subjected to heat shock (37°C, 9 hr) on day 2 of adulthood. Survival was assayed after 5 hr of recovery at 25°C (120 animals per treatment, N = 3). Asterisks mark Cochran-Mantel-Haenszel Test values of p<0.001. (C) Bars represent mean of the percentage of motile animals scored in age-synchronized day 5 Q35m or glp-1;Q35m animals (more than 45 animals per treatment, N = 3). Different shapes represent mean motility in independent experiments. Asterisks mark p-values<0.001 determined by One-Way ANOVA followed by Tukey's post hoc analysis. (D) Bars represent mean of means of the number of body bends per minute in age-synchronized day four unc-52(ts) animals. Animals were raised at 25°C till day 1 of adulthood, and shifted to the permissive temperature (15°C) thereafter. A total of 180 animals per strain were scored in three independent experiments. Different shapes represent mean motility in independent experiments. Asterisks mark Cochran-Mantel-Haenszel test values of p<0.001. Data are presented as mean ± SEM. See Supplementary file 8 for statistic details.

Figure 4 with 2 supplements
Inactivation of ptp-5.1 restores longevity in GSC(-) animals with perturbed endogenous siRNA.

(A) Scatter plot depicts comparisons of gene-by-gene siRNA counts from three paired glp-1 mutant and dcr-1 glp-1 double mutant samples. Gray- all genes. Orange- 132 genes whose levels increased by more than 1.5 folds in dcr-1 glp-1 double mutants compared to glp-1 single mutants at the transcriptome analysis. Purple- five overlapping genes between the transcriptome analysis and siRNA seq, which are candidate direct targets of endo-siRNA. Blue- ptp-5.1 (c24d10.1). See also Figure 4—figure supplement 1 and Supplementary files 4, 5, 6, and 7. (B) qRT-PCR of ptp-5.1 transcript on day 1 of adulthood. Asterisks mark Student’s t-test values of p value<0.05 of 4 independent experiments. Data are presented as mean ± SEM. The low levels of ptp-5.1 transcript in GSC(-) animals is consistent with the interpretation that in GSC(+) animals, most of the transcript is expressed in the germline. (C) Representative fluorescent images of dcr-1 glp-1 transgenic animals expressing an extrachromosomal array of a translational reporter fused to the ptp-5.1 gene, driven by ptp-5.1 upstream sequences. Note that the reporter is only detected in a fraction of the animals. Animals that expressed the reporter displayed a clear fluorescent signal specifically in two adjacent cells in the mid-intestine (see Figure 4—figure supplement 2B). A similar expression pattern of the transgene was observed in all genetic backgrounds (see Figure 4—figure supplement 2). Bars represent mean of the percentage of animals expressing the ptp-5.1::gfp transgene in the intestine. At least 250 animals per strain were scored in four independent experiments. Different colors represent independent experiments. Cochran-Mantel-Haenszel test P-values are indicated. Asterisks mark p<0.001. Somatic expression of the transgene was detected in all backgrounds in a fraction of the animals. Inactivation of the endo-siRNA pathway by the dcr-1 mutation increased the fraction of the animals expressing the reporter in their mid-intestine. (D) ptp-5.1(tm6122) extended the lifespan of dcr-1 glp-1 double mutants. Breslow (Generalized Wilcoxon) P-values for each mutant vs. the mutant; ptp-5.1(tm6122) double mutant are indicated. See Supplementary file 1 for additional lifespan data.

Figure 4—figure supplement 1
String analysis of 132 genes whose levels increased by more than 1.5 fold in dcr-1 glp-1 double mutants compared to glp-1 mutant.
Figure 4—figure supplement 2
The ptp-5.1::gfp transgene is expressed in the intestine of a fraction of the animals.

(A) Only a fraction of the transgenic animals of the indicated genotypes express the transgene. However, all animals that express the transgene express it to a similar extent and almost always in two adjacent intestinal cells in the mid-body of the animal. The reporter is expressed both in the cytoplasm and in the nucleus. (B) Representative X200 images of Day one dcr-1 glp-1 transgenic animal stained with the nuclear dye DAPI while maintaining the fluorescence of the GFP. DAPI staining and GFP fluorescence were individually captured and overlaid in the merged image, to demonstrate the localization of the GFP-expressing cells relative to the intestinal nuclei. Anterior part of the animals is to te left. Arrows indicate DAPI-stained intestinal nuclei.

Inactivation of ptp-5.1 improves proteostasis in GSC(-) animals with perturbed endogenous siRNA.

(A) Thermo-resistance of age-synchronized animals subjected to heat shock (37°C, 9 hr) on day 2 of adulthood upon 5 hr of recovery at 25°C (120 animals per treatment, N = 3). Asterisks mark Cochran-Mantel-Haenszel test values of p<0.001. (B) Bars represent mean of mean number of HSF-1::GFP nuclear foci per hypodermal cell. At least 140 cells per genotype were scored in a total of 3 independent experiments. Dots represent mean number of HSF-1 foci per cell with different shapes representing independent experiments. Asterisk marks p-value<0.05 determined by One-Way ANOVA followed by Tukey's post hoc analysis. Data are presented as mean ± SEM. See Supplementary file 8 for statistic details. (C) Fluorescence micrographs of representative hypodermal cells in day three adults, harboring a single copy of the Phsf-1::hsf-1::gfp transgene upon exposure to heat shock. Nuclear boundaries are circled. Exposures and contrast were adjusted for each picture independently to best emphasize foci amount.

Endo-siRNAs improve proteostasis and promote longevity of GSC(-) animals by enabling HSF-1 activation.

Model: Germline-less animals extensively remodel their transcriptome to promote longevity and proteostasis. HSF-1 is one of the central transcription factors that transcribe proteostasis and longevity-promoting genes. We find that endo-siRNAs are critical for HSF-1 activity in GSC(-) animals, and consequently for their longevity and improved proteostasis. These endo-siRNAs are important because they indirectly limit the level of the tyrosine phosphatase ptp-5.1. The inhibition of this tyrosine phosphatase is critical for HSF-1 activation in proteostasis challenging settings such as heat-shock and aging. Whereas the release of HSF-1 from ptp-5.1 inhibition is required for the proteostasis and longevity benefits in GSC(-) animals, it is not sufficient. To achieve effective remodeling of the proteostasis and longevity promoting networks, germline removal must coordinate between the removal of ptp-5.1-dependent inhibition of HSF-1 and additional cellular events that promote HSF-1 activity such as reducing the repressive chromatin marks at HSF1-regulated stress-responsive genes.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (C. elegans)N2Caenorhabditis Genetics CenterWild Type
Strain, strain background (C. elegans)CF1903Caenorhabditis Genetics Centerglp-1(e2144)outcrossed three times in C Kenyon's lab
Strain, strain background (C. elegans)YY470Caenorhabditis Genetics Centerdcr-1(mg375)an outcrossed version of YY11 dcr-1(m9375)
Strain, strain background (C. elegans)SHK77This paperdcr-1(mg375) glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)CF3152Cynthia Kenyon labrrf-3(pk1426)outcrossed three times in C Kenyon's lab
Strain, strain background (C. elegans)SHK55This paperrrf-3(pk1426);glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK80This paperergo-1(gg98)Strain outcrossed two times in S Henis-Korenblit lab. Total eight outcrosses
Strain, strain background (C. elegans)SHK87This paperglp-1(e2144);ergo-1(gg98)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)YY158Caenorhabditis Genetics Centernrde-3(gg66)
Strain, strain background (C. elegans)SHK328This paperglp-1(e2144); nrde-3(gg66)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK53This papersid-1(pk3321)Strain outcrossed four times in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK56This paperglp-1(e2144);sid-1(pk3321)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)OG497Caenorhabditis Genetics Centerunc-119(ed3);drSi13[hsf-1p::hsf-1::GFP::unc-54utr;Cb-unc-119+]
Strain, strain background (C. elegans)SHK299This paperunc-119(ed3);drSi13[hsf-1p::hsf-1::GFP::unc-54utr;Cb-unc-119+];glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK300This paperunc-119(ed3);drSi13[hsf-1p::hsf-1::GFP::unc-54utr;Cb-unc-119+];dcr-1(mg375)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK301This paperunc-119(ed3);drSi13[hsf-1p::hsf-1::GFP::unc-54utr;Cb-unc-119+];dcr-1(mg375) glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)AM140Caenorhabditis Genetics Center rmIs132 [unc-54p::Q35::YFP]
Strain, strain background (C. elegans)SHK409This paperrmIs132 [unc-54p::Q35::YFP];glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK412This paperrmIs132 [unc-54p::Q35::YFP];dcr-1 (mg375)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK410This paperrmIs132 [unc-54p::Q35::YFP];dcr-1 (mg375) glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)HE250Caenorhabditis Genetics Centerunc-52(e669su250)
Strain, strain background (C. elegans)SHK574This paperunc-52(e669su250);glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK575This paperunc-52(e669su250);dcr-1(mg375)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK576This paperunc-52(e669su250);dcr-1(mg375) glp-1(e2144)Strain created in S Henis-Korenblit lab
Strain, strain background
(C. elegans)
SHK415This paperptp-5.1(tm6122)Strain outcrossed three times in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK470This paperglp-1(e2144);ptp-5.1(tm6122)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK469This paperdcr-1(mg375);ptp-5.1(tm6122)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK471This paperdcr-1(mg375) glp-1(e2144);ptp-5.1(tm6122)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK405This paperunc-119(ed3);drSi13[hsf-1p::hsf-1::GFP::unc-54utr;Cb-unc-119+];glp-1(e2144);ptp-5.1(tm6122)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK406This paperunc-119(ed3);drSi13[hsf-1p::hsf-1::GFP::unc-54utr;Cb-unc-119+];dcr-1(mg375);ptp-5.1(tm6122)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK407This paperunc-119(ed3);drSi13[hsf-1p::hsf-1::GFP::unc-54utr;Cb-unc-119+];dcr-1(mg375) glp-1(e2144);ptp-5.1(tm6122)Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK619This paperbiuEx63[Pptp-5.1::genomic ptp-5.1::gfp+rol-6]Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK622This paperdcr-1(mg375); biuEx63[Pptp-5.1::genomic ptp-5.1::gfp+rol-6]Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK623This paperglp-1(e2144); biuEx63[Pptp-5.1::genomic ptp-5.1::gfp+rol-6]Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK624This paperglp-1(e2144); biuEx63[Pptp-5.1::genomic ptp-5.1::gfp+rol-6]Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK620This paperdcr-1(mg375) glp-1(e2144); biuEx63[Pptp-5.1::genomic ptp-5.1::gfp+rol-6]Strain created in S Henis-Korenblit lab
Strain, strain background (C. elegans)SHK621This paperdcr-1(mg375) glp-1(e2144); biuEx63[Pptp-5.1::genomic ptp-5.1::gfp+rol-6]Strain created in S Henis-Korenblit lab
Sequence-based reagentact-1 FWThis paperqPCR primersCCAATCCAAGAGAGGTATCCTTAC
Sequence-based reagentact-1 BWThis paperqPCR primersCATTGTAGAAGGTGTGATGCCAG
Sequence-based reagentF44E5.5 FWThis paperqPCR primersCAGAATGGAAAGGTTGAGATCCTCGCC
Sequence-based reagentF44E5.5 BWThis paperqPCR primersACTGTATTCTCTGGATTACGAGCTGCTTGA
Sequence-based reagenthsp-16.2 BWThis paperqPCR primersCTCTCCATCTGAGTCTTCTGAGATTGTTAACA
Sequence-based reagenthsp-16.2 FWThis paperqPCR primersCAATTCTTGTTCTCCTTGGATTGATAGCGT
Sequence-based reagenthsp-12.6 BWThis paperqPCR primersGATGGAGTTGTCAATGTCCTCGACGAC
Sequence-based reagenthsp-12.6 FWThis paperqPCR primersTTGTGCTCCATATGGATTTCAAGAAGTTCTCC
Sequence-based reagentptp-5.1 FWThis paperqPCR primersAAGGCTCCGTCTCCTGCACT
Sequence-based reagentptp-5.1 BWThis paperqPCR primersTCCAGAGACACTTGTTGCTATCGGAG
Sequence-based reagentbw_kpni_ptp-5.1_cdsThis papercloning primersGACAATGGTACCTTTCCAGGTCCCATCATACT
Sequence-based reagentfw_PstI_ptp-5.1_PromThis papercloning primersATGCCTGCAGCACC TACATTACGCCTGCGC
Antibodyanti-HSF-1, rabbit polyclonal AntibodyAbcamABE1044WB(1:1,000)
Antibodyanti-Tubulin mouse monoclonal ascites fluid B-5-1-2SIGMA-ALDRICHT5168WB(1:6000)
Antibodyanti-Tubulin, mouse monoclonalDHSBAA4.3WB(1:2,000), RRID:AB_579793
Commercial kitRNA spike-in kitAgilent5188–5279
Commercial kitmiRVana miRNA isolation kit (w/phenol)AmbionAM1560
Commercial assayC. elegans microarray 4 × 23,000AgilentG2519F-020186
Chemical compoundTRIzolAmbion15596026
Chemical compoundLinoleic acid sodium saltSigmaL8134
Chemical compoundMaxima SYBR GREENThermo ScientificK0221
Instrumentmicroarray scannerAgilentG2565BA
InstrumentCFX-96 real time systemBioRad
Software, algorithmAgilent Feature Extraction softwareAgilentversion 9.5.1.1Agilent Technologies, RRID:SCR_014963
Software, algorithmPartek Genomics Suite softwarePartekversion 6.6RRID:SCR_011860
Software, algorithmDAVIDRRID:SCR_001881
Software, algorithmSTRINGRRID:SCR_005223
Software, algorithmSPSSSPSSRRID:SCR_002865

Additional files

Supplementary file 1

Lifespan analysis of mutants with defective processing of endo-siRNA and inactivation of ptp-5.1.

https://cdn.elifesciences.org/articles/50896/elife-50896-supp1-v2.xlsx
Supplementary file 2

72 genes whose levels decreased by more than 1.5 fold in dcr-1 glp-1 double mutants compared to glp-1 single mutants (p-value<0.05).

https://cdn.elifesciences.org/articles/50896/elife-50896-supp2-v2.xlsx
Supplementary file 3

GO analysis of 72 genes whose levels decreased by more than 1.5 fold in dcr-1 glp-1 double mutants compared to glp-1 mutants (p-value<0.05).

https://cdn.elifesciences.org/articles/50896/elife-50896-supp3-v2.xlsx
Supplementary file 4

132 genes whose levels increased by more than 1.5 fold in dcr-1 glp-1 double mutants compared to glp-1 single mutants (p-value<0.05).

https://cdn.elifesciences.org/articles/50896/elife-50896-supp4-v2.xlsx
Supplementary file 5

Expression of secondary siRNAs of dcr-1 glp-1 vs. glp-1 mutants (Padj <0.05).

https://cdn.elifesciences.org/articles/50896/elife-50896-supp5-v2.xlsx
Supplementary file 6

DAVID analysis of 132 genes whose levels increased by more than 1.5 fold in dcr-1 glp-1 double mutants compared to glp-1 mutants (p-value<0.05).

https://cdn.elifesciences.org/articles/50896/elife-50896-supp6-v2.xlsx
Supplementary file 7

RNAi lifespan screen of dcr-1 glp-1 double mutant.

https://cdn.elifesciences.org/articles/50896/elife-50896-supp7-v2.xlsx
Supplementary file 8

Statistical data.

https://cdn.elifesciences.org/articles/50896/elife-50896-supp8-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/50896/elife-50896-transrepform-v2.docx

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