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mRNA decapping is an evolutionarily conserved modulator of neuroendocrine signaling that controls development and ageing

  1. Fivos Borbolis
  2. John Rallis
  3. George Kanatouris
  4. Nikolitsa Kokla
  5. Antonis Karamalegkos
  6. Christina Vasileiou
  7. Katerina M Vakaloglou
  8. George Diallinas
  9. Dimitrios J Stravopodis
  10. Christos G Zervas  Is a corresponding author
  11. Popi Syntichaki  Is a corresponding author
  1. Biomedical Research Foundation of the Academy of Athens, Center of Basic Research, Greece
  2. Department of Biology, School of Science, National and Kapodistrian University of Athens, Greece
  3. Department of Molecular Biology and Genetics, Democritus University of Thrace, Greece
Research Article
Cite this article as: eLife 2020;9:e53757 doi: 10.7554/eLife.53757
8 figures, 1 video, 1 table and 6 additional files

Figures

Neuronal restoration of dcap-1 rescues the short lifespan of dcap-1 mutants.

Lifespan of dcap-1 worms overexpressing a dcap-1::gfp fusion in neurons, epidermis, muscles or intestine at (A–D) 20°C and (E–H) 25°C. Statistical significance is calculated in comparison to dcap-1. ****p<0.0001. Log-rank (Mantel-Cox) test.

Figure 1—source data 1

Lifespan replicates of dcap-1(tm3163) worms that overexpress a dcap-1::gfp fusion tissue-specifically at 20°C.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig1-data1-v1.xlsx
Figure 1—source data 2

Lifespan replicates of dcap-1(tm3163) worms that overexpress a dcap-1::gfp fusion tissue-specifically at 25°C.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig1-data2-v1.xlsx
Neuronal overexpression of dcap-1 extends lifespan through mRNA decapping, DAF-16/FOXO and IIS.

Lifespan at 20°C of (A–D) wt worms overexpressing a dcap-1::gfp fusion in neurons, epidermis, muscles or intestine, (E) worms overexpressing neuronal or intestinal dcap-1::gfp in dcap-2 mutant genetic background, (F) worms overexpressing neuronal dcap-1::gfp in daf-16 mutant genetic background, (G) worms overexpressing neuronal dcap-1::gfp under post-developmental daf-16 RNAi knockdown, (H) worms overexpressing neuronal dcap-1::gfp during post-developmental daf-2 RNAi knockdown. *p<0.05, **p<0.01, ****p<0.0001. Log-rank (Mantel-Cox) test.

Figure 2—source data 1

Lifespan replicates of worms that overexpress a dcap-1::gfp fusion tissue-specifically in wtgenetic background at 20°C.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig2-data1-v1.xlsx
Figure 2—source data 2

Lifespan replicates of worms depleted of daf-2 or daf-16 at 20°C.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig2-data2-v1.xlsx
Figure 2—source data 3

Lifespan replicates of dcap-2(ok2023) worms that overexpress a dcap-1::gfp fusion tissue-specifically at 20°C.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig2-data3-v1.xlsx
Figure 3 with 7 supplements
Neuronal dcap-1 regulates ins-7 expression to control longevity.

(A) mRNA levels of lifespan-regulating ILPs in young (1 day old) and mid-aged (9 days old) wt, dcap-1 and neuronal dcap-1 overexpressing worms. (B) Fluorescence intensity of ins-7p::gfp reporter in whole worms or head neurons of wt and dcap-1 animals. (C–D) Total fluorescence intensity of ins-7p::gfp reporter in neurosecretion defective unc-31 or unc-13 mutants. (E) ins-7 mRNA levels in 9 days old worms overexpressing dcap-1::gfp in neurons (synEx293) or intestine (synEx328). (F) Total fluorescence intensity of ins-7p::gfp reporter in ins-7 mutants. (G) ins-7 mature mRNA/pre-mRNA ratio in ins-7 and dcap-1;ins-7 animals that carry a neuronally expressed unc-119p::ins-7 transgene. (H) Lifespan of wt and neuronal dcap-1 overexpressing worms sensitized for neuronal RNAi during post-developmental ins-7 RNAi knockdown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate mean ± SD. Unpaired t-test (A–G); Log-rank (Mantel-Cox) test (F).

Figure 3—source data 1

Lifespan replicates of worms exposed to ins-7 RNAi at 20°C.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig3-data1-v1.xlsx
Figure 3—source data 2

Figure 3A, E and G and Figure 3—figure supplement 6.

Numeric values of relative mRNA levels of insulin-like peptides in the indicated genetic backgrounds and ages.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig3-data2-v1.xlsx
Figure 3—source data 3

Figure 3B, C, D and F and Figure 3—figure supplement 2A.

Numeric values of relative fluorescence intensity of ins-7p::gfp and ges-1p::gfp reporters in the indicated genetic backgrounds.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Transcriptional activity of ins-7 promoter is significantly increased in dcap-1 mutants.

Representative confocal images (maximum projections) of wt or dcap-1(tm3163) worms that express gfp under the control of ins-7 promoter (array wwEX66) at various ages. Scale bar = 50 μm.

Figure 3—figure supplement 2
Mutation of dcap-1 does not affect the expression of a ges-1p::gfp transgene.

(A) Fluorescence intensity of ges-1p::GFP reporter in wild type and dcap-1 mutant worms at day 1 of adulthood. (B) Representative fluorescent images of wt and dcap-1 mutant worms expressing a ges-1p::gfp transgene. Scale bar = 50 μm.

Figure 3—figure supplement 3
Transcriptional activity of ins-7 promoter is not increased in dcap-1 mutants when unc-31 is missing.

Representative confocal images (maximum projections) of unc-31(e928) or dcap-1(tm3163);unc-31(e928) worms that express gfp under the control of ins-7 promoter (array wwEX66) at various ages. Scale bar = 50 μm.

Figure 3—figure supplement 4
Transcriptional activity of ins-7 promoter in unc-31 mutants is further induced upon daf-16 knockdown.

Total fluorescence intensity of ins-7p::gfp reporter in neurosecretion defective unc-31 and dcap-1;unc-31 1 day and 3-day-old adults under control conditions (C) or daf-16 knockdown (daf-16i).

Figure 3—figure supplement 5
Transcriptional activity of ins-7 promoter is significantly increased in dcap-1 mutants independently of unc-13.

Representative confocal images (maximum projections) of unc-13(e450) or dcap-1(tm3163);unc-13(e450) worms that express gfp under the control of ins-7 promoter (array wwEX66) at various ages. Scale bar = 50 μm.

Figure 3—figure supplement 6
Transcriptional activity of ins-7 promoter is not increased in dcap-1 mutants when ins-7 gene product is missing.

Representative confocal images (maximum projections) of ins-7(ok1573) or dcap-1(tm3163);ins-7(ok1573) worms that express gfp under the control of ins-7 promoter (array wwEX66) at various ages. Scale bar = 50 μm.

Figure 3—figure supplement 7
The ratio of eft-3 mature mRNA/pre-mRNA is not affected by dcap-1 mutation.

eft-3 (F31E3.5) mature mRNA/pre-mRNA ratio in ins-7 and dcap-1;ins-7 animals that carry a neuronally expressed unc-119p::ins-7 transgene.

Figure 4 with 2 supplements
DCAP-1 affects DAF-16 localization and stress resistance.

(A) Quantification of DAF-16a::GFP positive nuclei during early stages of heat shock response in 1-day-old wt and dcap-1 mutant worms. (B) Quantification of DAF-16a::RFP positive nuclei during early stages of heat shock response in 1-day-old wt and neuronal dcap-1::gfp overexpressing worms. (C) Survival of 1-day-old dcap-1 worms that express dcap-1::gfp in their neurons, after acute exposure to high temperature or high concentration of sodium arsenite. (D) Survival of 1-day-old wt worms overexpressing neuronal dcap-1::gfp after acute exposure to high temperature or high concentration of sodium arsenite. (E) Survival of wt worms overexpressing neuronal dcap-1::gfp during sustained exposure to low concentration of sodium arsenite. (F) Survival of 9 days old wt worms overexpressing neuronal dcap-1::gfp after acute exposure to high temperature. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate mean ± SD. Unpaired t-test.

Figure 4—source data 1

Numerical values of DAF-16-positive nuclei plotted in panels A and B.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig4-data1-v1.xlsx
Figure 4—source data 2

Numerical values of % survival plotted in panels C, D, E and F.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Translocation of DAF-16 to the nucleus is impaired in dcap-1 mutants.

Representative confocal images (maximum projections) of wt and dcap-1(tm3163) worms that express a daf-16a::gfp fusion under the control of the native daf-16 promoter, after mild heat shock at 35°C. Arrowheads point to nuclei. Scale bar = 50 μm.

Figure 4—figure supplement 2
Translocation of DAF-16 to the nucleus is facilitated in neuronal dcap-1 overexpressing worms.

Representative confocal images (maximum projections) of wt and neuronal dcap-1 overexpressing worms (synEx293) that express a daf-16a::rfp fusion under the control of the native daf-16 promoter, after mild heat shock at 35°C. Arrowheads point to nuclei. Scale bar = 50 μm.

Figure 5 with 1 supplement
Neuronal DCP1 affects lifespan and adult wing expansion in D. melanogaster.

(A) DCP1 mRNA levels in heads of two fly strains subjected to neuronal DCP1 knockdown. (B, C) Lifespan of flies subjected to neuronal DCP1 knockdown. (D) Lifespan of flies that neuronally overexpress a DCP1:eGFP fusion post-developmentally. (E, F) Malformations of adult flies subjected to neuron-specific DCP1 knockdown during their development (see text). Arrowheads point to wing morphology, arrows to the difference in cuticle tanning and asterisks to the crossing of postscutellar bristles. **p<0.01, ****p<0.0001. Error bars indicate mean ± SD. Unpaired t-test (A); Log-rank (Mantel-Cox) test (B–D).

Figure 5—source data 1

Lifespan replicates of flies with neuronal DCP1 knockdown or overexpression.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig5-data1-v1.xlsx
Figure 5—source data 2

Numeric values of relative mRNA levels plotted in panel 5A and figure supplement 2.

https://cdn.elifesciences.org/articles/53757/elife-53757-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
The penetrance of unexpanded wings phenotype does not correlate with the efficiency of DCP1 knockdown.

DCP1 mRNA levels in heads of flies with expanded or non-expanded wings, subjected to neuronal DCP1 knockdown.

Neuronal DCP1 affects wing disk morphology.

(A) Phalloidin staining of wing imaginal discs derived from animals subjected to neuron-specific DCP1 knockdown. Asterisks indicate abnormalities in epithelial folding. Green: phalloidin, Magenta: DAPI. (B) Fluorescent images of brain (left) and imaginal disc (right) dissected from elav >DCP1:eGFP larvae. Arrow shows autofluorescence in the trachea. (C) Phalloidin staining of wing discs derived from flies subjected to DCP1 knockdown only in the posterior part of wing disc epithelium (using engrailed expression driver). Anterior is to the left and posterior to the right. Scale bar = 50 μm.

Figure 7 with 1 supplement
Schematic representation of thermoinducible DCP1 knock down at different developmental stages.

The crucial period where DCP1 activity affects adult wing expansion is traced between the 1-day and the 3-day pupa stages.

Figure 7—figure supplement 1
Abnormalities in wing discs of larvae subjected to neuron-specific DCP1 knockdown at 29°C.

Phalloidin staining of wing imaginal discs derived from 3rd instar larvae subjected to neuron-specific DCP1 knockdown at 29°C. Yellow arrows indicate aberrant F-actin accumulation and red asterisks abnormalities in epithelial folding. Scale bar = 50 μm.

Putative model of DCP1-mediated regulation of neuroendocrine signalling that controls lifespan, stress response and development.

DCP1 activity or availability in neuronal cells controls the equilibrium between translation and storage/degradation of mRNAs encoding for insulin-like peptides (ILPs) or other neuropeptides. High levels of DCP1 promote decapping, storage and degradation, while low levels favour mRNA translation and secretion the resulting ILPs or neuropeptides through DCVs. Secreted ILPs/neuropeptides bind to Insulin/IGF-like receptor or their corresponding receptor in distal cells and control the activity of downstream effectors (like FOXO transcription factor), ultimately affecting lifespan, stress response and developmental events.

Videos

Video 1
Neuronal overexpression of dcap-1 delays the onset of age-dependent movement impairment. Video of 20 days old worms.

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (Caenorhabditis elegans)dcap-1www.wormbase.orgCELE_Y55F3AM.12WormBase ID:
WBGene00021929
Gene (Caenorhabditis elegans)dcap-2www.wormbase.orgCELE_F52G2.1WormBase ID:
WBGene00003582
Gene (Caenorhabditis elegans)daf-16www.wormbase.orgCELE_R13H8.1WormBase ID:
WBGene00000912
Gene (Caenorhabditis elegans)ins-7www.wormbase.orgCELE_ZK1251.2WormBase ID:
WBGene00002090
Gene (Drosophila melanogaster)DCP1www.flybase.orgCG11183FlyBase ID:
FBgn0034921
Strain, strain background (Escherichia coli)OP50Caenorhabditis Genetics Center (CGC)OP50For standard NGM plates
Strain, strain background (Escherichia coli)HT115Caenorhabditis Genetics Center (CGC)HT115(DE3)For RNAi plates
Strain, strain background (Caenorhabditis elegans)C. elegans strains used in this studyThis studySupplementary file 3
Strain, strain background (Drosophila melanogaster)D. melanogaster strains used in this studyThis studySupplementary file 4
Recombinant DNA reagentPrimers used in this studyThis studySupplementary file 5
Recombinant DNA reagentpromoterless dcap-1::gfpBorbolis et al., 2017
doi: 10.1098/rsob.160313
PS#302pBluescript KS(+);4181 bp dcap-1::gfp
Recombinant DNA reagentunc-119p::dcap-1::gfpBorbolis et al., 2017
doi: 10.1098/rsob.160313
PS#293pBluescript KS(+);2200 bp unc-119p;4181 bp dcap-1::gfp
Recombinant DNA reagentrab-3p::dcap-1::gfpThis studyPS#345pBluescript KS(+); 1200 bp rab-3p;4181 bp dcap-1::gfp
Recombinant DNA reagentges-1p::dcap-1::gfpBorbolis et al., 2017
doi: 10.1098/rsob.160313
PS#328pBluescript KS(+);1541 bp ges-1p;4181 bp dcap-1::gfp
Recombinant DNA reagenthlh-1p::dcap-1::gfpBorbolis et al., 2017
doi: 10.1098/rsob.160313
PS#326pBluescript KS(+);3100 bp hlh-1p; 4181 bp dcap-1::gfp
Recombinant DNA reagentcol-10p::dcap-1::gfpBorbolis et al., 2017
doi: 10.1098/rsob.160313
PS#364pBluescript KS(+);2000 bp col-10p; 4181 bp dcap-1::gfp
Recombinant DNA reagentges-1p::gfpThis studyPS#176pPD95.77;1541 bp ges-1p
Recombinant DNA reagentunc-119p::ins-7This studyPS#478pBluescript SK II; 2200 bp unc-119p; 1082 bp ins-7
Recombinant DNA reagentUAS:DCP1:eGFPThis studyPS#394pUASTattB; 1116 bp dcp1;
924 bp egfp
Recombinant DNA reagentdaf-16 RNAiThis studyPS#48pPD129.36(L4440);1721 bp genomic DNA
Recombinant DNA reagentdaf-2 RNAiThis studyPS#36pPD129.36(L4440);1393 bp genomic DNA
Recombinant DNA reagentins-7 RNAiThis studyPS#452pPD129.36(L4440);743 bp genomic DNA
Commercial assay or kitQIAprep spin Miniprep Kit (50)QIAGENQIA.27104
Commercial assay or kitQiaquick PCR purification kitQIAGENQIA.28104
Commercial assay or kitNucleospin RNA XSMacherey-Nagel740902.50
Commercial assay or kitFIREScipt RT cDNA Synthesis kitSOLIS BIODYNE06-15-00200
Commercial assay or kitMaxima H Minus First Strand cDNA Synthesis KitThermoFisher ScientificK1682
Commercial assay or kitKAPA SYBR FAST 2X MasterMix, UniversalKapa BiosystemsKK4602
Commercial assay or kitDNA oligos synthesisIDT
Chemical compoundTri ReagentSigma-AldrichT9424
Chemical compoundFUdRSigma-AldrichF0503
Software, algorithmPrism 8Graphpad SoftwareN/Awww.graphpad.com/scientific-software/prism/
Software, algorithmFijiImage JN/Aimagej.net/Fiji
Software, algorithmIllustrator CS6Adobewww.adobe.com/products/illustrator.html
Software, algorithmPhotoshop CS6Adobewww.adobe.com/products/photoshop.html
Software, algorithmHCI ImagingHAMAMATSUhcimage.com/
Software, algorithmLeica Application Suite Advanced FluorescenceLeica

Additional files

Supplementary file 1

Neuronal overexpression of a dcap-1::gfp fusion results in its aggregation to P-body like structures.

Representative fluorescent images of wild type and dcap-2(ok3032) worms that express a dcap-1::gfp fusion under the control of the pan-neuronal unc-119 promoter. Arrowheads point to P-body like structures. Scale bar = 20 μm.

https://cdn.elifesciences.org/articles/53757/elife-53757-supp1-v1.jpg
Supplementary file 2

mRNA levels of bursicon and rickets.

Relative mRNA levels of bursicon and its receptor rickets in heads dissected form newly eclosed adults subjected to neuron-specific DCP1 knockdown at 29°C.

https://cdn.elifesciences.org/articles/53757/elife-53757-supp2-v1.jpg
Supplementary file 3

List of C. elegans strains used in this study.

https://cdn.elifesciences.org/articles/53757/elife-53757-supp3-v1.xlsx
Supplementary file 4

List of D. melanogaster strains used in this study.

https://cdn.elifesciences.org/articles/53757/elife-53757-supp4-v1.xlsx
Supplementary file 5

List of primers used in this study.

https://cdn.elifesciences.org/articles/53757/elife-53757-supp5-v1.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/53757/elife-53757-transrepform-v1.docx

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