miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging
- Max Planck Institute for Biology of Ageing, Germany
- Department of Chemical Engineering, Texas Tech University, United States
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Germany
- Laboratory of Aging Biology, Department of Biochemistry and Tissue Biology, University of Campinas (UNICAMP), Brazil
- Experimental Medicine Research Cluster (EMRC), University of Campinas (UNICAMP), Brazil
- Obesity and Comorbidities Research Center (OCRC), University of Campinas (UNICAMP), Brazil
- State Key Laboratory of Cell Biology, Innovation Center for Cell Signaling Network, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
mir-1 mutants exhibit improved motility upon polyQ35 challenge.
(A) Schematic showing the mir-1 locus, deletion alleles mir-1(gk276) and mir-1(dh1111) (see Materials and methods). (B) Representative images of N2 wild-type (WT) and mir-1 mutant animals expressing …
Figure 1—figure supplement 1
Characterization of mir-1 mutant physiology under normal conditions and proteotoxic models.
(A) MicroRNA-seq analysis of miR-1 in wild-type (WT) worms of indicated ages (Zhou et al., 2019). Normalized miR-1 reads of three independent experiments for samples of days 1, 7, 14, and 21 of …
Figure 2 with 1 supplement
v-ATPase subunits are downstream mediators of mir-1-induced motility improvement.
(A) Computational screen for potential miR-1 targets using in silico predictions microRNA.org, TargetScanWorm (TargetScan), and PicTar identifies 68 shared candidates. (B) STRING network analysis of …
Figure 2—figure supplement 1
Bioinformatic and proteomic screens implicate v-ATPase subunits as downstream mediators of mir-1-induced motility improvement.
(A) v-ATPase subunits containing one or two predicted miR-1 binding sides (BS) in their 3′UTR. v-ATPase genes identified in the bioinformatic screen by all three databases are labeled 'B,' by two …
Figure 3 with 1 supplement
miR-1 directly regulates vha-13 via its 3′UTR in muscle tissue.
(A) RT-qPCR of vha-5, vha-10, vha-13, and vha-19, as well as daf-16 and hlh-30 mRNA levels in wild-type (WT) (N2) and mir-1(gk276) mutants on day 4 of adulthood. Mean ± SEM, N = 3–4, one-way ANOVA, …
Figure 3—figure supplement 1
miR-1 regulates vha-13 via its 3' UTR and its regulation may be evolutionarily conserved.
(A) Quantification of pharyngeal fluorescent intensity of endogenous 3xFlag::mNeonGreen::VHA-5 in indicated genotypes in late L4s maintained at 25°C. One representative experiment of N = 3, mean ± …
Figure 4 with 1 supplement
mir-1 mutation enhances lysosomal biogenesis and acidification.
(A) Fluorescent image comparing HLH-30::mNeonGreen nuclear localization in the hypodermis of the mir-1(gk276) and wild-type (WT) (N2) backgrounds at day 4 of adulthood, maintained at 25°C. Scale bar …
Figure 4—figure supplement 1
mir-1 mutation does not affect total expression of HLH-30 in the pharynx.
(A) Fluorescent image comparing HLH-30::mNeonGreen intensity in the pharynx of the mir-1(gk276) and wild-type (WT) (N2) backgrounds in L4s (top) or day 4 adults (bottom), grown at 25°C. Scale bar 20 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Caenorhabditis elegans) | mir-1 | WormBase | WBGene00003260 | |
| Gene (Caenorhabditis elegans) | vha-13 | WormBase | WBGene00013025 | |
| Gene (Caenorhabditis elegans) | vha-5 | WormBase | WBGene00006914 | |
| Gene (Caenorhabditis elegans) | hlh-30 | WormBase | WBGene00020930 | |
| Gene (Caenorhabditis elegans) | daf-16 | WormBase | WBGene00000912 | |
| Strain, strain background (Caenorhabditis elegans) | N2 Bristol | CGC | N2 Bristol RRID:WB-STRAIN:WBStrain00000001 | N2 WT |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I | This paper | AA2508 RRID:WB-STRAIN:WBStrain00035886 | |
| Strain, strain background (Caenorhabditis elegans) | mir-1(dh1111) I | This paper | AA4575 | |
| Strain, strain background (Caenorhabditis elegans) | rmIs132[unc-54p::Q35::YFP] I | CGC | AM140 RRID:WB-STRAIN:WBStrain00000182 | N2;Q35 |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276); rmIs132[unc-54p::Q35::YFP] I | This paper | AA4403 | gk276;Q35 |
| Strain, strain background (Caenorhabditis elegans) | mir-1(dh1111) rmIs132[unc-54p::Q35::YFP] I | This paper | AA4577 | dh1111;Q35 |
| Strain, strain background (Caenorhabditis elegans) | N2; dhEx965[mir-1p::mir-1, myo-2p::mCherry] | This paper | AA3275 | mir-1 transgene |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; rmIs132[unc-54p::Q35::YFP]; dhEx965[mir-1p::mir-1, myo-2p::mCherry] | This paper | AA4810 | |
| Strain, strain background (Caenorhabditis elegans) | N2; dhEx1206[myo3p::flag::HA::mCherry::vha-13cDNA::unc-54 3′UTR, myo-2p::GFP] | This paper | AA4865 | myo3p::vha-13::unc-54 3′UTR |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; dhEx1206[myo-3p::flag::HA::mCherry::vha-13cDNA::unc-54 3′UTR, myo-2p::GFP] | This paper | AA4866 | |
| Strain, strain background (Caenorhabditis elegans) | N2; dhEx1207[myo-3p::flag::HA::mCherry::vha-13cDNA::vha-13 3′UTR, myo-2p::GFP] | This paper | AA5067 | myo-3p::vha-13 wt 3′UTR |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; dhEx1207[myo-3p::flag::HA::mCherry::vha-13cDNA::vha-13 3′UTR, myo-2p::GFP] | This paper | AA5068 | |
| Strain, strain background (Caenorhabditis elegans) | vha-13(syb586[3xFlag::mNeonGreen::vha-13]) V | SunyBiotech | PHX586 | vha-13 wt 3′UTR |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; vha-13(syb586[3xFlag::mNeonGreen::vha-13]) V | This paper | AA4813 | |
| Strain, strain background (Caenorhabditis elegans) | vha-13(syb586[3xFlag::mNeonGreen::vha-13]) V; dhEx965[mir-1p::mir-1, myo-2p::mCherry] | This paper | AA4850 | vha-13 wt 3′UTR + mir-1 transgene |
| Strain, strain background (Caenorhabditis elegans) | vha-13(syb587,syb504[3xFlag::mNeonGreen::vha-13 miR-1 BS1 mutated]) V | SunyBiotech | PHX587 | vha-13 MUT1 3′UTR One miR-1 BS mutated |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; vha-13(syb587,syb504[3xFlag::mNeonGreen::vha-13 miR-1 BS1 mutated]) V | This paper | AA4184 | |
| Strain, strain background (Caenorhabditis elegans) | vha-13(syb2180,syb587,syb504[3xFlag::mNeonGreen::vha-13 miR-1 BS1,2 mutated]) V | SunyBiotech | PHX2180 | vha-13 MUT2 3′UTR Two miR-1 BSs mutated |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; vha-13(syb2180,syb587,syb504[3xFlag::mNeonGreen::vha-13 miR-1 BS,2 mutated]) V | This paper | AA5123 | |
| Strain, strain background (Caenorhabditis elegans) | vha-5(syb1093[3xFlag::mNeonGreen::vha-5]) IV | SunyBiotech | PHX1093 | |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; vha-5(syb1093[3xFlag::mNeonGreen::vha-5]) IV | This paper | AA5069 | |
| Strain, strain background (Caenorhabditis elegans) | hlh-30(syb809[hlh-30::mNeonGreen]) IV | SunyBiotech | PHX809 | |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; hlh-30(syb809[hlh-30::mNeonGreen]) IV | This paper | AA5195 | |
| Strain, strain background (Caenorhabditis elegans) | rmIs133[P(unc-54) Q40::YFP] | CGC | AM141 RRID:WB-STRAIN:WBStrain00000183 | N2;Q40 |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; rmIs133[P(unc-54) Q40::YFP] | This paper | AA3112 | gk276;Q40 |
| Strain, strain background (Caenorhabditis elegans) | N2; pkIs2386[Punc-54::alphasynuclein::YFP + unc-119(+)] | This paper | AA1311 | |
| Strain, strain background (Caenorhabditis elegans) | mir-1(gk276) I; pkIs2386[Punc-54::alphasynuclein::YFP + unc-119(+)] | This paper | AA5270 | |
| Strain, strain background (Escherichia coli) | OP50 | CGC | ||
| Strain, strain background (Escherichia coli) | DH5α | Life Technologies | ||
| Other | RNAi | Source BioScience, C. elegans RNAi Collection – Ahringer | Multiple | Supplementary file 1e |
| Other | RNAi | Source BioScience, C. elegans ORF-RNAi Resource – Vidal | Multiple | Supplementary file 1e |
| Other | hlh-30 RNAi | Source BioScience, C. elegans RNAi Collection – Ahringer | W02C12.3 | hlh-30i |
| Other | daf-16 RNAi | Source BioScience, C. elegans RNAi Collection – Ahringer | R13H8.1 | daf-16i |
| Other | vha-13 RNAi | Source BioScience, C. elegans RNAi Collection – Ahringer | Y49A3A.2 | vha-13i |
| Other | vha-5 RNAi | Source BioScience, C. elegans RNAi Collection – Ahringer | F35H10.4 | vha-5i |
| Recombinant DNA reagent | Plasmid L3781 | FireLab | Worm Expression vector | |
| Recombinant DNA reagent | Plasmid pDEST R4-R3 | Invitrogen | MultiSite Gateway destination vector | |
| Recombinant DNA reagent | Plasmid myo3p::flag::HA::mCherry::vha-13cDNA::unc-54 3′UTR | This paper | BA4331 | Vector pDEST R4-R3 |
| Recombinant DNA reagent | Plasmid myo3p::flag::HA::mCherry::vha-13cDNA::vha-13 3′UTR | This paper | BA4334 | Vector pDEST R4-R3 |
| Recombinant DNA reagent | Plasmid pPD122.11 | FireLab | Worm Expression vector | |
| Sequence-based reagent | Primer | This paper | Supplementary file 1h | |
| Commercial assay, kit | TaqMan assay for mir-1 | Life Technologies | Assay ID 000385 | |
| Antibody | Anti-GFP (mouse monoclonal) | Living Colors | JL-8 RRID:AB_10013427 | 1:2000 |
| Antibody | Anti-Histone H3 (rabbit polyclonal) | Abcam | ab1791 RRID:AB_302613 | 1:5000 |
| Antibody | Anti-α-Tubulin (mouse monoclonal) | Sigma | T6199 RRID:AB_477583 | 1:5000 |
| Antibody | Anti-FLAG (mouse monoclonal) | Sigma | F1804 RRID:AB_262044 | 1:2000 |
| Antibody | Anti-mouse IgG (goat polyclonal coupled with horseradish peroxidase) | Invitrogen | G-21040 RRID:AB_2536527 | 1:5000 |
| Antibody | Anti-rabbit IgG (goat polyclonal coupled with horseradish peroxidase) | Invitrogen | G-21234 RRID:AB_2536530 | 1:5000 |
| Chemical compound, drug | LysoTracker Red | Life Technologies | LysoTracker Red DND-99 |
Additional files
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Supplementary file 1
Raw data.
(a) Life span and heat stress data. (b) Aggregate and behavior data. (c) CeleST data. (d) List of candidate genes with in silico predicted miR-1 binding sites using microRNA.org, TargetScanWorm, and PicTar. (e) RNAi motility screen. (f) Proteomic analysis of differentially regulated proteins in mir-1 vs. wild-type. (g) Microscopy data. (h) Primers.
- https://cdn.elifesciences.org/articles/66768/elife-66768-supp1-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66768/elife-66768-transrepform-v1.docx
