C. elegans orthologs MUT-7/CeWRN-1 of Werner syndrome protein regulate neuronal plasticity
- Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Taiwan
- Department of Biological Science and Technology, National Chiao Tung University, Taiwan
- Department of Cell and Tissue Biology, University of California, San Francisco, United States
Figures
Figure 1
The two C. elegans orthologs of human WRN required for olfactory learning behavior.
(A) Alignment of C. elegans MUT-7 and CeWRN-1 with human WRN. The 3′−5′ exonuclease domain of nematode MUT-7 has been predicted to show 29% amino acid sequence identity to that of human WRN, as …
Figure 2 with 2 supplements
Different intracellular roles of MUT-7 mediate olfactory learning.
(A) Different localizations of MUT-7 in AWCs associated with different positions of the GFP tag. MUT-7 with an upstream (a and b) or downstream (c and d) GFP tag showed localization throughout the …
Figure 2—figure supplement 1
Pairwise comparison of odr-1 22G RNA levels.
The expression of odr-1 22G RNA in naïve and odor-trained populations was quantified by RT-qPCR by using the odr-1.7 probe. Each RT-qPCR result was normalized to the endogenous sn2343 gene, whose …
Figure 2—figure supplement 2
Prolonged odor exposure decreases odr-1 mRNA expression.
The expression of odr-1 mRNA in naïve and odor-trained worms was quantified by SYBR Green based quantitative PCR. The fold change is the ratio calculated between the mRNA level in odor-trained worms …
Figure 3
MUT-7 is phosphorylated by EGL-4 in the nucleus after prolonged odor exposure.
(A) Schematic representation of the seven predicted phosphorylation sites of PKG in MUT-7 in the top panel. Different point mutations were introduced by site-directed mutagenesis, and the constructs …
Figure 4 with 3 supplements
CeWRN-1 function in the AWC nucleus is required for olfactory learning.
(A) The BiFC screen showed in vivo specific protein interactions in the AWC nucleus after prolonged odor exposure. The BiFC fluorescent signals were scored in naïve and odor-trained worms. Each …
Figure 4—figure supplement 1
Images of BiFC screening results.
All representative fluorescent images related to Figure 4A from naïve and odor-trained worms are shown.
Figure 4—figure supplement 2
Localization of CeWRN-1 in AWC neurons.
Fluorescent images of wild-type animals expressing GFP under a putative 2.5 kb Cewrn-1 promoter showed the same localization obtained with the AWC promoter-driving mCherry.
Figure 4—figure supplement 3
Expression of MUT-7::HA::GFP in the whole cell body of the AWC neuron.
An influenza hemagglutinin (HA) peptide was inserted between MUT-7 and GFP in the CterGFP-MUT-7 construct. This construct was introduced into wild-type animals, and GFP was observed in the whole AWC …
Figure 5 with 3 supplements
Models of the involvement of MUT-7 and CeWRN-1 in long-term olfactory learning in AWC.
(A) In wild-type AWC neurons, prolonged odor exposure causes EGL-4 to enter the nucleus. odr-1 22G RNAs are generated by cytoplasmic MUT-7 and shuttled into the nucleus by NRDE-3. Once the small RNA …
Figure 5—figure supplement 1
Loss of MUT-7 significantly decreases brood size.
Animals were synchronized in the larva one stage and seeded on NGM culture plates. The number of eggs was scored from the young adult stage onward every 24 hr for 3 days. A t-test was used to …
Figure 5—figure supplement 2
Loss of MUT-7 significantly affects germline cell death.
Oocyte development was visualized from the young adult stage onward at 12, 24, 48, and 96 hr. The t-test was used to compare the total numbers of cell corpses per gonad arm between the indicated …
Figure 5—figure supplement 3
Images of cell corpses in wild-type and mut-7(pk204) animals.
Cell corpses in each strain are indicated by red arrows.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Caenorhabditis elegans) | N2 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00000001 | Genotype: wild type |
| Strain, strain background (C. elegans) | Cewrn-1(gk99) | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain 00035559 | Genotype: wrn-1(gk99) II |
| Strain, strain background (C. elegans) | hpl-2(tm1489) | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00030670 | Genotype: hpl-2(tm1489) III |
| Strain, strain background (C. elegans) | mut-7(ne4255) | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00040468 | Genotype: mut-7(ne4255) III |
| Strain, strain background (C. elegans) | mut-7(pk204) | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00028942 | Genotype: mut-7(pk204) III |
| Strain, strain background (C. elegans) | nrde-3(gg66) | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00040756 | Genotype: nrde-3(gg66) X |
| Recombinant DNA reagent | pceh-36prom3-pPD95.75 vector | Oliver Hobert Lab | AWC-specific promoter ceh-36prom3 | |
| Recombinant DNA reagent | pCewrn-1::GFP | This study | Bi-Tzen Juang Lab | 2.5 kb upstream of the Cewrn-1 start site of translation |
| Recombinant DNA reagent | pAWC::GFP::MUT-7 | Noelle D. L'Etoile Lab Juang et al., 2013 | ||
| Recombinant DNA reagent | pAWC::4XNLS::mCherry::MUT-7 | This study | Bi-Tzen Juang Lab | 4XNLS::mCherry obtained from pGC302 (Addgene) |
| Recombinant DNA reagent | pAWC::4XNLS::GFP::MUT-7 | This study | Bi-Tzen Juang Lab | 4XNLS::GFP obtained from pGC240 (Addgene) |
| Recombinant DNA reagent | pAWC::MUT-7::GFP | This study | Bi-Tzen Juang Lab | MUT-7 was amplified from yk443 plasmid |
| Recombinant DNA reagent | pAWC::CeWRN-1::GFP | This study | Bi-Tzen Juang Lab | Cewrn-1 cDNA from yk1276 plasmid |
| Recombinant DNA reagent | ODR-1::GFP | This study | Noelle D. L'Etoile Lab | AID::3xFLAG, 1 kb downstream of odr-1.b stop codon |
| Recombinant DNA reagent | pAWC::GFP::MUT-7(W812Amber) | This study | Bi-Tzen Juang Lab | Site-directed mutagenesis reaction Technologies |
| Recombinant DNA reagent | podr-3::NLS::VC155::EGL-4 | This study | Bi-Tzen Juang Lab | Add NLS sequence and BiFC analysis |
| Recombinant DNA reagent | podr-3::VC155::EGL-4. | This study | Bi-Tzen Juang Lab | BiFC analysis |
| Recombinant DNA reagent | pAWC::VN173::MUT-7 | This study | Bi-Tzen Juang Lab | BiFC analysis |
| Recombinant DNA reagent | pAWC::NLS::VN173::MUT-7 | This study | Bi-Tzen Juang Lab | Add NLS sequence and BiFC analysis |
| Recombinant DNA reagent | pAWC::VN173::MUT-7 | This study | Bi-Tzen Juang Lab | BiFC analysis |
| Recombinant DNA reagent | pAWC::MUT-7::HA::VC155 | This study | Bi-Tzen Juang Lab | Add HA taq |
| Recombinant DNA reagent | pAWC::MUT-7::c-Myc::VN173 | This study | Bi-Tzen Juang Lab | Add c-Myc taq |
| Recombinant DNA reagent | pAWC::HPL-2::VC155 | Noelle D. L'Etoile Lab | Juang et al., 2013 | |
| Recombinant DNA reagent | pAWC::HIS-71::VN173 | Noelle D. L'Etoile Lab | Juang et al., 2013 | |
| Recombinant DNA reagent | pAWC::CeWRN-1::VN173 | This study | Bi-Tzen Juang Lab | BiFC analysis |
| Commercial kit | QuikChange Lightning Site-Directed Mutagenesis Kit | Agilent Technologies | Agilent:210518 | |
| Commercial kit | TURBO DNA-free Kit | Thermo Fisher: Invitrogen | Catalog number::AM1907 | |
| Commercial kit | Multiscribe Reverse Transcriptase | Thermo Fisher: Applied Biosystems | Catalog number: 4311235 | |
| Commercial kit | TaqMan Universal PCR Master Mix | Thermo Fisher: Applied Biosystems | Catalog number: 4326708 | |
| Commercial kit | iScrpt cDNA Synthesis Kit | Bio-Rad | Catalog number: 1708890 | |
| Commercial kit | iTaq Universal SYBR Green Supermix | Bio-Rad | Catalog number: 1725120 | |
| Software, algorithm | GraphPad | Prism | GraphPad Prism 8.0.1 | |
| Software, algorithm | MetaMorph | Molecular Devices | Version 7.8.13.0 |
Author response table 1
| Strains (worms with transgenes) | BiFC signals in the nucleus of the AWC neuron (%) | |
|---|---|---|
| naïve worms | odor trained worms | |
| Wildtype ex[CeWRN-1 and MUT-7(all S/T to A)]* | 3% (n=68) | 4% (n=81) |
Additional files
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Supplementary file 1
Asymmetric expression of str-2 in AWC neurons.
The cell fate of the AWC neuron was examined by quantifying the asymmetric expression of pstr-2::DsRed. Animals were scored in three categories according to pstr-2::DsRed expression either in AWC (0AWCpstr-2 ON), in only one AWC (1AWCpstr-2 ON), or in both AWCs (2AWCpstr-2 ON).
- https://cdn.elifesciences.org/articles/62449/elife-62449-supp1-v2.docx
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Supplementary file 2
Nuclear accumulation of EGL-4 after prolonged butanone exposure.
Integrated GFP-tagged EGL-4 (termed pyIs500) was expressed in wild-type, hpl-2(tm1489), and Cewrn-1(gk99) animals. The nuclear expression of EGL-4 was scored in naïve and odor-trained animals.
- https://cdn.elifesciences.org/articles/62449/elife-62449-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62449/elife-62449-transrepform-v2.docx
