A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions
- Department of Molecular Genetics, University of Toronto, Canada
Figures
Figure 1 with 2 supplements
Functional validations of GFP::3xFLAG and 3xFLAG tagged Argonautes.
(A) Maximum likelihood evolutionary tree of A. thaliana AGO1 (AtAGO1), D. melanogaster PIWI (DmPIWI), and C. elegans Argonautes. (B) Workflow for characterizing C. elegans Argonautes. (C) Functional …
Figure 1—figure supplement 1
Epitope tag locations and Western blot validations.
(A) Gene diagrams of each Argonaute. The green arrow points to the GFP::3xFLAG tag insertion site. The number corresponds to the distance in amino acids from the first methionine. The letter …
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Figure 1—figure supplement 1—source data 1
This file contains original western blots of AGO IPs used in creating Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/83853/elife-83853-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Additional analyses of tagged Argonautes.
(A) Gene diagrams of WAGO-5 and WAGO-11 as in Figure 1—figure supplement 1 (left). GFP driven under the promoter of WAGO-11 (right). GFP is observed in the distal gonadal sheath of L4 …
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Figure 1—figure supplement 2—source data 1
This file contains original western blots of AGO IPs used in creating Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/83853/elife-83853-fig1-figsupp2-data1-v2.zip
Figure 2
Argonautes associate with different types of sRNAs and target different categories of genetic features.
(A) 5′ nucleotide and length of sRNAs present in each Argonaute IP shown in bar graph form. The pie charts depict which type of genetic element (biotype) the sRNAs correspond to, as listed. AS = …
Figure 3
Analysis of miRNAs in AGO IPs and ago mutants reveals novel miRNAs.
(A) Clustering diagram of miRNAs enriched in AGOs. Each blue line represents an individual mature miRNA sequence. miRNAs are categorized by conservation, with those conserved to C. briggsae …
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Figure 3—source data 1
This file contains original western blots of AGO IPs used in creating Figure 3.
- https://cdn.elifesciences.org/articles/83853/elife-83853-fig3-data1-v2.zip
Figure 4
Analysis of piRNAs in AGO IPs and ago mutants reveals new piRNAs.
(A) Genomic loci of annotated miRNAs that are enriched in PRG-1 IPs and suspected to be piRNAs. Ruby motifs highlighted above. (B) Violin plots depicting the fold change of all detected piRNAs in ago…
Figure 5 with 3 supplements
Analysis of endo-siRNA binding AGOs reveals functional categorization of AGOs to regulate distinct genetic elements.
(A) Clustering diagram of AGO protein-coding gene targets. Each blue line represents a transcript for which endo-sRNAs were enriched twofold or more relative to input in both IPs. 22G-RNAs (defined …
Figure 5—figure supplement 1
Metagene analysis of sRNAs.
Metagene profiles for the protein-coding gene targeting sRNAs enriched in association with each AGO. The number of targets is indicated in parenthesis. To account for differences in the size of …
Figure 5—figure supplement 2
Additional analysis of the endo-siRNA binding AGO IPs.
(A) A Venn diagram showing the overlaps of pseudogene targets of the AGO clusters as highlighted by the color scheme in Figure 5A. (B) A Venn diagram showing the overlaps of transposon targets of …
Figure 5—figure supplement 3
Loss of ago genes results in differential effects of sRNA levels associated with each AGO.
Box plots showing the expression levels (y-axis) of the sRNAs that target the protein-coding targets for which sRNAs were enriched in each AGO IP (x-axis) in the indicated argonaute mutant (panels). …
Figure 6 with 2 supplements
AGOs are differentially expressed in the germline and differentially regulate germline gene expression to promote fertility.
(A) Expression patterns of germline AGOs in L4 (left) and adult (right) germlines. Inset image zoomed in at an individual germ cell nucleus. Yellow arrow points to sperm within the spermatheca. …
Figure 6—figure supplement 1
Additional analysis of AGO mRNA expression.
(A) mRNA levels of AGOs in different developmental stages. Data is an aggregate of public datasets available on WormBase version WS273. (B) Illustration of hermaphrodite germline with sections that …
Figure 6—figure supplement 2
The mortal germline of wago mutants is reversible and associated with reduced germline proliferation.
(A) A temperature shift experiment where wago mutants were placed at 20°C for one generation, then transferred to 25°C for three generations and then back to 20°C. Brood size was measured every …
Figure 7 with 8 supplements
AGOs are expressed in multiple somatic tissues and several are required for normal immunity towards pathogens.
(A) AGOs expressed in the intestine. Adult worms shown. Brackets indicate the type of sRNA AGOs associate with. Intestines are outlined in yellow. Arrowhead indicates intestine cell nuclei. Scale …
Figure 7—figure supplement 1
AGO expression in embryos.
Images of embryos from left to right: 2 cell stage, 4 cell stage, ~40 cell stage, ~200 cell stage (end of gastrulation), comma/bean elongation stage, threefold stage/pretzel stage. Scale bar = 50 µm.
Figure 7—figure supplement 2
AGO expression in L1 larvae.
Images of L1 larvae. Scale bar = 50 µm.
Figure 7—figure supplement 3
AGO expression in L2 larvae.
Images of L2 larvae. Scale bar = 50 µm.
Figure 7—figure supplement 4
AGO expression in L3 larvae.
Images of L3 larvae. Scale bar = 50 µm.
Figure 7—figure supplement 5
AGO expression in L4 larvae.
Images of L4 larvae. Scale bar = 50 µm.
Figure 7—figure supplement 6
AGO expression in adult worms.
Images of adult worms. Scale bar = 50 µm.
Figure 7—figure supplement 7
AGO expression in males.
Images of adult males. Scale bar = 50 µm.
Figure 7—figure supplement 8
Representative images of developmental stages of the RDE-1 transcriptional reporter.
Top row of each panel is DIC + GFP images; bottom row is GFP images shown in grayscale. (A) embryos, (B) L1, (C) L2, (D) L3, (E) L4, and (F) adult. Scale bar = 50 µm. Note that nuclear localization …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (C. elegans) | rde-1 | WormBase | WBGene00004323 | |
| gene (C. elegans) | alg-1 | WormBase | WBGene00000105 | |
| gene (C. elegans) | alg-2 | WormBase | WBGene00000106 | |
| gene (C. elegans) | alg-3 | WormBase | WBGene00011910 | |
| gene (C. elegans) | alg-4 | WormBase | WBGene00006449 | |
| gene (C. elegans) | alg-5 | WormBase | WBGene00011945 | |
| gene (C. elegans) | wago-1 | WormBase | WBGene00011061 | |
| gene (C. elegans) | wago-3/ppw-1 | WormBase | WBGene00004094 | |
| gene (C. elegans) | wago-4 | WormBase | WBGene00010263 | |
| gene (C. elegans) | wago-5 | WormBase | WBGene00022877 | |
| gene (C. elegans) | wago-6/sago-2 | WormBase | WBGene00018921 | |
| gene (C. elegans) | wago-7/ppw-1 | WormBase | WBGene00004093 | |
| gene (C. elegans) | wago-8/sago-1 | WormBase | WBGene00019666 | |
| gene (C. elegans) | wago-9/hrde-1 | WormBase | WBGene00007624 | |
| gene (C. elegans) | wago-10 | WormBase | WBGene00020707 | |
| gene (C. elegans) | wago-11 | WormBase | WBGene00021711 | |
| gene (C. elegans) | wago-12/nrde-3 | WormBase | WBGene00019862 | |
| gene (C. elegans) | csr-1 | WormBase | WBGene00017641 | |
| gene (C. elegans) | C04F12.1/vsra-1 | WormBase | WBGene00007297 | |
| strain, strain background (C. elegans; hermaprhodites and males) | Bristol N2 | WormBase | ||
| strain, strain background (E. coli) | OP50 | Caenorhabditis Genetics Center | ||
| strain, strain background (E. coli) | HT115 | Caenorhabditis Genetics Centers | ||
| genetic reagent (C. elegans) | List of strains | This study, Supplementary file 8; Caenorhabditis Genetics Center | ||
| antibody | Anti-mouse IgG, HRP-linked Antibody (horse polyclonal) | Cell Signaling Technology | 7076 S | 1:1000 for western blots |
| antibody | Monoclonal ANTI-FLAG M2 antibody (mouse monoclonal) | Sigma | F1804 | 1:2000 for western blots 5 μg per 50 μl of Dynabeads in 200 μl for IPs |
| antibody | GFP-Trap_MA (alpaca recombinant nanobody) | ChromoTek | gtma | 20 μl of beads per 5 mg total protein in 500 μl |
| antibody | RFP-Trap_MA (alpaca recombinant nanobody) | ChromoTek | rtma | 20 μl of beads per 5 mg total protein in 500 μl |
| sequence-based reagent | List of oligonucleotides | This study, Supplementary file 8 | ||
| sequence-based reagent | High Throughput Sequencing Data (Conine et al., 2010) | https://www.ncbi.nlm.nih.gov/geo/ | GSE18731 | |
| sequence-based reagent | High Throughput Sequencing Data (Vasale et al., 2010) | https://www.ncbi.nlm.nih.gov/geo/ | GSE18714 | |
| sequence-based reagent | High Throughput Sequencing Data (Claycomb et al., 2009) | https://www.ncbi.nlm.nih.gov/geo/ | GSE18165 | |
| sequence-based reagent | High Throughput Sequencing Data (Gu et al., 2009) | https://www.ncbi.nlm.nih.gov/geo/ | GSE18215 | |
| sequence-based reagent | High Throughput Sequencing Data (Sapetschnig et al., 2015) | https://www.ncbi.nlm.nih.gov/geo/ | GSE66344 | |
| sequence-based reagent | High Throughput Sequencing Data | Phillips et al., 2012 | Table S3 | |
| sequence-based reagent | High Throughput Sequencing Data | Ortiz et al., 2014 | Table S1 | |
| sequence-based reagent | High Throughput Sequencing Data | Tzur et al., 2018 | Table S3 | |
| sequence-based reagent | High Throughput Sequencing Data | This study; https://www.ncbi.nlm.nih.gov/geo/ | GSE208702 | |
| commercial assay or kit | NEBuilder HiFi DNA Assembly Cloning Kit | New England Biolabs | E5520 | |
| commercial assay or kit | NEBNext Multiplex Small RNA Library Prep Kit for Illumina | New England Biolabs | E7560 | |
| chemical compound, drug | Q5 High-Fidelity DNA Polymerase | New England Biolabs | M0491 | |
| chemical compound, drug | T4 DNA Ligase | New England Biolabs | M0202 | |
| chemical compound, drug | Tri Reagent | Molecular Research Centre | TR118 | |
| chemical compound, drug | Phenol Chloroform Isoamyl Alcohol | Sigma-Aldrich | P2069 | |
| chemical compound, drug | cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Roche | 11836170001 | |
| chemical compound, drug | RNA 5' Polyphosphatase | Epicentre | RP8092H | |
| chemical compound, drug | Phosphatase Inhibitor Cocktail 2 | Sigma-Aldrich | P5726 | |
| chemical compound, drug | Phosphatase Inhibitor Cocktail 3 | Sigma-Aldrich | P0044 | |
| chemical compound, drug | DTT | BioShop Canada | DTT001 | |
| chemical compound, drug | NP-40 | BioBasic | NDB0385-500ML | |
| chemical compound, drug | Levamisole hydrochloride | Fisher Scientific | AC187870100 | |
| chemical compound, drug | Bovine Serum Albumin | BioBasic | 9048-46-8 | |
| chemical compound, drug | SUPERase• In RNase Inhibitor | ThermoFisher Scientific/Invitrogen | AM2696 | |
| chemical compound, drug | T4 Polynucleotide Kinase | New England Biolabs | M0201 | |
| chemical compound, drug | Glycogen | Ambion | AM9510 | |
| chemical compound, drug | RNAse and DNAse Away | BioBasic | DB0339 | |
| chemical compound, drug | Dynabeads Protein G | ThermoFisher Scientific/Invitrogen | 10003D | |
| chemical compound, drug | GB-Magic Protein A/G Immunprecipitation Magnetic Beads | GeneBiosystems | 22202B | |
| chemical compound, drug | Protein Assay Reagent A | Bio-Rad | 5000113 | |
| chemical compound, drug | Protein Assay Reagent B | Bio-Rad | 5000114 | |
| chemical compound, drug | Protein Assay Reagent S | Bio-Rad | 5000115 | |
| chemical compound, drug | Nitrocellulose blotting membrane | GE Healthcare | 10600016 | |
| chemical compound, drug | Luminata Classico Western HRP substrate | Millipore | WBLUC0500 | |
| chemical compound, drug | RNase I | ThermoFisher Scientific/Ambion | AM2295 | |
| software, algorithm | MEGA X | https://www.megasoftware.net/ | ||
| software, algorithm | CRISPOR | http://crispor.tefor.net/ | ||
| software, algorithm | SnapGene | https://www.snapgene.com/ | ||
| software, algorithm | GraphPad Prism | https://www.graphpad.com/scientific-software/prism/ | ||
| software, algorithm | STAR | Dobin et al., 2013 | ||
| software, algorithm | Rstudio | https://www.rstudio.com/ | ||
| software, algorithm | Custom Computational Pipeline | https://github.com/ | https://github.com/ClaycombLab/Seroussi_2022 | |
| other | Bolt Precast Bis-Tris Plus Gradient Gels (4–12%) | ThermoFisher Scientific | NW04120BOX | This study: Materials and Methods |
| other | Hybond C Membrane | GE/Amersham Biosciences | CA95038-380L | This study: Materials and Methods |
| other | Wheaton Steel Dounce Homogenizer | VWR | 62400–675 | This study: Materials and Methods |
Additional files
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Supplementary file 1
A table summarizing previously published data on C. elegans AGOs.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp1-v2.xlsx
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Supplementary file 2
A table summarizing small RNA library information.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp2-v2.xlsx
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Supplementary file 3
Tables summarizing the enrichment of reads, biotypes, and AGO targets in all libraries.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp3-v2.xlsb
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Supplementary file 4
A table summarizing sRNAs depleted in ago mutants.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp4-v2.xlsx
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Supplementary file 5
A table summarizing the mirDeep2 analysis results in predicting novel high confidence miRNAs.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp5-v2.xlsx
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Supplementary file 6
A table summarizing the 466 unannotated 21U sRNA sequences enriched in PRG-1 IPs and depleted in prg-1 mutants.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp6-v2.xlsx
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Supplementary file 7
Gene Ontology analysis results of AGO targets.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp7-v2.xlsx
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Supplementary file 8
Strains and primers used in this study.
- https://cdn.elifesciences.org/articles/83853/elife-83853-supp8-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/83853/elife-83853-mdarchecklist1-v2.pdf
