The <i>Pseudomonas aeruginosa</i> ribonuclease Ribocin cleaves eukaryotic ribosomes at helix 69 to inhibit host translation

Update 1 Dec 2020: Vasquez-Rifo A, Ricci EP, Ambros V (2020) Pseudomonas aeruginosa cleaves the decoding center of Caenorhabditis elegans ribosomes. PLOS Biology 18(12): e3000969. https://doi.org/10.1371/journal.pbio.3000969 View update Figures Abstract Pseudomonas aeruginosa employs host translation inhibition as a virulence-enhancing strategy. We previously showed that the bacterium induces cleavage of Caenorhabditis elegans large ribosomal RNA at helix 69 (H69), part of a central intersubunit bridge and the ribosomal decoding center. In this study, we demonstrate that a previously uncharacterized ribonuclease, Ribocin, is necessary and sufficient for H69 cleavage. Recombinant Ribocin cuts H69 in worm and mammalian ribosomes, indicating that H69 cleavage by P. aeruginosa is phylogenetically conserved. In worms, mammalian cells, and rabbit reticulocyte lysates, H69 cleavage results in translation inhibition. Furthermore, Ribocin contributes to bacterial virulence toward C. elegans, triggers a major host response to translation inhibition, and operates in parallel with Exotoxin A-mediated translation inhibition. These findings unveil the first known nuclease that cleaves eukaryotic ribosomes at H69 and expand the understanding of host translation-inhibition by establishing targeted rRNA cleavage as a mechanism of host attack. Citation: Vasquez-Rifo A, Susorov D, Sholi EH, Demo G, Jami Y, Sha J, et al. (2026) The Pseudomonas aeruginosa ribonuclease Ribocin cleaves eukaryotic ribosomes at helix 69 to inhibit host translation. PLoS Biol 24(5): e3003790. https://doi.org/10.1371/journal.pbio.3003790 Academic Editor: Matthew K. Waldor, Brigham and Women’s Hospital, UNITED STATES OF AMERICA Received: December 16, 2025; Accepted: April 22, 2026; Published: May 20, 2026 Copyright: © 2026 Vasquez-Rifo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All data is available in the manuscript and Supporting information files. Raw image data can be found in S1 Raw Images. Funding: This research was supported by funding from the US National Institutes of Health (https://www.nih.gov) R35GM127094 to A.A.K., R35GM131741 to V.A. and R35 GM153408 to J.A.W. and F31HL180041 to E.H.S., and from the Pew Charitable Trusts (https://www.pew.org) 00027360 to A.V-R. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: AS, ammonium sulfate; CDI, contact-dependent growth inhibition; cDNA, complementary DNA; CMMB, carboxylate-modified magnetic beads; CV, column volumes; FBS, fetal bovine serum; H69, helix 69; IC50, half-maximal inhibitory concentration; LB, Lysogeny Broth; PDB, Protein Data Bank; qPCR, real-time quantitative PCR; QS, quorum sensing; RRL, rabbit reticulocyte lysates; SK, slow killing; TFP, tandem fractionation procedure Introduction Protein synthesis—an essential process for gene expression and cell growth—is a primary target of antagonistic ecological interactions. For example, the fungal sarcin and plant ricin toxins inactivate ribosomes by cleaving (sarcin) or depurinating (ricin) the essential and conserved sarcin—ricin loop (H95) of large ribosomal RNA, acting as anti-predator strategies [1,2]. Pathogenic bacteria can also depurinate H95 (e.g., Shiga toxin [3]) or inhibit eukaryotic translation by inactivating host translation factors [4] (e.g., Diphtheria toxin, Exotoxin A). Lastly, bacterial protein synthesis is targeted in microbial interactions. For example, bacterial colicin E3 and CdiA-CTECL nucleases attack other bacteria’s ribosomes by cleaving small subunit rRNA at h44 [5–7], and alternative strategies rely on small molecules or peptides that inhibit translation by binding to the ribosome or translation factors [8,9]. Discovering and understanding the strategies of microbe-induced inhibition of host translation is critical for elucidating the mechanisms of disease pathogenesis and developing antimicrobial therapies. Pseudomonas aeruginosa is a bacterium that infects a wide range of hosts. In humans, it causes potentially fatal infections, such as hospital-acquired pneumonia, which are particularly severe in immunocompromised patients [10,11]. To investigate these infections, the interaction between P. aeruginosa PA14 and the nematode C. elegans serves as an established model system that has provided insights into host immunity and bacterial pathogenesis. Under “slow killing” (SK) co-culture conditions, bacteria colonize the intestines of adult worms and kill them over the course of approximately 3 days [12]. Under these conditions, the bacteria express virulence-promoting gene expression programs through quorum-sensing (QS) regulatory pathways [13,14]. In response, the worms activate multiple behavioral, stress-response, and innate immune pathways [15–17]. P. aeruginosa potently induces host translation inhibition. A well-studied strategy is via the Exotoxin A protein (ToxA), which ADP-ribosylates eukaryotic elongation factor 2 [18–20]. However, ToxA exerts minimal effects during infection of Caenorhabditis elegans [21,22]. We recently reported evidence suggesting a second translation inhibition strategy [23]. Specifically, upon interaction with virulent P. aeruginosa, C. elegans experiences a rapid accumulation of ribosomes with 26S large ribosomal RNA cleaved at the loop of helix 69 (H69). H69 forms part of the ribosomal decoding center and is critical for translation [24–26]. In previous studies [23,27], we showed that H69 cleavage is regulated by QS, promoted by the R-body gene cluster, and opposed by host-response pathways. However, the H69 nuclease remained unknown, and it was unclear whether the nuclease was encoded by the host or the bacterium [23]. In this study, we identify and mechanistically characterize the H69 nuclease. We find that a P. aeruginosa nuclease, which we named Ribocin, cleaves eukaryotic ribosomes to inhibit host translation, activate a host-response to translation inhibition, and promote virulence in a bacterium–animal pathogenic interaction. Results Identification of H69 nuclease candidates S100 lysates from infected worms—but not from uninfected worms—contain a factor that cleaves H69 in C. elegans ribosomes [23]. To identify the H69 nuclease, we established an in vitro H69-cleavage assay and performed two different tandem fractionation procedures, each yielding a single peak fraction with H69-cleavage activity (Fig 1A). We then used label-free LC–MS/MS to measure protein composition and abundance in fractions with peak H69-cleavage activity and in several adjacent fractions with intermediate or no cleavage activity. This analysis identified 230 candidate proteins enriched by both fractionation procedures (Fig 1A). A score-based metric quantified the correlation between candidate protein abundance and H69-cleavage activity in peak and adjacent fractions and was used to prioritize proteins for further testing (Fig 1B and S1 Table; see Materials and methods). (A) Scheme of the two tandem fractionation procedures. Venn diagram showing the number of protein candidates identified in each procedure and their overlap; p-value from the hypergeometric test is shown. (B) Scatterplot of absence and pattern scores for the 230 candidate nuclease proteins in (A). Circle size denotes the number of candidates at each score combination. Higher scores indicate a better fit to expectation. The PA14_21120 candidate is shown in purple. (C) Boxplot of H69 cleavage levels for mutants of 21 candidate proteins; n ≥ 2 biological replicates. The data underlying this figure can be found in S1 Data. “*” indicates a p-value 0) in all active fractions (S7, S8, IW, IF) were selected for score ranking. Two weighted scores were assigned per protein: the “absence score” (SA) and the “pattern score” (SP). To calculate the scores, the “peptide count” values of a given protein are indicated by “P” followed by the underscored fraction name (i.e., PS6 to PS8, PIF, PIW, PUF, PUW). The “absence score” assigns 1 point when each of the following equations holds true: PS6 = 0, PUF = 0, PUW = 0. The score varies from 0 to 3. The “pattern score” awards 3 points when each of the following equations holds true: PS7 > PS6, PS7 > PS8, PIW > PIF, PIW > PIF2, PIW > PUW, PIF > PUF. The score varies from 0 to 18. A protein candidate with perfect fit to the activity expectations has SA = 3 and SP = 18. To rank the candidates based on the two scores, the score variables were normalized (0–1) and the taxicab distance (δ) to SA = 3, SP = 18 was calculated, as follows: δ = (1/3) × |SA − 3| + (1/18) × |SP − 18|. The obtained scores for the protein candidates and their ranking are presented in S1 Table. The presented candidate scoring and ranking system is a novel experimental design feature for fractionation experiments that may be useful in other activity-based approaches to identify biological functions. Protein sequence and structure analyses Ribocin homologs were identified using BLASTp and the NCBI ClusteredNR database. The obtained homolog sequences were inspected for domain annotations using NCBI’s Conserved Domain Database. Multiple sequence alignment was carried out using Clustal Omega [61] and the ESPript 3 server [62]. Phylogenetic analysis was performed using “Simple Phylogeny” [63,64]. The predicted structure of the RbcN protein was obtained from the AlphaFold database version 2 [28]. The search for RbcN homologs in the Protein Data Bank (PDB) and structural alignments were performed using the protein structure comparison Dali server [29]. Ribocin expression in E. coli The full-length coding sequence (cds) of the P. aeruginosa PA14 rbcN gene was cloned into pET28a expression plasmids. Two plasmids were constructed: one with the wild-type rbcN cds and the other with an H60A point mutant (where histidine 60 is replaced with alanine). The plasmids were transformed into E. coli BL21, and bacterial cultures were grown in LB medium to ~0.4 OD600, then either induced (with IPTG) or maintained as uninduced control cultures (no IPTG added). The bacteria were then plated on NGM plates. Synchronized young adult worms were then placed on the plates to initiate the experiments. The designed plasmids were synthesized by Twist Bio and are listed in S5 Table. Production of recombinant Ribocin The coding sequence (cds) of the P. aeruginosa PA14 rbcN gene was cloned into pET28a expression plasmids. In the constructs, the rbcN signal peptide was removed, and an N-terminal 6x-His tag was incorporated. Two plasmids were created: one containing the wild-type rbcN cds and the other harboring the H60A point mutant (histidine 60 replaced with alanine). The designed plasmids were synthesized by Twist Bio and are listed in S5 Table. These plasmids were transformed into E. coli BL21 cells. Bacterial cultures were grown to an optical density of 0.8 and induced with a final IPTG of 1 mM. Cells were resuspended in lysis buffer [50 mM Tris (pH 8), 2 mM EDTA (pH 8)] and lysed by sonication using 5 cycles of 40 s bursts at 50% amplitude. Bacterial inclusion bodies were isolated by centrifugation at 20,000 g for 20 min at 4 °C before resuspending in 10 mL of solubilization buffer containing 100 mM Tris (pH 8), 2 mM EDTA (pH 8), 7 M guanidinium hydrochloride, and 150 mM reduced L-glutathione. Solubilization proceeded with stirring under inert atmosphere (argon) for 2 hours at room temperature. The protein was refolded under standard oxidative refolding conditions by diluting dropwise into 500 mL of (0.6 mM) oxidized L-glutathione and 500 mM L-arginine (pH 8) [65]. The refolded recombinant RbcN was clarified by centrifugation at 10,000 g for 20 min then subjected to a Ni-NTA column (Cytiva Life Sciences). Bound protein was washed with 10 column volumes (CV) of wash buffer [50 mM Tris (pH 8), 100 mM NaCl, 10 mM Imidazole] and eluted in 2 CV of wash buffer supplemented with 200 mM Imidazole. Recombinant RbcN was buffer exchanged into 50 mM HEPES pH 7.5 with 50 mM KCl and stored at −80 °C. In vitro translation assays In vitro translation assays in rabbit reticulocyte lysates (Green Hectares) were conducted as previously described [34] with the modification that rabbit reticulocyte lysates not treated with micrococcal nuclease (Green Hectares) were used, prepared as in [36]. Briefly, translation mixtures containing 1% nanoluciferase substrate furimazine (Promega) were preincubated with RbcN or a control buffer for 5 min at 30 °C. Then, nanoluciferase reporter mRNA was added to 30 nM and luminescence signal was recorded in kinetic mode using a Tecan Spark instrument. For bacterial translation, translation-competent E. coli extract (NEBExpress, New England Biolabs) was prepared according to manufacturer recommendations without addition of T7 polymerase as in [57]. RRL assay conditions were replicated in this E. coli extract with the modification that the reporter mRNA contained a Shine-Dalgarno sequence upstream of the nanoluciferase coding region. To estimate the IC50 of Ribocin, the maximal rate of translation for the in vitro translation assays was calculated and a three-parameter log-logistic model was fitted using the drc package (version 3.0) in R [66]. After 30 min, all translation reactions were terminated by addition of TRIzol (Invitrogen). Total RNA was extracted and subjected to capillary electrophoresis as described. Electroporation of recombinant RbcN protein into human cells Human HEK-293T (293T) cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Pen-strep). The mammalian cells were electroporated using a Neon instrument system (Thermo Fisher) using a 10 μL tip kit and following the manufacturer’s protocol. The 293T cells were transfected in 1 μM recombinant RbcN solution and the following parameters: 1,150 V, 20 ms and 2 pulses. Luciferase assays in vivo To assess the effect of RbcN on translation in vivo, 293T cells were electroporated with nanoluciferase mRNA and RbcN. Electroporated cells (~40,000/well) were plated in a 96-well plate (Corning) with 100 μL of DMEM supplemented with 10% FBS, without antibiotics. Three hours post-transfection, the media was replaced with 25 μL Opti-MEM + 1% nanoluciferase substrate endurazin (Promega), and luminescence recorded using a Tecan Spark instrument. Puromycin incorporation assays To measure translation in C. elegans worms a procedure was derived from the SunSET method [67]. A fixed number of adult worms were put on SK plates with a P. aeruginosa strain lawn for a designated time (e.g., 5 or 12 h), collected and incubated for 1 hour in a second SK plate carrying the same bacterial strain and 5 mM puromycin (Santa Cruz Biotech). The worms sample were then washed with M9 buffer, freeze-thawed with liquid nitrogen, and boiled in Laemmli buffer. Total protein was quantified on a Typhoon FLA 9500 instrument (GE Healthcare Life Sciences) using the TotalStain Q PVDF fluorescent total protein staining kit (Azure Biosystems). The worm samples were separated by electrophoresis on 4%–20% gels and blots developed with primary monoclonal anti-puromycin antibody (12D10, Sigma, RRID: AB_2566826) or monoclonal anti-tubulin (T6074, Sigma) and goat anti-mouse HRP-conjugated secondary antibody (1721011, Biorad, RRID: AB_11125936) secondary antibody. The blot chemiluminescence was measured using the SuperSignal West Femto Maximum Sensitivity Substrate (34095, ThermoFisher) and the Amersham Imager 600 instrument (GE Healthcare Life Sciences). The mean signal intensity for puromycin was measured using Fiji ImageJ software (version 1.54f) and normalized to that of tubulin. Western blot analyses To measure the abundance of the RbcN protein, a polyclonal anti-RbcN antibody (α-RbcN, Sino Biological) was developed. Purified recombinant RbcN (described in the section Production of recombinant Ribocin) was used as the antigen. Three rabbits were immunized, and the resulting serum was affinity-purified using protein A and RbcN. For Western blots, the bacterial samples were separated by electrophoresis on 10%–20% tricine gels (Novex, EC6625BOX) and blots were developed with α-RbcN serum (1:500 dilution) and HRP-conjugated donkey anti-rabbit antibody (NA934, Cytiva, RRID: AB_772206) secondary antibody. Chemiluminescence and image analysis was conducted as for the “Puromycin incorporation assays.” Western membranes were stained for total protein with a fluorescent dye (TotalStain Q - PVDF kit, Azure Biosystems) following the manufacturer’s protocol and imaged using a Typhoon FLA 9500 instrument (GE Healthcare Life Sciences). Real-time quantitative PCR To measure RNA abundance in bacterial and worm samples, total RNA was isolated (guanidinium thiocyanate, phenol/chloroform, and isopropanol precipitation). Complementary DNA (cDNA) was synthesized by reverse transcription (Protoscript) using random primers hexamers (ThermoFischer). Real-time quantitative PCR (qPCR) was performed using a SYBR green mixture (FastSYBR mixture, CWBio) in a QuantStudio 3 instrument (Thermo). Sequences of the primers used are found in S5 Table. For the qPCRs with C. elegans samples, six reference genes (act-1, tba-1, pmp-3, idhg-1, mdh-1, iscu-1) that have been previously proposed as reference genes [68,69] were evaluated using the samples and the geNorm algorithm [70]; act-1 was selected as the most reliable reference gene. For the qPCRs with P. aeruginosa samples, the clpX (PA14_41230) gene was used as reference gene [71]. In all cases, relative gene expression was calculated using the ΔΔCt method. Worm survival analysis Worms were exposed to P. aeruginosa under SK conditions (described in the section C. elegans—P. aeruginosa interaction assays). The time course of worm death was manually determined by prodding the worms with a wire pick. The collected survival data was analyzed using R (Survival package version 3.5 and Survminer package version 0.4.9) with the Kaplan–Meier (K-M) method. Statistical comparisons between survival curves were done using the log-rank test and Tarone–Ware test (rho = 0.5) using Survminer. The survival assays were performed with three or more independent biological replicates, and the results were consistent across all independent replicate experiments. Microscopy Transgene fluorescence (GFP or mCherry) was examined using a Zeiss Imager Z1 microscope, an Axiocam 503 camera, and Zen Blue software. Transgenic worms (irg-1::GFP) were grown on E. coli HB101 at 20 °C, synchronized by hypochlorite treatment, and exposed to P. aeruginosa strains at the young adult stage. Worm images were acquired using a 10× objective and constant exposure settings (200 ms). The images were further processed and quantified using Fiji/ImageJ [72,73]. Statistical analysis All experiments were performed with at least two biological replicates. All the statistic methods were conducted using the R language (version 4.2.2) and associated software packages. All the performed t-tests were two-tailed. The plotted figures were prepared using R and the ggplot2 software package (version 3.4.1). Supporting information S1 Fig. Features and Conservation of the Ribocin (RbcN) protein. (A) Protein sequence alignment of PA14_21120 and its ortholog PA3318 in P. aeruginosa strain PA01. The signal peptide sequence is underlined. (B) Unrooted phylogenetic tree of Ribocin homologs. Each node indicates the name of a bacterial taxon carrying an RbcN homolog. Arrow indicates P. aeruginosa Ribocin. The data underlying this panel can be found in S2 Data. (C) AlphaFold-predicted structure of RbcN (PA14_21120), colored by pLDDT score. The unstructured signal peptide is colored yellow. (D) Location in the AlphaFold RbcN structure of four conserved amino acids. H60 and K75 in red, T159 in green, and Y161 in blue color. https://doi.org/10.1371/journal.pbio.3003790.s001 (TIF) S2 Fig. Alignment of Ribocin (RbcN) and sequence homologs. Protein sequence alignment of P. aeruginosa RbcN and the sequence homologs shown in S1B Fig. Identical or similar amino acids are shown in red font and boxed in blue. https://doi.org/10.1371/journal.pbio.3003790.s002 (TIF) S3 Fig. Recombinant RbcN protein purity and activity. (A, B) Coomassie-stained gels of the recombinant RbcN isolation procedure for wild-type (wt) (A) and H60A mutant (B) protein. Following refolding, RbcN-containing samples were passed through a Ni-NTA column. The protein profiles of flow-through, wash, and eluate samples are shown. The full-length RbcN protein is indicated by arrows. L, protein ladder. kDa, kilodalton. (C) Denaturing agarose gel for an mRNA assayed for cleavage by recombinant RbcN (wt or H60A). Arrowheads indicate RNA ladder markers (× 103 nucleotides). https://doi.org/10.1371/journal.pbio.3003790.s003 (TIF) S4 Fig. Effect of Ribocin on prokaryotic ribosomes. (A, B) RNA profiles of Escherichia coli ribosomes in translationally-competent 30S lysate after treatment with recombinant RbcN wild-type (wt) or H60A mutant RbcN at the indicated concentrations. The individual RNA profiles are overlayed in (A). “M” indicates a 15-nucleotide (nt) marker. R.F.U. represents relative fluorescence units. https://doi.org/10.1371/journal.pbio.3003790.s004 (TIF) S5 Fig. Effects of rbcN on translation and mRNA abundance. (A) Quantification of the mScarlet-I3::PEST fluorescence upon worm exposure to cycloheximide (CHX) for 3.5 hours (h). (B) Relative abundance of the mScarlet-I3::pest mRNA by RT-qPCR for the conditions shown in (A). (C) Fluorescence microscopy images of mScarlet-I3::pest worms (strain VT4414) exposed to E. coli expressing rbcN wild-type (wt) or H60A for 3 hours. Scale bar measures 50 μm for all images. (D) Quantification of the mScarlet-I3::PEST mean fluorescence intensity for worms exposed to E. coli expressing rbcN wt for 7 or 12 h. (E) Relative abundance of the mScarlet-I3::pest mRNA and pre-mRNA measured by RT-qPCR for the 7 h condition shown in (D). (F) Relative mRNA abundance of four intestine-specific genes (ges-1, ifb-2, vha-6, vit-6) by RT-qPCR for the 7 h conditions shown in (D). The intermediate filament ifb-2 is induced by host responses. The data underlying this figure can be found in S1 Data. “n.s.” denotes not significantly different. “***” indicates a p-value < 0.001 for two-sided Welch t test comparisons. https://doi.org/10.1371/journal.pbio.3003790.s005 (TIF) S6 Fig. Effect of rbcN and toxA on bacterial virulence. (A–D) Survival curves of adult worms exposed to PA14 (wt, in red) or ΔrbcN. (A,C); ΔtoxA (B); or ΔrbcN ΔtoxA (D). The p-value from a log-rank test curve comparison and n values (individual animals) are shown. The data underlying this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003790.s006 (TIF) S7 Fig. Effect of rbcN on the host ZIP-2/IRG-1 pathway. Fluorescence microscopy images of irg-1::GFP worms exposed to E. coli heterologously expressing the rbcN gene, either wild-type (wt) or H60A mutant. Scale bar measures 50 μm for all images. https://doi.org/10.1371/journal.pbio.3003790.s007 (TIF) S8 Fig. Characterization of the ΔtoxA strain. RT-qPCR of relative toxA mRNA abundance in the ΔtoxA strain and PA14 wt. “**” indicates a p-value < 0.01 for a two-sided Welch t test comparison. The data underlying this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003790.s008 (TIF) S9 Fig. H69 cleavage of rabbit ribosomes in vitro. (A) Total RNA profiles of 80S rabbit ribosomes treated with S100 lysate from PA14-exposed worms or mock-treated. Arrows indicate the H69-cleaved fragments with their estimated nucleotide size (×103). (B) Sanger sequencing trace indicating the cloned cleavage site (vertical line) from rRNA of S100-treated rabbit ribosomes shown in (A). (C) Cleavage site (arrowhead) indicated in the Helix 69 rRNA secondary structure. “M” denotes a 15-nt marker. R.F.U. stands for relative fluorescence units; nt, nucleotide; H69, helix 69. https://doi.org/10.1371/journal.pbio.3003790.s009 (TIF) S1 Table. Scores and ranking of H69 nuclease protein candidates. https://doi.org/10.1371/journal.pbio.3003790.s010 (XLSX) S2 Table. Taxonomic classification of species with RbcN homologs. https://doi.org/10.1371/journal.pbio.3003790.s011 (XLSX) S3 Table. Structural Ribocin homologs identified using the Dali server. https://doi.org/10.1371/journal.pbio.3003790.s012 (XLSX) S4 Table. Caenorhabditis elegans strains used in the present study. https://doi.org/10.1371/journal.pbio.3003790.s013 (XLSX) S5 Table. Oligonucleotides and plasmids used in the present study. https://doi.org/10.1371/journal.pbio.3003790.s014 (XLSX) S6 Table. Bacterial strains used in the present study. https://doi.org/10.1371/journal.pbio.3003790.s015 (XLSX) S7 Table. Proteins identified by mass spectrometry analysis of fractionated samples. https://doi.org/10.1371/journal.pbio.3003790.s016 (XLSX) S1 Data. Spreadsheet containing the data points used for all plotted graphs in the manuscript (Figs 1–5, S4–S6, S8, and S9). https://doi.org/10.1371/journal.pbio.3003790.s017 (XLSX) S2 Data. Phylogenetic tree file for the RbcN homologs (S1B Fig). https://doi.org/10.1371/journal.pbio.3003790.s018 (TREE) S1 Raw Images. Uncropped images for Figs 2 and S3. Reference to main figure panels is made in the figure. Please refer to the respective legend. https://doi.org/10.1371/journal.pbio.3003790.s019 (PDF) Acknowledgments We would like to acknowledge members of the Ambros, Korostelev and Mello laboratories for their feedback on this research project. We acknowledge the Mitani lab as the source of multiple tested Caenorhabditis elegans strains. 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