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• W4365794537 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode Caenorhabditis elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work, we systematically analyzed every C. elegans AGO using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss. Editor's evaluation This impressive study presents the most comprehensive analysis of the Argonautes, their small RNA partners, their targets, and their biological functions in any species to date. The work provides new insights into Argonaute-based pathways, includes extensive validation of existing models, and describes overall a treasure-trove of reagents and datasets for future exploration of the vast Argonaute world in C. elegans. https://doi.org/10.7554/eLife.83853.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Small RNA-mediated gene regulatory pathways (collectively referred to as RNA interference [RNAi]) have been identified in organisms from all domains of life (Swarts et al., 2014). These pathways utilize an array of molecular mechanisms in the epigenetic modulation of gene expression and exert their influence on nearly every step in the lifecycle of a transcript, from transcription to translation (Meister, 2013; Wu et al., 2020). At the cellular level, small RNA (sRNA) pathways are key contributors to regulating genome and transcriptome homeostasis, both under normal conditions and stress responses. At the organismal level, sRNA pathways are key regulators of gene expression programs that direct development and differentiation, and mis-regulation of sRNA pathways or components can lead to conditions such as cancer and infertility (Wu et al., 2020). The central effectors of sRNA pathways are the highly conserved Argonaute (AGO) family of proteins. AGOs are the core components of ribonucleoprotein complexes called RISCs (RNA induced silencing complexes) and are guided in a sequence-specific manner by sRNAs (18–30 nucleotides long) to complementary target transcripts (Dueck and Meister, 2014). AGOs have a bilobed structure consisting of four major domains: PAZ, MID, PIWI, and a low-complexity N-terminal domain. The PAZ and MID domains possess pockets to coordinate 3′ and 5′ end sRNA binding, respectively (Sheu-Gruttadauria and MacRae, 2017). The PIWI domain resembles RNaseH and has the capacity to direct endonucleolytic cleavage of the target RNA if the active site harbors a tetrad of catalytic amino acids (DEDD/H, Nakanishi et al., 2012). Relatively few AGOs possess this catalytic tetrad, and many AGOs recruit additional proteins to elicit other gene regulatory outcomes, such as mRNA de-capping, de-adenylation, or chromatin modulation. In Caenorhabditis elegans, at least four types of endogenous sRNAs—miRNAs, piRNAs, 22G-RNAs, and 26G-RNAs—and as many as 27 AGO-like genes have the potential to contribute to complex networks of gene regulation in different tissues throughout development. miRNAs and piRNAs are genomically encoded and transcribed by RNA polymerase II, while the 22G-RNAs and 26G-RNAs are generated by the activity of different RNA-dependent RNA polymerases (RdRPs). miRNAs are known to associate and function with the conserved AGOs ALG-1, ALG-2, and ALG-5 (Hutvagner et al., 2004; Brown et al., 2017; Corrêa et al., 2010; Vasquez-Rifo et al., 2013). The Piwi-interacting RNAs (piRNAs, also called 21U-RNAs in C. elegans) bind to the PIWI AGO PRG-1 and are thought to maintain germline genome integrity by silencing foreign or deleterious nucleic acids such as transgenes (Lee et al., 2012; Shirayama et al., 2012). Although piRNA pathways in other animals play a more prominent role in regulating transposable elements than in C. elegans, the functions of the piRNA pathway are broadly and consistently required in animal germlines to ensure fertility (Ozata et al., 2019). Two additional types of endogenous sRNAs present in C. elegans are the 26G-RNAs and 22G-RNAs (named for their predominant length and 5′ nucleotide). Because these sRNAs are generated by RdRPs, they are thought to exploit perfect complementarity to their targets. The 26G-RNAs are synthesized by the RdRP RRF-3, which generates dsRNA that is processed into 26G-RNAs by the endonuclease DICER and the phosphatase PIR-1 (Chaves et al., 2021). 26G-RNAs are classified into two groups: those of spermatogenic origin (class I, associated with ALG-3 and ALG-4, Han et al., 2009; Conine et al., 2010) and those of oogenic and embryonic origin (class II, associated with ERGO-1, Vasale et al., 2010; Han et al., 2009). The 22G-RNAs are generated by the RdRPs RRF-1 and EGO-1, independent of DICER. Currently, 22G-RNAs are divided into two main groups, those that are bound by CSR-1 and target germline expressed protein-coding transcripts to protect them from silencing, along with fine-tuning gene expression in the embryo (Claycomb et al., 2009; Cecere et al., 2014; Seth et al., 2013; Wedeles et al., 2013; Singh et al., 2021; Gerson-Gurwitz et al., 2016; Nguyen and Phillips, 2021; Charlesworth et al., 2021), versus those that are bound by other WAGO class AGOs (such as WAGO-1 and HRDE-1) that silence protein-coding genes, pseudogenes, transposable elements, and cryptic loci (Gu et al., 2009). 22G-RNAs are generally thought to act as secondary, amplified sRNAs that are synthesized after a transcript is targeted by a primary sRNA/AGO complex, with the main exception being the majority of CSR-1 associated 22G-RNAs. Primary sRNAs take several forms: piRNAs (PRG-1), 26G-RNAs (ALG-3, ALG-4, and ERGO-1), and exogenous-siRNAs produced by DICER during exogenous RNAi (exoRNAi) (RDE-1). C. elegans AGOs have generally been studied on a case-by-case basis, with agos being uncovered via genetic screens, or selected for study based on phenotype (e.g., Tabara et al., 1999). Such approaches are limited because not all phenotypes to which agos may contribute have been tested, and redundancy among the agos could confound their recovery in genetic screens. To date, only one study has taken a systematic approach to understanding AGO functional relationships, examining the requirement for each ago in exogenous RNAi (Yigit et al., 2006). A lack of antibodies against individual AGOs and difficulties with transgenic approaches have also hampered the development of a cohesive set of reagents to study AGO function. Indeed, several C. elegans AGOs have yet to be studied, and others remain only partially characterized. In this study, we have undertaken a systematic analysis of the C. elegans AGOs. We employed CRISPR-Cas9 genome editing to introduce GFP::3xFLAG epitope tags in the endogenous loci of each ago using these strains to examine spatiotemporal expression profiles throughout development, combined with sequencing sRNAs from AGO complexes and ago mutants to define the core of C. elegans sRNA pathways. We systematically assessed fertility of ago mutants and employed phenotypic assays directed by our expression and sRNA sequencing data, enabling us to uncover new roles for specific AGOs in maintaining germline integrity and in regulating immune responses to bacterial and viral pathogens. Collectively, our findings provide a foundation for understanding the full scope of sRNA pathway activity in C. elegans. With these AGO tools and knowledge of sRNA binding partners and targets, our findings provide a deeper understanding of sRNA functions throughout development and under varied environmental conditions in C. elegans. Results Systematic analysis of C. elegans Argonautes Previous studies identified 27 ago genes in C. elegans (Yigit et al., 2006); however, some have been reclassified as pseudogenes (e.g., prg-2). To define an updated set of ago genes to study, we searched the genome (WormBase version WS262) for genes that contain PAZ and PIWI domains, have a predicted protein size of ~100 kDa, and bear homology to known AGOs. Twenty-one genes met these criteria, and we ultimately characterized 19 of these AGOs (Supplementary file 1, Figure 1A and B). Construction of a phylogenetic tree for these 19 AGOs in relation to Arabidopsis thaliana AGO1 and Drosophila melanogaster PIWI places seven AGOs in the AGO clade: ALG-1, ALG-2, ALG-3, ALG-4, ALG-5, ERGO-1, and RDE-1; a single AGO in the PIWI clade: PRG-1; and 13 AGOs in the WAGO clade: CSR-1, C04F12.1 (renamed VSRA-1 for Versatile Small RNAs Argonaute-1, see below), WAGO-1, PPW-2/WAGO-3, WAGO-4, SAGO-2/WAGO-6, PPW-1/WAGO-7, SAGO-1/WAGO-8, HRDE-1/WAGO-9, WAGO-10, and NRDE-3/WAGO-12 (Figure 1A). It is important to note that CSR-1 exists as two isoforms, a long isoform (CSR-1a) and a short isoform (CSR-1b), that differ by 163 amino acids at their N terminus (Nguyen and Phillips, 2021; Charlesworth et al., 2021). Here, we generally use ‘CSR-1’ to refer to both isoforms of the protein and designate specific isoforms in the text and figures as relevant. Figure 1 with 2 supplements see all Download asset Open asset 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 validations of tagged ALG-1, ALG-5, PRG-1, WAGO-1, PPW-2, and WAGO-4 strains. Brood size was determined at 25°C for each indicated genotype. N ≧ 5 worms per condition. (D) Functional validation of tagged ALG-2 and CSR-1 strains. Brood size was determined at 20°C for each indicated genotype. For the ALG-2 tag validations, the brood size was determined when worms were fed dsRNA of alg-1. N = 5 worms per condition. (E) Functional validations of tagged ALG-3 and ALG-4 strains. Brood size was determined at 25°C for each indicated genotype. N = 10 worms per condition. (F) Functional validation of the tagged HRDE-1 strain via a Mortal germline assay at 25°C. N = 5 worms per condition. (G) Functional validations of tagged RDE-1 and SAGO-1 strains. Worms were fed bacteria expressing dsRNA of dpy-11 or an empty vector (EV) RNAi control. The length ratio of dpy-11 dsRNA fed P0 worms compared to the average length on EV was determined. N = 30 worms per condition. (H) Functional validations of tagged ERGO-1 and NRDE-3 strains. Worms were fed bacteria expressing dsRNA of dpy-11 or an EV control. The length ratio of the F1s of the dpy-11 dsRNA fed worms compared to the average length on EV was determined. N = 30 worms per condition. (C–H) *p-value<0.05, **p-value<0.01, ***p-value, n.s. = not significant. One-way ANOVA with Tukey’s post hoc multiple comparison test. All error bars represent standard deviation. We used CRISPR-Cas9 genome editing to introduce a GFP::3xFLAG tag into the N-terminus or within the first exon of the endogenous gene loci of all 21 agos (Dickinson et al., 2015; Figure 1—figure supplement 1A). We detected GFP expression for both the transcriptional reporter and GFP::3xFLAG::AGOs for 19 AGOs by confocal microscopy. (Note that both isoforms of CSR-1 were tagged in the strain used here, JMC101.) We verified that the GFP::3xFLAG::AGOs were full-length proteins by Western blot analysis (Figure 1—figure supplement 1B). We were unable to detect WAGO-5 and WAGO-11 fusion protein expression by microscopy or Western blot analysis under common laboratory conditions (Figure 1—figure supplement 2A). RNA-tiling-array data (Celniker et al., 2009) showed that both wagos are expressed at low levels with poor sequencing coverage (Figure 1—figure supplement 2B), and RT-PCR only detected low amounts of wago-11 mRNA (Figure 1—figure supplement 2C). wago-5 is targeted by WAGO-1-associated 22G-RNAs, suggesting it is silenced by the WAGO pathway (Figure 1—figure supplement 2D). During this project, the designation of wago-11 was changed to ‘pseudogene’ in WormBase (version WS275). Our data support that these wagos are pseudogenes; therefore we excluded these WAGOs from further analysis. We tested the function of the tagged AGOs by assessing phenotypes associated with ago loss of function (Yigit et al., 2006; Batista et al., 2008; Guang et al., 2008; Han et al., 2009; Buckley et al., 2012; Brown et al., 2017; Xu et al., 2018; Figure 1C–H, Figure 6D, and E, Figure 7D, and E). While previous studies have defined loss-of-function defects for several agos, some had no known loss-of-function defects (Supplementary file 1). Of all the AGOs tested, the GFP::3xFLAG tagged AGOs WAGO-1 and NRDE-3 did not behave as wild-type in phenotypic assays (Figure 1C and H). Therefore, we tagged WAGO-1 and NRDE-3 with only 3xFLAG (Figure 1—figure supplement 2E) for the purpose of small RNA cloning and used the GFP::3xFLAG tagged strain for analysis of expression patterns. Both 3xFLAG tagged AGOs were functional in phenotypic assays (Figure 1C and H). We note that although PPW-2 (also known as WAGO-3) and WAGO-1 were recently shown to be N-terminally processed by the protease DPF-1, both GFP::3xFLAG::PPW-2 and 3xFLAG::WAGO-1 strains behaved as wild-type, consistent with previous reports (Gudipati et al., 2021; Schreier et al., 2022). The tagged C04F12.1/VSRA-1 and WAGO-10 were not tested for a specific phenotype as there are no known phenotypes for C04F12.1/vsra-1 and wago-10 mutants. As we observed that the GFP tag may interfere with function in some instances (e.g., WAGO-1 and NRDE-3), we tagged C04F12.1/VSRA-1 and WAGO-10 with 3xFLAG at the same position as the GFP::3xFLAG tags for the purpose of sRNA cloning out of an abundance of caution (Figure 1—figure supplement 2E). AGO sequencing reveals sRNA association The sRNAs associated with each AGO provide important insight into the transcripts the AGOs may regulate. To identify the sRNAs that interact with each AGO, we performed immunoprecipitation (IP) followed by high-throughput sequencing of sRNAs for each of the tagged AGOs in duplicate. In parallel, we sequenced total sRNAs from the same lysates as the IPs (‘Input’ samples). We conducted IPs on worm populations at the L4 to young adult transition (58 hr post L1 synchronization), because all but a few AGOs are expressed at this stage. The only exceptions were ALG-3, ALG-4, and WAGO-10, which are only expressed during spermatogenesis (Charlesworth et al., 2021). Therefore, we conducted ALG-3, ALG-4, and WAGO-10 IPs during the mid L4 stage (48 hr post L1 synchronization), during which spermatogenesis occurs. For consistency, we treated all libraries with 5′ polyphosphatase to enable detection of 5′ tri-phosphorylated small RNA species (22G-RNAs), along with 5′ mono-phosphorylated species (miRNAs, piRNAs, 26G-RNAs) (Supplementary file 2). We assessed the length and 5′ nucleotide distribution of sRNAs associated with each AGO and mapped this total set of reads to genomic features (Figure 2A). We also determined how many miRNAs, piRNAs, and other genomic features (protein-coding genes, transposable elements, pseudogenes, and long intergenic noncoding RNAs, or lincRNAs that are targeted by antisense endogenous siRNAs including, but not limited to, 22G-RNAs and 26G-RNAs) were enriched over twofold in both IP replicates relative to the input samples (Supplementary file 3). For this enrichment analysis, we did not place any constraints on sRNA length or 5′ nucleotide and considered all genome mapping antisense reads. We defined 22G-RNA reads as 20-24 nt with no 5′ nucleotide bias and 26G-RNAs as 25-27 nt with no 5′ nucleotide bias. These stringent criteria led to the assignment of a high-confidence set of AGO-enriched sRNAs. We refer to transcripts for which antisense siRNAs are enriched over twofold as the ‘targets’ of AGO/sRNA complexes. Figure 2 Download asset Open asset 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 = antisense, S = sense. The ‘Other’ category encompasses: miRNA AS, piRNA AS, protein-coding S, pseudogene S, repetitive elements S, lincRNA S, rRNA S/AS, snoRNA S/AS, snRNA S/AS, tRNA S/AS, ncRNA S/AS, and antisense lncRNAs (ancRNAs/anr loci) (Nam and Bartel, 2012). The average of two biological replicates is shown. The GFP::3xFLAG tagged Argonautes were used for IPs except for C04F12.1/VSRA-1, WAGO-10, and NRDE-3, where a 3xFLAG tag was used. All IPs were performed on Young Adult samples except for ALG-3, ALG-4, and WAGO-10, which were conducted on L4 staged animals. The CSR-1 strain tags both isoforms. (B) A table summarizing the percentage of reads in each set of AGO IPs corresponding to genetic element types in (A). A subset of the AGO clade primarily associated with miRNAs (Corrêa et al., 2010; Vasquez-Rifo et al., 2013; Brown et al., 2017). For ALG-1, miRNAs comprised ~98% of all associated reads (47 miRNAs enriched); ALG-2, ~98% (81 enriched), ALG-5, ~68% (37 enriched); and RDE-1, ~53% (103 enriched). RDE-1 is involved in exoRNAi (Tabara et al., 1999); however, we observed that it also associates with 22G-RNAs targeting protein-coding genes (~21%, 536 enriched genes). We expect that these 22G-RNAs possess a 5′ mono-phosphate, given that RDE-1 was previously shown to associate with multiple types of sRNAs that are likely DICER products (Corrêa et al., 2010). We also detected abundant miRNAs associated with the rest of the AGO clade AGOs ALG-3, ALG-4, and ERGO-1, with ~55% (26 enriched), ~40% (2 enriched), and ~26% (33 enriched) of reads corresponding to miRNAs, respectively. These three AGOs were previously described as genetically required for 26G-RNA accumulation, and ERGO-1 was shown to physically associate with 26G-RNAs (Conine et al., 2010; Vasale et al., 2010). Indeed, ALG-3, ALG-4, and ERGO-1 all associate with 26G-RNAs with ~17, 32, and 15% of reads corresponding to 26G-RNAs respectively (Figure 2A and B). The endo-siRNAs in ALG-3 and ALG-4 IPs are primarily antisense to protein-coding genes (~22%, 2561 enriched and ~39%, 2848 enriched, respectively) and pseudogenes (~1%, 66 enriched and 1.2%, 97 enriched, respectively), while ERGO-1 targets primarily protein-coding genes (~31%, 411 enriched), pseudogenes (~5%, 39 enriched), and lincRNAs (~3%, 30 enriched) (Figure 2A and B). PRG-1, the only PIWI homolog in C. elegans, PRG-1, is known to associate with, and be required for piRNA stability (Wang and Reinke, 2008; Batista et al., 2008; Das et al., 2008). It is the major piRNA-associated AGO, and ~64% of the reads in PRG-1 IPs correspond to piRNAs (5932 enriched) (Figure 2A and B). We also detected 22G-RNAs (~21%) that primarily targeted protein-coding genes enriched in PRG-1 complexes (150 gene targets enriched). Previous studies predicted that all WAGOs would associate with 22G-RNAs (Guang et al., 2008; Gu et al., 2009; Buckley et al., 2012; Xu et al., 2018), and we observed that 22G-RNAs are the most abundant class of small RNAs associated with the WAGOs, many of which are antisense to protein-coding genes, ranging from ~26% of reads in SAGO-1 IPs to ~93% of reads in CSR-1 IPs. Groups of WAGOs associate more prominently with sRNAs that target specific genomic features including pseudogenes, lincRNAs, and repetitive and transposable elements. Pseudogenes are primarily targeted by HRDE-1 (~19% of reads in the IP are antisense to these elements), NRDE-3 (~17%), WAGO-1 (~12%), PPW-1 (~12%), SAGO-2 (~11%), SAGO-1 (~11%), and PPW-2 (~10%) (Figure 2A and B). lincRNAs are primarily targeted by NRDE-3 (~43% of reads in the IP are antisense to these elements), SAGO-1 (~35%), C04F12.1/VSRA-1 (~30%), SAGO-2 (~7%), and PPW-1 (~6%) (Figure 2A and B). sRNAs antisense to repetitive and transposable elements are most abundant in PPW-2 (~19% of reads in the IP are antisense to these elements), WAGO-1 (~18%), HRDE-1 (~15%), and PPW-1 (~14%) complexes (Figure 2A and B). miRNAs miRNAs associate with ALG-1, ALG-2, ALG-5, and RDE-1 A total of 437 miRNAs are annotated by mirBase 22.1 (encompassing 253 families with individual 5p or 3p strands). We detected reads for 402 miRNAs across our AGO Input and IP samples. Of these, 190 were found to be enriched over twofold in the AGO IPs. We observed miRNAs in association with the known miRNA binding AGOs, ALG-1, ALG-2, ALG-5, and RDE-1, and in association with the 26G-RNA binding AGOs ALG-3, ALG-4, and ERGO-1 (Figure 3A). Of the miRNAs enriched in the AGO IPs, some were enriched in association with only one AGO while others were enriched in multiple AGOs (Figure 3A). For example, 103 miRNAs were enriched in RDE-1 complexes, with 55 being exclusive to RDE-1. RDE-1 was the only AGO where the majority (63/103) of enriched miRNAs were not conserved with those of the related nematode, Caenorhabditis briggsae, suggesting that newly evolved miRNAs may be routed initially into the RDE-1 pathway before subsequently integrating into the ALG-1/2/5 pathway (Figure 3L). Figure 3 Download asset Open asset 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 designated in burgundy on the left, and by whether they are the canonical guide strand, as designated in green on the left. (B) The number of mismatches in the precursor miRNA sequences for which a mature miRNA was enriched in the indicated AGOs. Each black dot represents a single precursor miRNA duplex. The red dot and lines indicate the average and standard deviation. (C) Fold change of miRNAs enriched in AGO IPs in the corresponding ago mutant. (D) Fold change of all detected miRNAs in ago mutants compared to wild-type. (E) Western blots of co-IP experiments of ERGO-1 and ALG-1 and ALG-2. GFP::3xFLAG::ERGO-1 was crossed to HA::ALG-1 or HA::ALG-2 strains and IPed using anti-GFP antibodies. (F) As in (E) but GFP::3xFLAG::ERGO-1 IPs were conducted either with anti-GFP or anti-FLAG antibodies with or without RNase treatment. Asterisks indicate IgG. (G) Scatter plots showing enrichment of miRNAs in IP and IP +RNase-treated samples of GFP::3xFLAG::ERGO-1. The top graph shows the results of an anti-GFP IP and the bottom graph shows the results of an anti-FLAG IP (one replicate per condition). (H) Bar plots showing quantification of sRNA types in IP and IP +RNase-treated samples of GFP::3xFLAG::ERGO-1 (anti-GFP IP on the left; anti-FLAP IP on the right). (I) Survival of worms of the indicated genotype beyond the L4 stage (bottom). N ≧ 100. (J) An example of a novel miRNA within an intron of the gene tat-1 as determined by mirDeep2 analysis of ALG-1 IPs. (K) Analysis of the levels of predicted novel miRNAs in wild-type, alg-1 and alg-2 mutants. Predicted novel miRNAs are provisionally named. Error bars represent standard deviation. (L) A summary of miRNA pathway observations. 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 Download elife-83853-fig3-data1-v2.zip Adjusting the number and position of mismatches in the precursor miRNA (pre-miRNA) duplex of a transgenic let-7 miRNA has been shown to shift the balance between ALG-1 and RDE-1 loading of the transgenic let-7. These experiments demonstrated that ALG-1 preferentially associates with miRNAs from mismatched precursors while RDE-1 prefers perfectly matching precursors (Steiner et al., 2007). Therefore, we examined the number of mismatches in precursor miRNA duplexes that are enriched in ALG-1, ALG-2, ALG-5, and RDE-1 complexes (Figure 3B). miRNAs loaded into RDE-1 showed a lower average of mismatches in their precursors, and miRNAs derived from precursors with no mismatches were only bound by RDE-1, although some miRNAs derived from mismatched precursors were also loaded into RDE-1. These data suggest that endogenous miRNAs with higher complementarity in their precursor duplex are preferentially loaded into RDE-1. Given that ALG-1 and ALG-2 are thought to be required for the stability of miRNAs (Brown et al., 2017), and sRNAs are generally unstable in the absence of their AGO binding partner, we asked whether these AGOs are required for the stability of their enriched miRNAs or for miRNAs in general, by sequencing sRNAs from ago mutants and wild-type worms. We found that the AGO-enriched miRNAs were substantially depleted in alg-1, alg-2, alg-5, and rde-1 mutants (Figure 3C), and five PRG-1-enriched ‘miRNAs’ were over 60 times lower in abundance in prg-1 mutants (see below). Loss of ALG-1 had the most substantial effect on global miRNA levels, leading to a greater than twofold decrease (Figure 3D), indicating that alg-1 is genetically required for the stability of most miRNAs and potentially explaining why alg-1 mutants have more severe phenotypes than the other miRNA-associated ago mutants (Bukhari et al., 2012; Brown et al., 2017). Loss of RDE-1 also led to a substantial depletion of miRNAs overall, while loss of ALG-2 or ALG-5 did not result in major changes, which could reflect redundancy or differences in function for these AGOs. Loss of several WAGOs also led to a decrease in global miRNA levels, and we speculate that this is due to indirect effects on protein-coding gene regulation, rather than a direct influence on the miRNA pathway. miRNA and ERGO-1 26G-RNA pathways intersect To understand the association of ALG-3, ALG-4, and ERGO-1 with both 26G-RNAs and miRNAs, we tested whether the GFP::3xFLAG tag interfered with proper sRNA loading, using existing ERGO-1 IP-sRNA sequencing data performed with an ERGO-1-specific antibody (Vasale et al., 2010). In this study, the authors detected a subset of ERGO-1-associated miRNAs, but dismissed this as a nonspecific interaction. We reanalyzed these data using our custom computational pipeline and identified 26 miRNAs that were enriched twoold over input and significantly overlapped with our IP data set, suggesting that miRNA association with ERGO-1 is not a result of the GFP::3xFLAG tag, but that miRNA enrichment may be a property of ERGO-1 IPs. We examined our AGO expression and localization data, and observed that ERGO-1 expression closely overlaps with ALG-1 and ALG-2, indicating that these AGOs could physically interact in vivo (Figures 6A and F and 7A, Figure 7—figure supplements 1–8). To determine whether ERGO-1 physically interacts with one or more of the miRNA-binding AGOs, we crossed GFP::3xFLAG::ERGO-1 worms to HA::ALG-1 and HA::ALG-2 tagged strains (Brown et al., 2017) and performed co-IP experiments. We found that ERGO-1 physically interacted with both ALG-1 and ALG-2 (Figure 3E) in an RNA-dependent manner (Figure 3F). These data suggest that the ERGO-1/ALG-1 or -2 interactions are due to AGO associations on shared target transcripts. Further supporting this model, we sequenced sRNAs associated with ERGO-1 IPs after RNase treatment and observed that miRNAs were substantially reduced, while 26G-RNAs remained enriched, compared to non-RNase treated ERGO-1 IPs (Figure 3G and H). Collectively, these data suggest that the miRNA enrichment present in the ERGO-1 IPs may be indirect due to an interaction between ERGO-1 and ALG-1 or ALG-2 on target transcripts. They also imply that co-regulation of target transcripts by 26G-RNAs and miRNAs could occur. Finally, these observations highlight an important consideration for interpreting IP/sRNA sequencing data: the association of multiple AGOs on common transcripts could result in skewed sRNA enrichment patterns, thus additional experiments such as those described above and examination of ago mutant sRNA sequencing data are warranted. To explore the functional and developmental consequences of physical interaction between ERGO-1 and ALG-1, we created alg-1; ergo-1 double mutants. Loss of alg-1 results in heterochronic phenotypes; however, loss of ergo-1 has no obvious phenotypic impact, aside from enhanced ability to perform exogenous RNAi (Eri phenotype). alg-1; ergo-1 double mutants appeared sickly, being smaller and more pale than wild-type animals, with many dying prematurely, and only ~4% of the animals surviving past the L4 stage, compared to ~45% and 100% for alg-1 and ergo-1 single mutants, respectively (Figure 3I). These data indicate that alg-1 and ergo-1 genetically interact to ensure survival into adulthood, and are consistent with the idea that coordinated regulation of targets by these AGOs is required for development. Novel miRNAs associated with ALG-1/2 The sequencing depth of our IP experiments allowed us to identify novel and lowly expressed miRNAs that may have previously eluded detection, been mis-annotated or otherwise not appreciated as bona fide miRNAs because they are not known to associate with a classical (miRNA) AGO. We used the miRNA prediction program mirDeep2 to analyze sequencing data from the miRNA binding AGO IPs (Friedländer et al., 2008). We" @default.
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• W4365794537 date "2023-02-13" @default.
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• W4365794537 title "Author response: A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions" @default.
• W4365794537 doi "https://doi.org/10.7554/elife.83853.sa2" @default.
• W4365794537 hasPublicationYear "2023" @default.
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