Dual roles of EGO-1 and RRF-1 in regulating germline exo-RNAi efficiency in Caenorhabditis elegans

  1. Katsufumi Dejima
  2. Keita Yoshida
  3. Shohei Mitani

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Abstract

RNA interference (RNAi) is widely used in life science research and is critical for diverse biological processes, such as germline development and antiviral defense. In Caenorhabditis elegans , RNA-dependent RNA polymerases, with redundant involvement of EGO-1 and RRF-1, facilitate small RNA amplification in germline exogenous RNAi (exo-RNAi). However, their coordination during the regulation of exo-RNAi processes in the germline remains unclear. Here, we examined non-null mutants of the ego-1 gene and found that ego-1(S1198L) animals exhibited germline exo-RNAi defects with normal fertility, abnormalities in germ granules, and synthetic temperature-dependent sterility with rrf-1 . The exo-RNAi defects in ego-1(S1198L) were partially restored by inhibiting hrde-1 , cde-1 , and znfx-1 . Similar defects were observed in wild-type and ego-1(S1198L) heterozygous descendants derived from ego-1(S1198L) , but these were suppressed by ancestral inhibition of rrf-1 . These data reveal a dual role for EGO-1 in the positive regulation of germline exo-RNAi: it not only mediates target silencing through its RNA-dependent RNA polymerase activity, but also fine-tunes germ granule function or downstream processes, which are antagonized by RRF-1.

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    Reviewer #1

    A systemic analysis of the influence of these ego-1 alleles on fertility can provide valuable information on further studies on EGO-1's functions in fertility.

    We thank the reviewer for this insightful comment. We scored the brood size of all strains carrying a missense mutation at the ego-1 locus and added an extended figure showing their brood sizes as Fig. EV1A. Although the strain carrying gk721963, which was outcrossed six times with tmC18, showed a slightly reduced brood size, other strains showed no significant change in brood size compared to wild-type animals. The original strain carrying gk721963 has 24 homozygous mutations on chromosome I, where ego-1 is located. Of these, 15 mutations are in the region covered by tmC18, and 9 alleles are not covered. These background mutations may not be unremoved and affect fertility in concert with the ego-1 mutation. However, we believe that identifying the cause of this slight phenotype is very difficult and not essential to the overall analysis, so we have only presented the scored data for future studies on EGO-1's functions.

    The genotype of JMC231 is hrde-1(tor125[GFP::3xFLAG::hrde-1]) III. In line 245 and 551, HRDE-1::GFP is typed. typo?

    Thank you for pointing this out. We have corrected these for consistency.

    1. In Figure 4C, the fluorescence intensity in ego-1(S1198L) appears to be more than twice as high as the wild type animals, yet the mean intensity shows only mildly upregulated in Figure 4D. Is the images representative?

    Thank you for your comment. We agree that the fluorescence intensity in the original wild-type image may not have been representative. To address this concern, we have replaced the wild-type image in Fig. 3C (4C in the previous version) with an image that is more reflective of the average fluorescence intensity observed across the biological replicates.

    1. A brief introduction of tmC18 in the legend of Figure 6 would be friendly to readers.

    Thank you for your suggestion. We have added statements explaining tmC18 to the legend of Fig. 5 (Fig. 6 in the previous version) for clarity and to make the experiments more understandable.

    1. In the discussion section, a detailed summary of three recent published papers about the "phenotypic hangover" phenotype would help to understand how EGO-1 contribute to feeding RNAi. (Dodson & Kennedy, 2019; Lev et al., 2019; Ouyang et al., 2019).

    Thank you for the suggestion. We have incorporated a detailed summary of the "phenotypic hangover" phenotype in the discussion section.

    1. Has the authors examined the cellular localization of EGO-1(S1198L) ? Construction of gfp::ego-1(S1198L) animals would provide this information.

    We thank the reviewer for this insightful comment. We have generated the GFP::EGO-1(S1198L) strain and analyzed its subcellular localization and dynamics. These analysis revealed no abnormality in the expression, localization and dynamics of GFP::EGO-1(S1198L) compared to the wild type. The data are shown in Fig. EV3, and a section of the description about this is added to the third section of the Results.

    Reviewer #2

    Key conclusions are convincing, but data and stats need to be clarified in some cases (see below).

    Line 202-211: The found that znfx-1(-) partially restored sensitivity of S1198L mutants to pos-1 RNAi but did not significantly restore pop-1 RNAi. Later, section 228-243, they provide evidence that cde-1 and hrde-1 mutations partially restore sensitivity to pos-1, but not pop-1, RNAi. The authors should discuss what might be going on here.

    Thank you for your comment. We have added a discussion on the differential restoration of sensitivity to pos-1 and pop-1 RNAi in the presence of znfx-1, cde-1, and hrde-1 mutations, proposing that this variation may result from differences in the RNA metabolism of these target genes (Knudsen-Palmer et al., 2024). Additionally, we incorporated the results from the additional RNAi experiments targeting gld-1 and mpk-1 (as outlined in our response to Reviewer 3, Comment 3), which further support our proposed model. We hope this revision presents a more thorough analysis of the interplay between these mutations and RNAi sensitivity.

    Lines 276-279: Confusing as written. The authors do not show RNAi assays for germline genes with rrf-1(null) ego-1(S1198L) double mutants. They should show these data.

    Thank you for the feedback. We have added the RNAi assay data for germline genes with rrf-1(null) ego-1(S1198L) double mutants in Figure EV3C and D.

    For the wording, I suggest "RRF-1 compensates for partial loss of EGO-1 activity in S1198L with respect to 25{degree sign}C brood size (Fig. #), but not for germline exo-RNAi (Fig. #). Therefore, the defects..."

    Thank you for the suggestion. We have revised the wording as recommended.

    Minor comments Throughout, figure legends shown indicate the statistical test used, and the p value must be indicated (e.g., *** indicates p-value of #).

    The authors should use consistent nomenclature for the ego-1 null allele. In Fig. 5 it's listed as "" and elsewhere as tm521.

    Thank you for pointing this out. We corrected this in the revised manuscript.

    Line 90: Please include references for the ego-1 null germline phenotype.

    Thank you for your suggestion. We included two references demonstrating the ego-1 null germline phenotype in the revised manuscript.

    Line 107-109: Wording is confusing. I suggest "Disruption of the E granule, of which EGO-1 is a component, has recently been shown to upregulate sRNA targeting ..."

    Thank you for the suggestion. We have revised the wording as suggested.

    Line 118-120: Wording is unclear. I suggest "In addition we found that sid-1 and rde-11 transcripts in ego-1(S1198L) were downregulated, and this effect was suppressed in hrde-1, cde-1, and znfx-1 mutants."

    Thank you for the suggestion. We have revised the wording as suggested.

    Line 121-123: The meaning is unclear. Please clarify what "detached" means in this context.

    Thank you for the comment. We have revised the sentence to remove the term "detached" for clarity and have instead explicitly described the phenomenon, stating that the RNAi-defective (Rde) phenotype persists over generations in an RRF-1-dependent manner, even in the absence of the original ego-1(S1198L) mutation.

    Line 171-172: Substitute "in the genome" for "in terms of its genomic locus"

    Thank you for the suggestion. We have revised the wording as suggested.

    Line 207: Substitute "the pos-1 RNAi defect" for "the Rde phenotype of pos-1 RNAi"

    Thank you for pointing this out. We have revised the text as suggested.

    Line 269: Text says Fig 5A,B, shows restoration to "wt levels," but stats only show significant change from ego-1(S1198L). Stats showing comparison with wt should be shown, as well.

    Thank you for the comment. We have revised the text to clarify the expression levels and removed the statement about "restoration to wild-type levels" where statistical comparisons were not provided.

    The text refers to the wrong figure/panel in some places. Line310 references Fig. 6A-C as showing the phenotype of ego-1(+/-) heterozygotes and ego-1(+/+) homozygotes, but only the latter is shown in 6A-C. Heterozygotes are shown in Fig. 6D-F.

    Thank you for pointing this out. We have revised the statement accordingly.

    Line 350 should reference Fig. 7C, D (not Fig 3A).

    Thank you for your suggestion. We have corrected it to Fig. 6C, D (Fig. 7C, D in the previous version) as suggested.

    Line 380-381: Wording is awkward. I suggest "Additionally, this allele showed synthetic ts sterility with an rrf-1 deletion mutation."

    Thank you for pointing this out. We have revised the text as suggested.

    Figure 8: There is a typo in panel C: the allele shown is ego-1(null) not ego-1(S1198).

    Thank you for pointing this out. We have updated the allele to ego-1(null) in panel C.

    Reviewer #3

    1. The authors link the direct gene-silencing function of EGO-1 with temperature-sensitive sterility (Figure 8). However, the data in Figure 1 show that the RNAi resistance phenotype and ts-sterility are anti-correlated, the most RNAi-resistant ego-1 alleles are least ts-sensitive and vice versa. Therefore, motivating further experiments through the connection between exo-RNAi resistance and ts-sterility is not justified, e.g. "the temperature sensitive sterile phenotype is a hallmark of the mutator complex.... which is necessary for exo-RNAi-driven silencing". Also, the claim of the redundancy between ego-1 and rrf-1 in controlling ts-sterility is not justified. The ego-1(V1128E) and (C823Y) alleles show strong ts-sterility (Figure 1E), which is not compensated by RRF-1. Therefore, the specific nature of ego-1(S1198L) and (R539Q) mutations leads to a higher dependence of endogenous RNAi silencing processes on RRF-1. Remarkably, although the exo-RNAi resistance of these alleles is dominant (Figure EV2 A,B) and clearly distinct from ego-1 null heterozygous animals, the ts-sterility of ego-1 null heterozygouts and S1198L or R539Q heterozygouts is identical (Figure EV C).

    We thank the reviewer for the insightful comments. We have revised the second section of the Results to simplify the argument by removing descriptions related to WAGO 22G RNA and fertility. This revision ensures that our conclusions remain focused and directly address the observed genetic interactions. Additionally, we have expanded the Discussion to further clarify the specific nature of ego-1(S1198L) with respect to RRF-1.

    1. The experiments in Figures 6 and Figure 7C,D are the most important findings of this study, showing that EGO-1 has a role in the licensing of genes important for exo-RNAi in the germline (such as sid-1 and rde-11). The apparent persistence of RRF-1-dependent (and presumably HDRE-1-dependent) silencing of sid-1 and rde-11 in a genetically wild-type background that correlates with exo-RNAi resistance is remarkable, although not novel (it was shown for mutants defective in P-granules). The use of ego-1 missense viable background was instrumental in these experiments. However, it is not clear whether the specific nature of ego-1(S1198L) mutation also played a role, such as enhanced production of RRF-1-dependent endogenous silencing small RNAs. The ego-1(V1128E) allele is an apparent hypomorph, which is viable and exo-RNAi-resistant (Figure 1, EV2A). Performing an experiment shown in Figure 6 with this allele for five generations would be highly illuminating, and either outcome would be interesting.

    Thank you for this insightful comment. We agree that investigating whether the specific nature of the ego-1(S1198L) mutation contributes to the observed effects is essential. To address this, we performed the experiment shown in Figure 6 using the ego-1(V1128E) allele four generations and data is now shown in Fig. EV7.

    1. Conclusions from the experiments in Figures 3 and 4 are not convincing. The imaging data can be moved to supplemental materials. The suppression experiments shown in Figure 4A,B are weak. The effects of cde-1 mutation are hard to interpret, and these data can be omitted. The znfx-1 and hrde-1 loss does not affect resistance to pop-1. If the authors want to insist on their model, they should use several additional exo-RNAi target genes producing Emb (or other) phenotypes and repeat the experiments.

    Thank you for your valuable feedback. We agree with the concerns raised and have made the suggested changes, including moving the imaging data to Fig. EV4 and omitting the cde-1 data. Regarding the lack of suppression effects for pop-1, we acknowledge the need for further investigation and have performed additional exo-RNAi experiments with target genes gld-1 (Ste) and mpk-1 (Ste) to evaluate our model. Both znfx-1 and hrde-1 mutants significantly suppressed the Rde phenotype in ego-1(S1198L) when subjected to these RNAi, supporting our model. We have added these data in Fig. 3B and EV5A and moved the pop-1 RNAi data to Fig. EV5B.

    1. The exo-RNAi resistance and reduced sid-1 and rde-11 expression correlate. The reduction of these exo-RNAi factors is a plausible explanation for the epigenetic RNAi resistance shown in Figure 6. However, ego-1(S1198L); hrde-1(-) P0 is resistant to pop-1(RNAi) to a large extent (Figure 4B), while sid-1 and rde-11 expression is restored in this double compared to single ego-1(S1198L) (Figure 5B). Therefore, ego-1(S1198L) exo-RNAi resistance is not likely driven to any extent by the misregulation of other RNAi genes. The nature of the (S1198L) mutation is likely to play a major role. Also, surprisingly, rrf-1(-) addition to ego-1(S1198L) does not restore sid-1 and rde-11 expression. Why? The authors do not comment on this.

    Thank you for your detailed comment. To address your concerns, we will incorporate additional experimental data outlined in our response to Comment 3 and revised our description accordingly. Regarding the observation that rrf-1(-) addition to ego-1(S1198L) does not restore sid-1 and rde-11 expression, we hypothesize that this may result from the process by which the rrf-1 knockout was generated via CRISPR in an ego-1(S1198L) mutant background, where sid-1 and rde-11 expression was already reduced. This suggests that rrf-1 may not be required to maintain the reduced expression state once it is established. We will include these points in the revised manuscript.

    1. The discussion points about the nature of new EGO-1 missense mutations involving Alpha Fold predictions can be illustrated through Alpha Fold model figures.

    Thank you for your comment. We agree that illustrating the discussion points with Alpha Fold model figures would enhance clarity. We included an extended view figure based on Alpha Fold predictions to better visualize the structural implications of the EGO-1 mutations.

    1. The authors should consider a model where ego-1(S1198L) affects RRF-1 activity such that it is more active in the endogenous RNAi silencing processes at the expense of exo-RNAi. This could explain the reduced ts-sterility in ego-1(S1198L), which is RRF-1-dependent, similar to the better-investigated epigenetic inheritance of exo-RNAi resistance. However, the exact mechanism of ego-1(S1198L) cannot be explained by genetic methods and is beyond the scope of this study.

    Thank you for this insightful and critical comment. We agree that the interaction between ego-1(S1198L) and RRF-1 activity is an important aspect to consider. Based on the results from our additional experiments described above, we discussed about this possibility. We deeply appreciate your suggestion, as it provides valuable direction for interpreting our findings and developing a more comprehensive understanding of the mechanism.

    Minor comments:

    • Figure 8C typo: ego-(0) is meant to be shown.

    Thank you for pointing this out. We have updated the allele to ego-1(null) in panel C.

    • Pak and Fire, Science, 2007 should be cited in connection to secondary siRNA production. Ruby and Bartel, Cell, 2006 should be cited as the first study that identified 21U-RNAs.

    Thank you for pointing this out. We added citations to Pak and Fire (Science, 2007) in connection to secondary siRNA production and to Ruby and Bartel (Cell, 2006) as the first study identifying 21U-RNAs.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Mitani and colleagues' manuscript investigates the role of RNA-dependent RNA polymerase (RdRP) EGO-1 in regulating exogenous RNAi (induced by dsRNA delivery) efficiency in the germline of C. elegans. Since the null ego-1 mutation leads to sterility, the authors take advantage of several missense ego-1 mutant strains that are fertile but RNAi-resistant.

    Major comments:

    The authors recognize at least two distinct mechanisms of EGO-1 function in regulating exo-RNAi. The first is direct, since EGO-1 RdRP is required for the production of secondary small RNAs mediating exo-RNAi silencing (this mechanism has been studied for many years), and the second one is indirect, through the role of EGO-1 RdRP in the production of endogenous "licensing" small RNAs that allow germline gene expression, including expression of genes required for exo-RNAi response. In addition, the authors find that the chosen missense mutant strains show a dominant exo-RNAi resistance phenotype, unlike the recessive ego-1 null.

    Although the authors recognize the complex nature of ego-1 phenotypes and provide a helpful model in Figure 8, I find that not all conclusions are consistent with the presented data. A more rigorous data interpretation and presentation logic is required for publication. Also, some additional simple experiments can be done to enhance the rigor of conclusions.

    1. The authors link the direct gene-silencing function of EGO-1 with temperature-sensitive sterility (Figure 8). However, the data in Figure 1 show that the RNAi resistance phenotype and ts-sterility are anti-correlated, the most RNAi-resistant ego-1 alleles are least ts-sensitive and vice versa. Therefore, motivating further experiments through the connection between exo-RNAi resistance and ts-sterility is not justified, e.g. "the temperature sensitive sterile phenotype is a hallmark of the mutator complex.... which is necessary for exo-RNAi-driven silencing". Also, the claim of the redundancy between ego-1 and rrf-1 in controlling ts-sterility is not justified. The ego-1(V1128E) and (C823Y) alleles show strong ts-sterility (Figure 1E), which is not compensated by RRF-1. Therefore, the specific nature of ego-1(S1198L) and (R539Q) mutations leads to a higher dependence of endogenous RNAi silencing processes on RRF-1. Remarkably, although the exo-RNAi resistance of these alleles is dominant (Figure EV2 A,B) and clearly distinct from ego-1 null heterozygous animals, the ts-sterility of ego-1 null heterozygouts and S1198L or R539Q heterozygouts is identical (Figure EV C).
    2. The experiments in Figures 6 and Figure 7C,D are the most important findings of this study, showing that EGO-1 has a role in the licensing of genes important for exo-RNAi in the germline (such as sid-1 and rde-11). The apparent persistence of RRF-1-dependent (and presumably HDRE-1-dependent) silencing of sid-1 and rde-11 in a genetically wild-type background that correlates with exo-RNAi resistance is remarkable, although not novel (it was shown for mutants defective in P-granules). The use of ego-1 missense viable background was instrumental in these experiments. However, it is not clear whether the specific nature of ego-1(S1198L) mutation also played a role, such as enhanced production of RRF-1-dependent endogenous silencing small RNAs. The ego-1(V1128E) allele is an apparent hypomorph, which is viable and exo-RNAi-resistant (Figure 1, EV2A). Performing an experiment shown in Figure 6 with this allele for five generations would be highly illuminating, and either outcome would be interesting.
    3. Conclusions from the experiments in Figures 3 and 4 are not convincing. The imaging data can be moved to supplemental materials. The suppression experiments shown in Figure 4A,B are weak. The effects of cde-1 mutation are hard to interpret, and these data can be omitted. The znfx-1 and hrde-1 loss does not affect resistance to pop-1. If the authors want to insist on their model, they should use several additional exo-RNAi target genes producing Emb (or other) phenotypes and repeat the experiments.
    4. The exo-RNAi resistance and reduced sid-1 and rde-11 expression correlate. The reduction of these exo-RNAi factors is a plausible explanation for the epigenetic RNAi resistance shown in Figure 6. However, ego-1(S1198L); hrde-1(-) P0 is resistant to pop-1(RNAi) to a large extent (Figure 4B), while sid-1 and rde-11 expression is restored in this double compared to single ego-1(S1198L) (Figure 5B). Therefore, ego-1(S1198L) exo-RNAi resistance is not likely driven to any extent by the misregulation of other RNAi genes. The nature of the (S1198L) mutation is likely to play a major role. Also, surprisingly, rrf-1(-) addition to ego-1(S1198L) does not restore sid-1 and rde-11 expression. Why? The authors do not comment on this.
    5. The discussion points about the nature of new EGO-1 missense mutations involving Alpha Fold predictions can be illustrated through Alpha Fold model figures.
    6. The authors should consider a model where ego-1(S1198L) affects RRF-1 activity such that it is more active in the endogenous RNAi silencing processes at the expense of exo-RNAi. This could explain the reduced ts-sterility in ego-1(S1198L), which is RRF-1-dependent, similar to the better-investigated epigenetic inheritance of exo-RNAi resistance. However, the exact mechanism of ego-1(S1198L) cannot be explained by genetic methods and is beyond the scope of this study.
    • Data and the methods are presented in such a way that they can be reproduced.
    • Statistical analyses are adequate.

    Minor comments:

    • Figure 8C typo: ego-(0) is meant to be shown.
    • Pak and Fire, Science, 2007 should be cited in connection to secondary siRNA production. Ruby and Bartel, Cell, 2006 should be cited as the first study that identified 21U-RNAs.

    Significance

    General assessment:

    The strength of this study is in generating reagents suitable for performing experiments that were not feasible with the sterile null mutant. The major finding of the paper is the epigenetic inheritance of resistance to exo-RNAi by the wild-type descendants of ego-1 mutants, which is dependent on rrf-1. There are numerous weaknesses in the interpretation of other data, which are described in section 1. The study's limitation is the exclusive use of genetic approaches. The effect of the antimorphic point mutations on EGO-1 stability, localization, and interaction with other proteins could have provided more insight into the protein's function.

    • The most notable results presented in the paper are very similar to the findings of several groups published in 2019 (Lev et al., Ouyang et al, and Dodson and Kennedy) and, therefore, are not novel. The experimental setup is identical to Dodson and Kennedy; it just uses different mutants. The novel aspect is the opposite relationship between ego-1 and rrf-1, which has not been described before.
    • This research will be of interest to C. elegans researchers and those following epigenetic phenomena.
    • My expertise is in RNAi in C. elegans and epigenetics. I have sufficient expertise to evaluate all aspects of the paper.
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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    EGO-1 is a C. elegans RNA-directed RNA polymerase well known to amplify small-interfering (si) RNA in the germline and to be required for germline development. The authors screened several partial loss-of-function mutations in ego-1, identified in the million mutation project collection, and identified one that does not reduce brood size yet is RNAi defective (Rde). Null and most other ego-1 mutations are completely sterile and strongly Rde. The newly identified allele, which the authors call S1198L, does not disrupt fertility at moderate culture temperatures yet severely disrupts RNAi, indicating that sterility is separable from the Rde phenotype. S1198L mutants do have reduced fertility at elevated culture temperature; this phenotype is enhanced by a rrf-1 null mutation, suggesting these two RdRPs are redundantly required for fertility under conditions of temperature stress. Using S1198L, they explore the relationship between EGO-1 and expression or function of other components and regulators of the small RNA machinery as well as components of germ granules (RRF-1, HRDE-1, PGL-1, CDE-1/PUP-1, ZNFX-1). One very interesting characteristic of ego-1(S1198L) is that it has a dominant RNAi defect, unlike null alleles; therefore, the EGO-1(S1198L) protein may interfere with EGO-1 wt activity. It seems likely that this allele will be useful for exploring additional aspects of EGO-1 activity beyond those included in this report.

    Major comments

    Key conclusions are convincing, but data and stats need to be clarified in some cases (see below).

    Line 202-211: The found that znfx-1(-) partially restored sensitivity of S1198L mutants to pos-1 RNAi but did not significantly restore pop-1 RNAi. Later, section 228-243, they provide evidence that cde-1 and hrde-1 mutations partially restore sensitivity to pos-1, but not pop-1, RNAi. The authors should discuss what might be going on here.

    Lines 276-279: Confusing as written. The authors do not show RNAi assays for germline genes with rrf-1(null) ego-1(S1198L) double mutants. They should show these data. For the wording, I suggest "RRF-1 compensates for partial loss of EGO-1 activity in S1198L with respect to 25{degree sign}C brood size (Fig. #), but not for germline exo-RNAi (Fig. #). Therefore, the defects..."

    Minor comments

    Throughout, figure legends shown indicate the statistical test used, and the p value must be indicated (e.g., *** indicates p-value of #).

    The authors should use consistent nomenclature for the ego-1 null allele. In Fig. 5 it's listed as "" and elsewhere as tm521.

    Line 90: Please include references for the ego-1 null germline phenotype.

    Line 107-109: Wording is confusing. I suggest "Disruption of the E granule, of which EGO-1 is a component, has recently been shown to upregulate sRNA targeting ..."

    Line 118-120: Wording is unclear. I suggest "In addition we found that sid-1 and rde-11 transcripts in ego-1(S1198L) were downregulated, and this effect was suppressed in hrde-1, cde-1, and znfx-1 mutants."

    Line 121-123: The meaning is unclear. Please clarify what "detached" means in this context.

    Line 171-172: Substitute "in the genome" for "in terms of its genomic locus"

    Line 207: Substitute "the pos-1 RNAi defect" for "the Rde phenotype of pos-1 RNAi"

    Line 269: Text says Fig 5A,B, shows restoration to "wt levels," but stats only show significant change from ego-1(S1198L). Stats showing comparison with wt should be shown, as well.

    The text refers to the wrong figure/panel in some places.
    Line310 references Fig. 6A-C as showing the phenotype of ego-1(+/-) heterozygotes and ego-1(+/+) homozygotes, but only the latter is shown in 6A-C. Heterozygotes are shown in Fig. 6D-F.
    Line 350 should reference Fig. 7C, D (not Fig 3A).

    Line 380-381: Wording is awkward. I suggest "Additionally, this allele showed synthetic ts sterility with an rrf-1 deletion mutation."

    Figure 8: There is a typo in panel C: the allele shown is ego-1(null) not ego-1(S1198).

    Significance

    The paper addresses the mechanisms and activity of small RNA-mediated pathways, including in regulating gene expression and development. The work will be general interest to the large community studying small RNA-mediate gene expression and/or germline development in C. elegans and more broadly. The work is significant because it reveals distinct requirements for EGO-1 RdRP in exo-RNAi, germline development under conditions of temperature stress, and germline development more broadly.

    I am a C. elegans biologist with many decades of experience studying germline development and RNAi-related phenomena.

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    Referee #1

    Evidence, reproducibility and clarity

    The study conducted by Katsufumi Dejima and colleagues represents an advance in understanding the multiple roles of RdRPs in C. elegans germ cells. EGO-1 is an essential RdRP that is required for multiple aspects of C. elegans germline development and efficient RNAi of germline-expressed genes. Yet, currently there is a lack of sufficient genetic mutants to differentiate the multiple biological functions of EGO-1. In this study, the authors examined a large number of non-null alleles for ego-1 gene and identified four alleles that affect exogenous RNAi, while does not compromise fertility. The authors then focused on the allele ego-1(S1198L), examined its influence on germ granule compartments and investigated the molecular mechanism of EGO-1's involvement in feeding RNA interference. Together, their work reveal an extensive interdependent RdRP network that is responsible for regulating exo-RNAi in the germline.

    Overall, this is a well-executed study that uncovers the molecular mechanism of EGO-1' function in germline RNAi response and the multiple roles of EGO-1 and RRF-1 in regulating germline RNAi. The findings are poised to have an impact on RNAi research fields.

    I have a few comments below. While they are largely minor, addressing them would further enhance the manuscript's clarity and impact.

    1. A systemic analysis of the influence of these ego-1 alleles on fertility can provide valuable information on further studies on EGO-1's functions in fertility.
    2. The genotype of JMC231 is hrde-1(tor125[GFP::3xFLAG::hrde-1]) III. In line 245 and 551, HRDE-1::GFP is typed. typo?
    3. In Figure 4C, the fluorescence intensity in ego-1(S1198L) appears to be more than twice as high as the wild type animals, yet the mean intensity shows only mildly upregulated in Figure 4D. Is the images representative?
    4. A brief introduction of tmC18 in the legend of Figure 6 would be friendly to readers.
    5. In the discussion section, a detailed summary of three recent published papers about the "phenotypic hangover" phenotype would help to understand how EGO-1 contribute to feeding RNAi. (Dodson & Kennedy, 2019; Lev et al., 2019; Ouyang et al., 2019).
    6. Has the authors examined the cellular localization of EGO-1(S1198L) ? Construction of gfp::ego-1(S1198L) animals would provide this information.

    Significance

    Strength: Enough genetic alleles to differentiate the multiple biological functions of EGO-1.

    Limitations: Whether mutant alleles affect siRNA production is unknown.

    Advance: The multiple functions of RdRp protein were analyzed through genetic means.

    Audience: Basic research, small RNA community and C. elegans community

    My expertise: small RNA and germ granule.

  5. Version published to 10.1101/2024.09.24.614643 on bioRxiv