A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila

In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Here, using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, we show that Su(Ste) piRNAs are made in the early male germline via 5′-to-3′ phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.

biogenesis (Rev#1 points copied below; Rev#2 point #3, see also points #4, #5), and clarifying the mechanism experimentally should be a goal of the revision: Rev#1: "-how do the authors explain the requirement for Ago3 in the process?Ago3 is not a recipient of phased piRNAs.Does this mean that the amplification of the hoppel trigger piRNA through pingpong during embryonic/larval development is a requirement for the efficient triggering of the Su(Ste) transcript?Adding a comment here would be great.
-does the silencing of Stellate by Su(Ste) piRNAs generate Ago3-bound responder piRNAs from the Stellate transcripts in testes?" 2-All other reviewer comments about strengthening existing data, technical aspects, controls (including Rev#2 points #2, #7), methodological questions, and requests for clarifications, discussion, or text edits, should also be addressed.
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Melina Casadio, PhD Senior Editor, Nature Cell Biology ORCID ID: https://orcid.org/0000-0003-2389-2243Reviewers' Comments: Reviewer #1: Remarks to the Author: In this manuscript, Venkei and colleagues demonstrate that the 5' to 3' directed phased piRNA biogenesis pathway allows utilisation of a Y-encoded transcript as piRNA biogenesis substrate through a maternally deposited transposon piRNA that cleaves the 5' end of the Y-linked transcript.This is a great paper that combines clever genetics with high end quantitative molecular biology.I support publication after incorporation of the following comments: -title: I am not sure whether this is the most intuitive title for the broad readership of this journal.Also: utilisation of a Y-linked piRNA is not really the point as it is utilisation of a Y-linked piRNA precursor, no? -The authors write: "We detected expression of Su(Ste) piRNA precursor transcripts only from the genomic strand that produces transcripts antisense to Ste mRNAs (not shown)." Please add the original data here.
-The authors write: "smRNA-FISH can detect Ste mRNAs and Su(Ste) precursor transcripts but not mature piRNAs, because small RNAs are not retained in formaldehyde-fixed tissues."Could the authors add a citation for this statement?How did Plasterk and colleagues detect microRNAs in zebrafish by in situ hybridisation?-The authors write: "We conclude that maternal deposition of Su(Ste) piRNAs by XXY mothers suffices to silence Ste mRNA and bypasses the requirement for phased piRNA production pathway in early male germ cells."This would mean that the maternally deposited piRNAs are stable until the onset of Stellate transcription, is this correct?Or do the authors envision that the maternally deposited piRNAs are capable of processing the Su(Ste) transcript through ping-pong?If the latter is the case, would this mean that the sense piRNAs of Stellate are essential?A clarifying comment would be helpful in the manuscript.
-how do the authors explain the requirement for Ago3 in the process?Ago3 is not a recipient of phased piRNAs.Does this mean that the amplification of the hoppel trigger piRNA through ping-pong during embryonic/larval development is a requirement for the efficient triggering of the Su(Ste) transcript?Adding a comment here would be great.
-does the silencing of Stellate by Su(Ste) piRNAs generate Ago3-bound responder piRNAs from the Stellate transcripts in testes?-Figure 1: I have a hard time to see where the f and g panels are related to the boxed areas in b and c.
-Figure 2: was the quantification of Su(Ste) quantification done in a z-stack so that each cell is properly analysed?-Figure 5: please add a length scale for the 1360/Su(Ste) transcript.
-Figure 6a: why is the small RNA size profile cut at 23nt? Please include sizes down to 20nt.
-methods: Ct values were normalized to Gapdh by the ∆∆Ct method.Please add a citation -please add molecular weight marker for western blots.
Finally, one maybe crazy idea: If one would express at high levels one or two siRNAs that are between the 1360 trigger piRNA target site and the Stellate sequence, then this might block the phasing process, tehreby leading to the inability to silence Stellate.This is not an experiment that is required for this manuscript to be published, but it might be a neat test for the model.
Reviewer #2: Remarks to the Author: piRNAs are small non-coding RNAs that recognise complementary targets, such as transposons, leading to their silencing.In Drosophila, piRNAs are maternally deposited through the oocyte.The male germline uses the piRNA pathway to repress Stellate (Ste), which, if active, leads to the production of Ste crystals and impairs male fertility.piRNAs capable of targeting Ste are produced from the Suppressor of Stellate [Su(Ste)] locus on the Y chromosome, and thus are not maternally deposited.How Su(Ste) piRNAs are generated in testes is currently not understood.This manuscript by Venkei, Gainedinov et al. sheds light on the mechanism of Su(Ste) piRNA production in male germ cells.The authors used smRNA-FISH to visualise Ste and Su(ste) transcripts throughout Drosophila testis development and found that Su(Ste) is expressed earlier than its target Ste.Through temporally controlled knockdown and rescue experiments, they illustrate an early requirement of phased biogenesis components, and a late requirement of factors of the ping-pong amplification cycle.
The Su(Ste) promoter is localised within a 1360/Hoppel transposon insertion and as 1360/Hoppel piRNAs are maternally deposited, the authors hypothesised that cleavage by these piRNAs trigger phased piRNA biogenesis of the Su(Ste) transcript.To test this, they used XXY females expressing Su(Ste) from their Y chromosome.First, these females were able to produce piRNAs targeting Su(Ste) from the Y-linked Su(Ste) locus and pass these piRNAs onto their sons.Second, through sequencing of small and long RNAs from XXY ovaries, they found Su(Ste)-derived long 5'-monophosphorylated RNAs that displayed signatures consistent with anti-1360/Hoppel piRNA-guided cleavage and processing through the phased biogenesis pathway.This work addresses the wider question on how intergenerational information is transferred through the piRNA pathway.Very little has been known about Su(Ste) piRNA production and this study contributes to understanding their biogenesis mechanism.The manuscript is well composed and easy to follow.However, several points as outlined below have to be addressed to fully support the authors' conclusions.
Major comments: 1) The ping-pong machinery has previously been reported as unlikely to drive the generation of Su(Ste) piRNAs (PMID 20980675) -this should be acknowledged.In the same study, one abundant piRNA dominated both the Aub-and Ago3-loaded piRNAs mapping to Su(Ste), is this one also detected in the present study?If so, how does it relate to the phased biogenesis?
2) Su(Ste) has been described to produce sense transcripts giving rise to dsRNA.However, the authors did not detect any sense transcription, described as "data not shown".Given its importance, I think these data should be included and further expanded by stranded RNA-seq data.Similarly, I think it would be important to show both sense and antisense piRNAs (or the absence thereof) in Figure 6.Are any siRNAs detected?
3) Considering that Piwi seems not expressed in male germ cells, how can neither Aub or Ago3 be required for phased biogenesis (Extended Data Figure 2)?I don't follow how the Su(Ste)-derived piRNAs are retained to the later developmental stages if they are not loaded onto Aub or Ago3 during their biogenesis.If Aub/Ago3 act redundantly, can this be verified through a double-KD?4) Fig 4 shows that Ste transcription is derepressed if Aub/Ago3 are knocked-down, while Su(Ste) mRNA is still processed (Extended Data Figure 2).What happens with the resulting Su(Ste) piRNAs?Are they degraded after the cleavage as they are not loaded onto Aub or Ago3, what is the end results in Ste expression?
5) The rescue experiments (e.g., Figure 4g) are elegant.Similar could be done for Ago3.It may be interesting to also perform rescues in the context of Aub/Ago3 double-KDs.
6) I find the significance of the 5' monophosphorylated RNAs hard to interpret without putting the numbers in context.The authors claim that "For Su(Ste)-derived long RNAs overlapping both the upstream transposon insertion and the sequence complementary to Ste, the 5′ ends of ~40% of these long RNAs lay between nucleotides g10 and g11 of an antisense maternal 1360/Hoppel piRNA".Is 40% more than what you would expect by chance?To know this, we need to check how many of the 1360/Hoppel positions that constitute piRNA 5' ends (threshold?).Also, is the 10-nt overlap stronger in this region compared to the downstream Su(Ste) region or other genomic regions?Is there a correlation between piRNA and cleavage product abundances?Is there a "ping-pong"-like signature?Please do present the data more exhaustive to aid interpretation.5b: Is armi involved in degrading all 5' monophosphorylated long RNAs in the testis, or is this specific to the putative pre-pre-piRNAs in the 1360/Hoppel region?Please include suitable (negative and positive) controls.8) Figure 7a: I have two major concerns with this analysis.First, I am not sure how relevant it is that Su(Ste) display phased biogenesis in ovaries.Any 5' monophosphorylated long RNA is likely to undergo phased biogenesis in the ovaries.However, we still do not know what happens in testis.Could the testis data discussed in Figure 5 be used instead?If not, I think the limitations of the current analysis should be highlighted.Second, I find it difficult to see that "5′ ends of most Su(Ste) piRNAs in XXY ovaries concentrated in periodic peaks lying ~26 nt apart".Extended Figure 6a is more helpful but could be supplemented by checking for +1U signal, the hallmark of zuc-mediated cleavage.Phasing could be further quantified using cross correlation or Fourier transform.Is the phasing signature similar across both the 1360/Hoppel and Su(Ste) regions?

7) Figure
Minor comments: 1) Other factors, such as Spn-E, have been implicated in Ste suppression and this could be cited.
2) Extended Data Figure 1 and 2 have the same title.
3) Figure 2K: A t-test is not appropriate for discrete non-negative counts.4) Row 161: "Figure 3a-c,g" should likely be "Figure 3a,c,g"?In general, the panel order could be reorganised here and elsewhere to make the figures easier to digest.

5)
Please clarify what data is shown in Figure 7. How many cleavage sites are shown?How were they defined?6) Please provide an overview of the high-throughput data generated and their key metrics.
7) The bioinformatic method description is very light and replicating some analyses (e.g., Figure 7), would be near impossible unless the authors make their scripts available or significantly increase the amount of detail.
Reviewer #3: Remarks to the Author: This study elucidates the mechanism of the initial Y-linked piRNA biogenesis in Drosophila males, concerning the Ste-Su(Ste) piRNA-mediated suppression system.The smRNA-FISH analyses revealed that the Y-linked Su(ste) non-coding gene is expressed earlier than the X-linked Ste in male germ cells.A series of genetic experiments showed that Armi and Zuc are required for processing of Su(Ste) piRNA precursor transcripts in germline stem cells and spermatogonia.Most significantly, they showed that the maternal 1360/Hoppel-derived piRNAs initiated phased biogenesis of Su(Ste) piRNAs in males.This conclusion was further substantiated by studying XXY ovaries.The data are of high quality.The genetic experiments (knockdown, genetic mutation, and transgene) are elegant and informative.The conclusions are supported by the data.This study delineated the intergenerational mechanism for the maternal piRNA trigger that leads to silencing of Ste in male germ cells.
One minor comment on the last sentence in Discussion: "We speculate that this same mechanism may be used by mothers to protect their sons from selfish DNA in other species".This statement is too general.Do the authors mean other Drosophila species?It needs to be more specific or this speculation can be deleted.

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Responses to Reviewers' Critiques
We thank the Reviewers for their useful comments, which helped us substantially improve the manuscript.Reviewers' recommendations inspired us to conduct additional experiments testing the proposed model of Su(Ste) piRNA biogenesis and function.In particular, we are grateful for the suggestion that, in addition to XXY ovaries, we examine in more detail the molecular mechanism of Su(Ste) piRNA biogenesis in testis.These new data offer additional evidence for phased processing of Su(Ste) precursors in early spermatogenesis.
Our point-by-point responses are below shown in blue text.

Editor:
In particular, it would be essential to: 1-As per Rev#2 point #1, further addressing how the data fit with past work (ref 54) suggesting that the ping-pong machinery only may not drive the generation of Su(Ste) piRNAs will be important.
Response: Data reported in Nagao et al., RNA 2010 indeed support the model proposed in our study.We revised the text to incorporate these earlier findings: e.g., the fact that most Su(Ste) piRNAs in Aub and Ago3 are derived from the antisense Su(Ste) precursors, and the requirements (1) for armi in Su(Ste) piRNA biogenesis and (2) for both Aub and Ago3 in Ste repression.
Similarly, Reviewers #1 and #2 are not clear on the requirement for Aub or Ago3 for phased biogenesis (Rev#1 points copied below; Rev#2 point #3, see also points #4, #5), and clarifying the mechanism experimentally should be a goal of the revision: Response: The Editor's and the Reviewers' comments on the role of Aub and Ago3 in the proposed model encouraged us to conduct additional analyses and experiments (e.g., new data in Fig. 4) and to revise the text to better describe the role of the two proteins in Su(Ste) piRNA biogenesis and function.
Rev#1: "how do the authors explain the requirement for Ago3 in the process?Ago3 is not a recipient of phased piRNAs.Does this mean that the amplification of the hoppel trigger piRNA through ping-pong during embryonic/larval development is a requirement for the efficient triggering of the Su(Ste) transcript?Adding a comment here would be great.2-All other reviewer comments about strengthening existing data, technical aspects, controls (including Rev#2 points #2, #7), methodological questions, and requests for clarifications, discussion, or text edits, should also be addressed.

Response:
We have added FISH data for the sense Su(Ste) precursor transcript and generated the requested stranded RNA-seq data (Rev#1 comment and Rev#2 point 2) in Extended Data Fig. 1.The controls for the analyses of 5' monophosphorylated long RNAs now appear in Extended Data Fig. 4 (Rev#2 point 7).The text was revised to include additional analyses, clarification, or references to discuss the detection of small RNAs in formaldehyde-fixed tissues, stability of small RNAs, sense and antisense piRNAs from both Ste and Su(Ste) loci and the absence of siRNA production from Su(Ste), the probability of 10-nt overlap between piRNAs and 5′ monophosphorylated long RNAs.We thank the Reviewers for pointing out ways to strengthen our analyses and clarify our interpretations.
3-Finally, please pay close attention to our guidelines on statistical and methodological reporting (listed below) as failure to do so may delay the reconsideration of the revised manuscript.In particular please provide: -a Supplementary Figure including unprocessed images of all gels/blots in the form of a multipage pdf file.Please ensure that blots/gels are labeled and the sections presented in the figures are clearly indicated.

Response:
The revised manuscript adds a new Supplementary Figure 1 with uncropped Western blotting images from Fig. 3i and Extended Data Fig. 7.
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Response:
The new Supplementary Table 7 contains all numerical data from the main and Extended Data Figures.

Reviewer #1:
In this manuscript, Venkei and colleagues demonstrate that the 5' to 3' directed phased piRNA biogenesis pathway allows utilisation of a Y-encoded transcript as piRNA biogenesis substrate through a maternally deposited transposon piRNA that cleaves the 5' end of the Y-linked transcript.This is a great paper that combines clever genetics with high end quantitative molecular biology.

I support publication after incorporation of the following comments:
Response: We thank the Reviewer for their encouraging feedback.We have revised the manuscript to address the Reviewer's comments.
-title: I am not sure whether this is the most intuitive title for the broad readership of this journal.Also: utilisation of a Y-linked piRNA is not really the point as it is utilisation of a Y-linked piRNA precursor, no?
Response: The title was edited to "A maternally-programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila".
-The authors write: "We detected expression of Su(Ste) piRNA precursor transcripts only from the genomic strand that produces transcripts antisense to Ste mRNAs (not shown)."Please add the original data here.
Response: Fluorescence in situ hybridization (FISH) data for both sense and antisense Su(Ste) transcripts now appear in the new Extended Data Figs.1a, b.Consistent with the unlikely role of ping-pong amplification in the production of Su(Ste) piRNAs, only antisense Su(Ste) transcripts are detectable by FISH in germline stem cells and spermatogonia (GSC/SGs; Extended Data Fig. 1a).Few foci of sense Su(Ste) transcripts are detectable in spermatocytes, yet these foci appear several days after the transcription and processing of antisense Su(Ste) precursors occurs in GSC/SGs, suggesting that ping-pong between sense and antisense Su(Ste) precursors is unlikely.The requirement for the phased biogenesis pathway in earlier stages (GSC/SGs) also argues against the idea that potential ping-pong in spermatocytes is functionally critical.These data concur with the strand-specific RNA-seq data that we added to the revised manuscript: in whole testes, steady-state abundance of sense Su(Ste) transcripts is <1/10 th of the levels of antisense piRNA precursors (Extended Data Fig. 1c).We edited the text to include the discussion of these observations.
-The authors write: "smRNA-FISH can detect Ste mRNAs and Su(Ste) precursor transcripts but not mature piRNAs, because small RNAs are not retained in formaldehyde-fixed tissues." Could the authors add a citation for this statement?How did Plasterk and colleagues detect microRNAs in zebrafish by in situ hybridisation?

Response:
The revised text adds the reference to Pena et al., Nat Methods 2009 reporting that "in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5' phosphate."We speculate that Plasterk and colleagues detected the miRNA precursors-pre-miRNAs or pri-miRNAs-in their work.
-The authors write: "We conclude that maternal deposition of Su(Ste) piRNAs by XXY mothers suffices to silence Ste mRNA and bypasses the requirement for phased piRNA production pathway in early male germ cells." This would mean that the maternally deposited piRNAs are stable until the onset of Stellate transcription, is this correct?Or do the authors envision that the maternally deposited piRNAs are capable of processing the Su(Ste) transcript through ping-pong?If the latter is the case, would this mean that the sense piRNAs of Stellate are essential?A clarifying comment would be helpful in the manuscript.
Response: Argonaute-loaded small RNAs are one of the most stable RNA species in insect and mammalian cells.The median half-life of miRNAs in MEF cells is nearly 1.5 days (Kingston and Bartel, Genome Res 2019), and half-lives of all Ago2-loaded 2′-Omethylated small RNAs and many Ago1-loaded miRNAs in S2 cells are > 24 hours (Reichohlf et al., Mol Cell 2019).The remarkably slow turnover of small RNAs likely explains the efficient silencing of Ste mRNAs by the maternally deposited Su(Ste) piRNAs in sons of XXY mothers.In fact, the exceptional stability of Argonaute-protected small RNAs probably underlies the intergenerational inheritance of transposon-targeting piRNAs in animals with maternally deposited germplasm.We revised the text to add this discussion.
-how do the authors explain the requirement for Ago3 in the process?Ago3 is not a recipient of phased piRNAs.Does this mean that the amplification of the hoppel trigger piRNA through ping-pong during embryonic/larval development is a requirement for the efficient triggering of the Su(Ste) transcript?Adding a comment here would be great.Response: For a more intuitive arrangement, the revised Fig. 1 now groups Z projections by genotype, not by cell type as in the original version.We also added the clarification in the legend.Also please note that the images of entire apical tip are Zprojections to better represent the overall signals, whereas images for each cell type are single Z plane (to avoid signals from multiple cells in depth).This is clarified in the revised legend.

Response
-Figure 2: was the quantification of Su(Ste) quantification done in a z-stack so that each cell is properly analysed?
Response: The quantification is, in fact, based on the z-stack (max projection for signal intensity), which is reflected in the revised figure legend.
-Figure 5: please add a length scale for the 1360/Su(Ste) transcript.

Response:
The length scale was added to Fig. 5.
-Figure 6a: why is the small RNA size profile cut at 23nt? Please include sizes down to 20nt.
Response: Small RNAs of 20-30-nt in length are now included in Figs. 1 and 6.The data in the revised Fig. 1 also attest to the lack of siRNA production from Su(Ste) transcripts, supporting the idea that only antisense Su(Ste) transcripts are expressed in early spermatogenesis.
methods: Ct values were normalized to Gapdh by the ∆∆Ct method.Please add a citation Response: The reference was added.
-please add molecular weight marker for western blots.

Response:
The molecular weight was added to the revised Figures, and the uncropped images appear in Supplementary Figure 1.
Finally, one maybe crazy idea: If one would express at high levels one or two siRNAs that are between the 1360 trigger piRNA target site and the Stellate sequence, then this might block the phasing process, thereby leading to the inability to silence Stellate.This is not an experiment that is required for this manuscript to be published, but it might be a neat test for the model.

Response:
We thank the reviewer for suggesting an interesting test for the model.siRNA-guided slicing of transcripts however generates 5′ monophosphorylated cleavage products that are indistinguishable from RNA intermediates in the phased piRNA biogenesis (Han et al., Science 2015).siRNA-directed cleavage of piRNA precursor transcripts may thus result in increased phased piRNA production.In fact, siRNA slicing was recently proposed as an initiating event in de novo piRNA biogenesis in flies (Luo et al., bioRxiv 2022).

Reviewer #2:
Remarks to the Author: piRNAs are small non-coding RNAs that recognise complementary targets, such as transposons, leading to their silencing.In Drosophila, piRNAs are maternally deposited through the oocyte.The male germline uses the piRNA pathway to repress Stellate (Ste), which, if active, leads to the production of Ste crystals and impairs male fertility.piRNAs capable of targeting Ste are produced from the Suppressor of Stellate [Su(Ste)] locus on the Y chromosome, and thus are not maternally deposited.How Su(Ste) piRNAs are generated in testes is currently not understood.This manuscript by Venkei, Gainedinov et al. sheds light on the mechanism of Su(Ste) piRNA production in male germ cells.The authors used smRNA-FISH to visualise Ste and Su(ste) transcripts throughout Drosophila testis development and found that Su(Ste) is expressed earlier than its target Ste.Through temporally controlled knockdown and rescue experiments, they illustrate an early requirement of phased biogenesis components, and a late requirement of factors of the ping-pong amplification cycle.
The Su(Ste) promoter is localised within a 1360/Hoppel transposon insertion and as 1360/Hoppel piRNAs are maternally deposited, the authors hypothesised that cleavage by these piRNAs trigger phased piRNA biogenesis of the Su(Ste) transcript.To test this, they used XXY females expressing Su(Ste) from their Y chromosome.First, these females were able to produce piRNAs targeting Su(Ste) from the Y-linked Su(Ste) locus and pass these piRNAs onto their sons.Second, through sequencing of small and long RNAs from XXY ovaries, they found Su(Ste)-derived long 5'-monophosphorylated RNAs that displayed signatures consistent with anti-1360/Hoppel piRNA-guided cleavage and processing through the phased biogenesis pathway.
This work addresses the wider question on how intergenerational information is transferred through the piRNA pathway.Very little has been known about Su(Ste) piRNA production and this study contributes to understanding their biogenesis mechanism.The manuscript is well composed and easy to follow.However, several points as outlined below have to be addressed to fully support the authors' conclusions.

Response:
We appreciate these encouraging comments.We have revised the manuscript as detailed below.
Major comments: 1) The ping-pong machinery has previously been reported as unlikely to drive the generation of Su(Ste) piRNAs (PMID 20980675) -this should be acknowledged.

Response:
As mentioned in the response to the Editor, data reported in Nagao et al., RNA 2010 indeed support the model proposed in our study.We revised the text to incorporate these earlier findings: the fact that most Su(Ste) piRNAs in Aub and Ago3 are derived from the antisense Su(Ste) precursors, and the requirements (1) for armi in Su(Ste) piRNA biogenesis and (2) for both Aub and Ago3 in Ste repression.
In the same study, one abundant piRNA dominated both the Aub-and Ago3-loaded piRNAs mapping to Su(Ste), is this one also detected in the present study?If so, how does it relate to the phased biogenesis? 2) Su(Ste) has been described to produce sense transcripts giving rise to dsRNA.However, the authors did not detect any sense transcription, described as "data not shown".Given its importance, I think these data should be included and further expanded by stranded RNA-seq data.
Response: Please see our response to a similar comment from Reviewer #1.Briefly, FISH data for the sense Su(Ste) transcripts now appear in the new Extended Data Figs.Aub-bound piRNAs (Nagao et al., RNA 2010) detectable in spermatocytes, yet this observation agrees with the proposed model, as these foci appear several days after the transcription and processing of antisense Su(Ste) precursors commences in GSC/SGs.These data concur with the requested strand-specific RNA-seq data that we added to the revised manuscript: in whole testes, steady-state abundance of sense Su(Ste) transcripts is <1/10 th of the levels of antisense piRNA precursors (Extended Data Fig. 1c).
Similarly, I think it would be important to show both sense and antisense piRNAs (or the absence thereof) in Figure 6.Are any siRNAs detected?
Response: The updated Figs. 1 and 6 now includes 20-30-nt small RNAs and demonstrates the lack of siRNA production from Su(Ste) transcripts, supporting the idea that only antisense Su(Ste) transcripts are expressed in early spermatogenesis.We also added the more detailed analyses of sense and antisense Ste-and Su(Ste)-derived piRNAs in Figs. 1 and 6 and Extended Data Fig. 8b.
3) Considering that Piwi seems not expressed in male germ cells, how can neither Aub or Ago3 be required for phased biogenesis (Extended Data Figure 2)?I don't follow how the Su(Ste)derived piRNAs are retained to the later developmental stages if they are not loaded onto Aub or Ago3 during their biogenesis.If Aub/Ago3 act redundantly, can this be verified through a double-KD?
Response: As mentioned in the response to the Editor, the Reviewer's comment encouraged us to conduct additional analyses and experiments (see below) and revise the text to better describe the role of Aub and Ago3 in Su(Ste) piRNA biogenesis.
Data in Nagao et al. and in this manuscript show that both Aub and Ago3 are required for efficient silencing of Ste mRNAs.First, it had been already noted by the previous study that the majority of piRNAs in Aub and Ago3 are derived from antisense Su(Ste) precursor (Nagao et al., RNA 2010), i.e., both proteins are guided by piRNAs targeting Ste mRNAs.Second, in the absence of either Aub or Ago3, Ste mRNAs are derepressed (Nagao et al., RNA 2010, and Fig. 4 in this manuscript).These findings support the idea that both Aub and Ago3 are programmed with antisense Su(Ste) piRNAs during phased biogenesis in GSC/SGs.Aub and Ago3 therefore do not act redundantly, but additively: both proteins are required for efficient slicing of Ste mRNAs in spermatocytes.
In bam-driven rescue experiments, expression of transgenic Aub (Fig. 4g) and Ago3 (new data in Fig. 4i) starts at 4-cell SG stage, i.e., before the transcription of antisense Su(Ste) precursors reaches its peak.Su(Ste) piRNAs are thus loaded in Aub and Ago3 in early spermatogenesis and direct cleavage of Ste mRNAs later.We have updated the text and the figures accordingly (see new section "Ste Silencing Requires Expression of Both Aub and Ago3 in Spermatogonia").

Decision Letter, first revision:
Our ref: NCB-A49585A 14th June 2023 Dear Dr. Yamashita, Please accept our apologies for the delay in sending a decision to you, as Reviewer#2 was unable to re-review your revisions.We secured Reviewer#1 to assess on your responses to Reviewer#2's previous concerns.
Thank you for submitting your revised manuscript "A maternally-programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila" (NCB-A49585A).It has now been seen by the one of original referees, whom we have asked to comment on the responses to referee 2's points, and their comments are below.The reviewer finds that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Cell Biology, pending minor revisions to comply with our editorial and formatting guidelines.
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does the silencing of Stellate by Su(Ste) piRNAs generate Ago3-bound responder piRNAs from the Stellate transcripts in testes?"Response: Please see the detailed replies to the comments from Reviewer #1 and #2.Briefly, Nagao et al. and the data in this manuscript show that both Aub and Ago3 are required for efficient silencing of Ste mRNAs.First, Nagao et al. noted that the majority of piRNAs in Aub and Ago3 are derived from antisense Su(Ste) precursor, i.e., both proteins are guided by piRNAs targeting Ste mRNAs.Second, in the absence of either Aub or Ago3, Ste mRNAs are derepressed (Nagao et al., RNA 2010, and Fig. 4 in this manuscript).These findings support the idea that both Aub and Ago3 are programmed with antisense Su(Ste) piRNAs during phased biogenesis in GSC/SGs.Aub and Ago3 therefore do not act redundantly, but additively: both proteins are required for efficient slicing of Ste mRNAs in spermatocytes.The revised manuscript also includes analyses (Supplementary Text 3) showing that the majority of Ste-derived piRNAs explained by Su(Ste)-guided cleavage are loaded in Ago3, not Aub.These data support the Reviewer's hypothesis that Ago3-loaded responder piRNAs are produced when Ste mRNAs are sliced by Su(Ste) piRNAs.However, these responder piRNAs cannot further propagate Ping-Pong piRNA amplification because Su(Ste) transcripts are not present in Ste expressing cells.

:
In testis and ovaries, transposon(TE)-derived piRNAs indeed partition between Aub and Ago3: most antisense, 1U-enriched TE piRNA are bound to Aub; most sense, 10A-biased TE piRNAs are loaded in Ago3 (Brennecke et al., Cell 2007; Nagao et al., RNA 2010).By contrast, the majority of Su(Ste) piRNAs both in Ago3 and in Aub are antisense to Ste mRNAs and display a 1U-bias (as already shown by Nagao et al., RNA 2010; and new data in Extended Data Fig. 3e).Both Aub and Ago3 are required for Ste silencing (Nagao et al., RNA 2010; and Fig. 4 in this manuscript).These data support the idea that both Aub and Ago3 are programmed with antisense Su(Ste) piRNAs during phased processing of Su(Ste) precursors in GSC/SGs, and both Aub and Ago3 are required for efficient slicing of Ste mRNAs in spermatocytes.-does the silencing of Stellate by Su(Ste) piRNAs generate Ago3-bound responder piRNAs from the Stellate transcripts in testes?Response: As stated in the response to the Editor, the revised manuscript includes analyses (Supplementary Text 3) showing that the majority of Ste-derived piRNAs explained by Su(Ste)-guided cleavage are loaded into Ago3.These data support the Reviewer's hypothesis that Ago3-loaded responder piRNAs are produced when Ste mRNAs are sliced by Su(Ste) piRNAs.-Figure 1: I have a hard time to see where the f and g panels are related to the boxed areas in b and c.

Response:
Consistent with processing of Su(Ste) precursors by Zuc, 77 ± 1% of Su(Ste)-derived piRNAs begin with a uridine and have no enrichment of adenine at position 10 (21 ± 1%; new data in Extended Data Figure 3e).Su(Ste)-4 (5′-UCUCAUCGUCGUAGAACAAGCCC[…]-3′) is the most abundant Su(Ste)-derived piRNA both in our data (~1.5% of all reads) and in data from Nagao et al. (~60% of reads in Aub IP, ~5% of reads in Ago3 IP).The likely reason for the 3-40-fold difference in the fraction of Su(Ste)-4 reads in our and the published data is the ligation bias during small RNA library preparation.The bias was first reported in Hafner et al., RNA 2011, after the work by Nagao et al. was published.Most current small RNA library preparation procedures-including the one used in this work-minimize the ligation bias by increasing ligase concentration, extending reaction time, using randomized portions in adapters, and conducting ligation in presence of PEG-8000 (e.g., see AQ-seq in Kim et al., NAR 2019).Consistent with this explanation, the complexity of datasets generated in this study is greater than for those from Nagao et al., even after down-sampling the data to the same sequencing depth (Response Fig. 1, below).
1a, b.Consistent with the unlikely role of ping-pong amplification in the production of Su(Ste) piRNAs, only antisense Su(Ste) transcripts are detectable by FISH in germ stem cells and spermatogonia (GSC/SGs).Few foci of sense Su(Ste) transcripts are Response Figure 1: The complexity of data from Nagao et al., RNA 2010 and this manuscript.All datasets were down-sampled to 100,000 sequencing reads.
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