1. Author Response:

    Reviewer #3 (Public Review):

    The manuscript of Price et al. used TURBO-ID of multiple P granule components to identify new factors required for their assembly and function. They identify over 75 shared proteins, including 2 related TUDOR-domain proteins that they analyze in further detail through mutation and localization studies. The two proteins are necessary for the localization of several P granule components to the nuclear periphery, and they show in an ectopic tissue that EGGD-1 is sufficient to localize GLH-1 to the nuclear periphery.

    While the paper could go further to test physical interactions between EGGD-1 (and EGGD-2) with GLH-1 and the nuclear pore protein, this is not critical, although the authors should be cautious in their model that they have not proven that these associations are direct.

    We agree with the reviewer. We revised the statement and added references that support the model.

    It would be important to provide evidence that the STOP-IN cassette in eggd-2 is a true null. There may be downstream methionines that can be used. And the phenotype is weaker than eggd-1 so this could be an issue.

    This is a good point. Using two CRISPR guide RNAs, we generated a second allele of eggd-2 by deleting its full open reading frame. This allele and the allele bearing 17-nt insertion exhibited similar phenotypes: 1). No noticeable change in PGL-1::TagRFP localization in the pachytene region, 2). PGL-1::TagRFP failed to concentrate in the germ lineage in the embryos. We updated the information in the Figure 3—figure supplement 1 and text (line 292-297).

    All of the TURBO-ID strains have reduced viability. Since there is some concern that P granule composition could be affected in the tagged strains, showing the localization of other known components of P granules in these mutants (GLH-1, PGL-1) would be critical.

    Thanks for this suggestion. Please see the responses above.

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  2. Evaluation Summary:

    The authors use proximity labeling and genetic experiments to identify and functionally characterize new components of C. elegans P granules. The conclusions of the paper are well-supported by the data. This work will be of broad interest to developmental biologists, particularly those interested in the formation and function of germ cells.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    Cytoplasmic germ granules are a common feature of germ cells across species. Great effort has gone into trying to (1) identify proteins that localize to these condensates, (2) gain insights into how these granules assemble, and (3) characterize their functional significance. Pioneering studies on P granules in C. elegans have greatly expanded our understanding of these structures.

    In this paper, Price et al. use proximately labeling to identify previously unknown P granule components. They tag several known P granule protein genes with TurboID at their endogenous loci and perform biotinylation reactions in two different ways. Labeled proteins were subsequentially pulled down and identified using mass spectrometry. This approach successfully identified known P granule components and many new potential candidates. The authors focus their efforts on characterizing two related proteins EGGD-1 and EGGD-2 (also known as MIP-1 and MIP-2, based on recently published work). Knockdown of EGGD-1 and EGGD-2 using RNAi results in P granule defects. Cas-9 induced mutations confirm and extend this genetic analysis. The authors also perform structure/function analysis on EGGD-1 and define specific roles for its LOTUS and Intrinsically Disordered Region (IDR) domains in perinuclear P granule formation and function. Epistasis analysis shows that EGGD-1 acts upstream of GLH-1 in P granule assembly, and overexpression of EGGD-1 can drive granule formation outside of germ cells.

    Strengths

    The P granule proteome data presented here provides a useful resource for the community. The genetic analysis on eggd-1 provides important insights into the function of a new P granule component. The data are convincing, and the experiments are well-controlled.

    Weaknesses

    There are relatively few weaknesses and the general conclusions are supported by the data.

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  4. Reviewer #2 (Public Review):

    In this manuscript from Price et al, proximity labeling is used to define the proteome of the C. elegans P granule. This is an ideal method to use for this experiment because of the phase-separated nature of P granules, meaning that a traditional co-immunoprecipitation may not work to identify transiently interacting proteins. While many of the interacting proteins identified were previously known (validating the proximity labeling approach), the authors focused on two previously uncharacterized proteins, EGGD-1 and -2 (also known as MIP-1 and -2) which they find to disrupt PGL-1 and GLH-1 localization by RNAi and knockout mutants. The authors then proceed to do a careful structure-function analysis of EGGD-1 - demonstrating that the Lotus domains are important for recruitment of other P granule proteins while the IDRs are important for EGGD-1 localization to the nuclear periphery. Lastly, the manuscript demonstrates that EGGD-1 is sufficient to form perinuclear granules in somatic cells, and ectopically recruit GLH-1 to these granules. These data suggest that EGGD-1 directly interacts with nuclear pores (or at least nuclear pore associated proteins found in somatic cells) and does not require any other germline-specific P granule proteins for its perinuclear association. This is a nicely designed paper that is thorough and rigorous. There are no major weaknesses.

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  5. Reviewer #3 (Public Review):

    The manuscript of Price et al. used TURBO-ID of multiple P granule components to identify new factors required for their assembly and function. They identify over 75 shared proteins, including 2 related TUDOR-domain proteins that they analyze in further detail through mutation and localization studies. The two proteins are necessary for the localization of several P granule components to the nuclear periphery, and they show in an ectopic tissue that EGGD-1 is sufficient to localize GLH-1 to the nuclear periphery.

    While the paper could go further to test physical interactions between EGGD-1 (and EGGD-2) with GLH-1 and the nuclear pore protein, this is not critical, although the authors should be cautious in their model that they have not proven that these associations are direct.

    It would be important to provide evidence that the STOP-IN cassette in eggd-2 is a true null. There may be downstream methionines that can be used. And the phenotype is weaker than eggd-1 so this could be an issue.

    All of the TURBO-ID strains have reduced viability. Since there is some concern that P granule composition could be affected in the tagged strains, showing the localization of other known components of P granules in these mutants (GLH-1, PGL-1) would be critical.

    Read the original source
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