Fertilization triggers cytosolic functions and P-body recruitment of the RNA-binding protein Mei2 to drive fission yeast zygotic development

  1. Ayokunle Araoyinbo
  2. Clàudia Salat-Canela
  3. Aleksandar Vještica

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Compartmentalized regulation of RNAs is emerging as a key driver of developmental transitions, with RNA-binding proteins performing specialized functions in different subcellular compartments. The RNA-binding protein Mei2, which arrests mitotic proliferation and drives zygotic development in fission yeast, was shown to function in the nucleus to trigger meiotic divisions. Here, using compartment-restricted alleles, we report that Mei2 functions in the cytosol to arrest mitotic growth and initiate development. We find that Mei2 is a zygote-specific component of P-bodies that inhibits the translation of tethered mRNAs. Importantly, we show that P-bodies are necessary for Mei2-driven development. Phosphorylation of Mei2 by the inhibitory Pat1 kinase impedes P-body recruitment of both Mei2 and its target RNA. Finally, we establish that Mei2 recruitment to P-bodies and its cytosolic functions, including translational repression of tethered RNAs, depend on the RNA-binding domain of Mei2 that is dispensable for nuclear Mei2 roles. Collectively, our results dissect how distinct pools of an RNA-binding protein control developmental stages and implicate P-bodies as key regulators of gamete-to-zygote transition.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    'Please download the Response to Reviewers file, which contains Reviewers comments and authors responses and new data'

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In this work, the authors investigate the cytoplasmatic roles of Mei2, an RNA-binding protein in fission yeast, in particular its interactions with processing bodies (PBs) in the cytoplasm. The manuscript rests heavily on microscopy data, using a combination of time-resolved microscopy and molecular mutation and tagging techniques.

    Mei2 is known for its role in the nucleus of zygotic cells. Here, it is shown that Mei2 co-localizes with the PB markers Dcp2 and Edc3. This happens in zygotes but not in gametes (e.g. when fusion is blocked in fus1 mutants) (Fig 4E).
    This co-localization in PBs is counteracted by Pat1-driven phosphorylation of Mei2. Phosphorylation by Pat1 is known to suppress Mei2 activity. Mei3 inhibits Pat1; in a mei3 mutant Mei2 cannot accumulate in PBs, the same happens with a non-phosphorylatable mei2 allele (Fig. 5). In a pat1Δ mutant, constitutively active Mei2 is compatible with growth if it stays in the nucleus (mei2-NLS), but not if Mei2 is forced to the cytoplasm (mei2-NES) (Fig. 3G). This indicates that it is the cytoplasmic function of Mei2 that is critical.

    Forcing Pat1 to be cytoplasmic (Pat1-NES) allowed normal vegetative growth and mating (Fig. 3A-C), whereas nuclear Pat1 (Pat1-NLS) produced premature mating (Fig. 3A,B). Thus, cytoplasmic Pat1 phosphorylation of Mei2 is critical for controlling the transition from mitotic growth to fusion and zygote formation.

    Mei2 shuttles between the nucleus and cytoplasm, and one of its RNA-binding domains (RRM1) drives nuclear import, while both RRM1 and RRM3 are required for export to the cytoplasm (Fig. 2 and S2). Little was known previously of the role of RRM1.

    They present evidence that this localization to PBs is required for development. Knocking out the RNA helicase Ste13 (ortholog of S. cerevisiae Dhh1 which is a PB component) reduces PB formation (Fig. 6A). Even a non-phosphorylatable mei2 allele (i.e. it cannot be inactivated by Pat1) is incapable of driving sporulation in a ste13Δ background (Fig. 6B-D). This demonstrates that Mei2 activity is dependent on PBs.

    The study is well conceived and performed, and the conclusions mostly well backed by data. Experimental and statistical procedures are well described, and the number of replicates is sufficient.

    There are some minor questions however:

    In the literature, Mei2 is described as appearing as a nuclear dot in zygotic cells, but invisible in mitotic cells. Here, the authors demonstrate a Mei2 dot already 30 minutes before fertilization (Fig. 2A). Is the reason for this a more sensitive microscopic technique, or something else?

    The authors claim that the RRM1 RNA-binding region of Mei2 is essential for cytoplasmic Mei2 function and recruitment to PBs. This contrasts with previous publications (Watanabe 1994, Watanabe 1997, Otsubo 2014), as pointed out by the authors, where RRM1 appears to be dispensable for development. How do the authors argue about this discrepancy?

    Significance

    Overall, this paper presents major advances in our understanding of the cytoplasmic functions of this intensely studied RNA-binding protein, Mei2, in the transitions between the mitotic and meiotic cell cycles.

    It builds on the original observations of Mei2 as an essential protein for fusion and meiosis (Watanabe EMBO J 1988), being RNA-binding (Watanabe Cell 1994), and forming a nuclear dot in meiotic cells (Yamashita Cell 1998). These were followed by e.g. reports how Pat1 phosphorylation regulates Mei2 degradation (Matsuo J Cell Sci 2007) and its binding to RNA (Shen J Mol Cell Biol 2022). The present manuscript gives a broader view of the functions of Mei2 beyond its previously described role in the nucleus, and characterizes its interactions with the other players in fusion and meiosis.

    These findings will be of great interest not only to the fission yeast community, but to a wide range of scientists specializing in meiosis and fertilization, and to the RNA biologists at large. Since Mei2 is conserved across many branches of the eukaryotic tree as an RNA-binding protein, albeit with somewhat different functions in e.g. plants, the work has general relevance.

    I have read this manuscript with a background in general yeast cell and molecular biology, including post-transcriptional regulation. I am no microscopy expert, however I find the experimental setup with fluorescent tagging, combinations of mutations in key components in the pathway, and high resolution microscopy data from time series, convincing.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Araoyinbo et al. present a wealth of detailed data analyzing the cellular behavior of mainly three proteins, the RNA-binding protein Mei2, the kinase Pat1 and its inhibitor the protein Mei3 during mating and subsequent initiation of meiosis in fission yeast. This analysis involve also the detailed testing of potential models about how these protein act on each other to fulfil their different functions, such as to block remating on zygotes, the initiation of zygotic S-phase and the initiation of meiosis and sporulation. These data converge to a model whereby Pat1 inhibition by Mei3 expression upon cell fusion unleashes Mei2 function in the cytoplasm. This is due to the subsequent dephosphorylation of Mei2, and its RNA-recognition motif RRM1 interacting with and recruiting Mei2-bound RNAs to P-bodies, where their translation is most likely repressed (at least the translation of a synthetic mRNA - Mei2 pair is repressed when the pair is targeted to P-bodies). Together, this study provides detailed insights into how the meiotic cycle is induced upon mating of fission yeast cells but not in gametes).

    Overall, this is a very carefully controlled study and the data are very convincing and very interesting. It makes a compelling case for the model proposed and makes many original observations and far reaching observations, such as the role of nucleo-cytoplasmic compartmentalization and P-bodies in implementing developmental decisions. Since the notion that P-bodies have a function at all has been strongly questioned in recent years, this study will be very useful for the field.

    The only limitations that I have concerns the readability of the manuscript. It is extremely dense and that makes it a laborious read. Furthermore, the manuscript is not particularly well motivated, such that it is not very obvious what questions the authors are after. This becomes more or less clear only slowly as the reader progresses, or in the second read. Therefore, this very nice piece of work may escape people who are not working on fission yeast mating and meiosis, which would be a pity. I therefore recommend working on better motivating the study and its different parts for a general audience, streamlining the fission yeast intricacies and explaining more precisely what is conceptually learnt from these studies, on a broad sense and possibly in a way that would be relevant beyond the model used. This paper is opening a reach area of research and it would be unfortunate to not make that point more clearly.

    Significance

    Overall, this is a very carefully controlled study and the data are very convincing and very interesting. It makes a compelling case for the model proposed and makes many original observations and far reaching observations, such as the role of nucleo-cytoplasmic compartmentalization and P-bodies in implementing developmental decisions. Since the notion that P-bodies have a function at all has been strongly questioned in recent years, this study will be very useful for the field.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The study by Araoyinbo et al. explores the role of the RNA-binding protein Mei2 in fission yeast zygotic development. It highlights Mei2's cytosolic functions, its interaction with P-bodies, and nucleocytoplasmic shuttling. Mei2's regulation by Mei3 and Pat1, and the importance of its RNA recognition motifs (RRM1 and RRM3) are also discussed.

    The main conclusion of the manuscript is somewhat unexpected from previous studies about Mei2. Particularly, the cytoplasmic function of Mei2 is a novel point in this field.

    Lots of experiments have been done to make the scenario of the manuscript. The experiments and results are technically sound, and I potentially agree with the interpretation by the authors. It would require some more explanation as well as additional experiments to conclude in the way the authors wish to do.

    Major points

    1. Page 4. "Taken together, these results show that fertilization, and Mei3 expression in particular, promote Mei2 nuclear export." It is also possible that Mei2-NLS-GFP was degraded somewhere in the cell (as Mei2 may be still shuttling even if NLS was fused) upon mating (120 min onwards in Fig2D) rather than exported to the cytoplasm. In mei3∆ (Fig 2E) Mei2-NLS-GFP might be somehow escaped from the degradation. Also, nuclear signal of Mei2 is very bright but cytosolic signal seems vague. I wonder the entire results in the manuscript could be interpreted from the viewpoint of degradation/protein stability/protein amount, rather than regulation of localization such as nuclear import and export.
    2. Page 4. "We conclude that RRM1 promotes nuclear import of Mei2." This may be true, but is it also possible that RRM1 inhibits nuclear export of Mei2? This type of possible dual explanation can be applied to the entire manuscript. This is expected to be neutralized or clarified at each point.
    3. Page 5. "Thus, diminishing nuclear Pat1 levels does not compromise its roles during growth and mating." It is interesting for me to find that Pat1-NLS induced ectopic meiosis. This is a fine finding. I wonder just addition of NLS (basic residues) at the C-terminus of Pat1 might deteriorate the activity of Pat1, apart from localization shift. Is it possible to exclude this possibility by making NES-Pat1-NLS-3GFP fusion, in which NLS and NES are fused doubly and distally, because proximal double fusion such as Pat1-NLS-NES-3GFP might just mutually cancel the NLS NES activities.
    4. In general in the Results section. What confused me is when each event occurred. Nutritional conditions, -N but not yet conjugated, after conjugation, premeiotic S or meiotic prophase (or even later). It is particularly hard to catch the story when the timing issue and the location issue (nuclear and cytosolic localization, NLS and NES...) are discussed at the same time. Explanation in chronological order, hopefully at the earlier stages such as explanation for Figures 2 and 3, would be appreciated. The model shown in Figure 8 is quite helpful for my understanding.

    Minor points

    1. "Fertilization" in the title, and "Mei2 is expressed in gametes" in the main text on pare 2. Authors try to generalize fission yeast mating as fertilization of higher organisms as both are events in which two haploids conjugate. I personally do not agree with this type of explanation. This is mainly because S. pombe conjugation (mating) is a part of sexual differentiation and therefore is biologically distinct from fertilization of higher organisms. S. pombe grows and divides in the haploid state, which is distinct from general gametes. To avoid such confusion, I would propose authors to neutralize expression throughout the manuscript.
    2. I found quite a few "surprising(ly)", which are hopefully neutralized, as it is somewhat emotional.

    Significance

    General assessment: strengths and limitations:

    Strengths: It provides novel understanding of molecular mechanisms of meiotic initiation of fission yeast. Technically sound. Lots of experiments. Limitations: The story is very confusing and difficult to catch. Explanation can be simplified.

    Advance: compare the study to existing published knowledge: does it fill a gap? What kind of advance does it make (conceptual, clinical, fundamental, methodological, incremental,,,,)? It is a big advancement. It is conceptually novel regarding how meiosis is initiated in fission yeast.

    Audience: which communities will be interested/influenced, what kind of audience (broad, specialized, clinical, basic research, applied science, fields and subfields,,,) It is mainly for audience of basic research, biology, molecular mechanism of gene explanation, meiosis or yeast cellular events. For non-yeast researchers, this manuscript is probably very hard to read/understand, although the authors tried to generalize yeast-specific events with general words.

    Describe your expertise:

    Yeast genetics, Meiosis, Cell biology, Gene expression regulation

  5. Version published to 10.1101/2024.12.29.630664 on bioRxiv