1. Cell Biology
  2. Developmental Biology
Download icon

Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins

  1. Chih-Yung S Lee
  2. Andrea Putnam
  3. Tu Lu
  4. ShuaiXin He
  5. John Paul T Ouyang
  6. Geraldine Seydoux  Is a corresponding author
  1. Johns Hopkins University School of Medicine, United States
Research Article
  • Cited 34
  • Views 5,378
  • Annotations
Cite this article as: eLife 2020;9:e52896 doi: 10.7554/eLife.52896

Abstract

RNA granules are protein/RNA condensates. How specific mRNAs are recruited to cytoplasmic RNA granules is not known. Here we characterize the transcriptome and assembly of P granules, RNA granules in the C. elegans germ plasm. We find that P granules recruit mRNAs by condensation with the disordered protein MEG-3. MEG-3 traps mRNAs into non-dynamic condensates in vitro and binds to ~500 mRNAs in vivo in a sequence-independent manner that favors embryonic mRNAs with low ribosome coverage. Translational stress causes additional mRNAs to localize to P granules and translational activation correlates with P granule exit for two mRNAs coding for germ cell fate regulators. Localization to P granules is not required for translational repression but is required to enrich mRNAs in the germ lineage for robust germline development. Our observations reveal similarities between P granules and stress granules and identify intrinsically-disordered proteins as drivers of RNA condensation during P granule assembly.

Data availability

Sequencing data have been deposited in GEO under accession number GSE139881,GSE139878, GSE139879 and GSE139880.Description of iCLIP analysis and additional python codes are deposited in Github :https://github.com/fishhead1978/iCLIP_2019

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Chih-Yung S Lee

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  2. Andrea Putnam

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  3. Tu Lu

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5697-300X
  4. ShuaiXin He

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  5. John Paul T Ouyang

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  6. Geraldine Seydoux

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    For correspondence
    gseydoux@jhmi.edu
    Competing interests
    Geraldine Seydoux, serves on the Scientific Advisory Board of Dewpoint Therapeutics, Inc.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8257-0493

Funding

National Institutes of Health (R37 HD37047)

  • Geraldine Seydoux

Howard Hughes Medical Institute

  • Geraldine Seydoux

National Institutes of Health (T32 GM007445)

  • John Paul T Ouyang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Timothy W Nilsen, Case Western Reserve University, United States

Publication history

  1. Received: October 19, 2019
  2. Accepted: January 23, 2020
  3. Accepted Manuscript published: January 24, 2020 (version 1)
  4. Version of Record published: February 7, 2020 (version 2)

Copyright

© 2020, Lee et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,378
    Page views
  • 866
    Downloads
  • 34
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Cell Biology
    2. Microbiology and Infectious Disease
    Yong Fu et al.
    Research Article

    Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

    1. Cell Biology
    Michelina Kierzek et al.
    Tools and Resources

    Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically-encoded FRET biosensors. Moreover, FASTM is compatible with opto-chemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling.