1. Developmental Biology
  2. Structural Biology and Molecular Biophysics
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Engineering a conserved RNA regulatory protein repurposes its biological function in vivo

  1. Vandita D Bhat
  2. Kathleen L McCann
  3. Yeming Wang
  4. Dallas R Fonseca
  5. Tarjani Shukla
  6. Jacqueline C Alexander
  7. Chen Qiu
  8. Marvin Wickens
  9. Te-Wen Lo
  10. Traci M Tanaka Hall  Is a corresponding author
  11. Zachary T Campbell  Is a corresponding author
  1. University of Texas Dallas, United States
  2. National Institute of Environmental Health Sciences, National Institutes of Health, United States
  3. Ithaca College, United States
  4. University of Wisconsin-Madison, United States
Research Article
  • Cited 7
  • Views 1,805
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Cite this article as: eLife 2019;8:e43788 doi: 10.7554/eLife.43788

Abstract

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

Article and author information

Author details

  1. Vandita D Bhat

    Department of Biological Sciences, University of Texas Dallas, Richardson, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Kathleen L McCann

    Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7144-4851
  3. Yeming Wang

    Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Dallas R Fonseca

    Department of Biology, Ithaca College, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Tarjani Shukla

    Department of Biological Sciences, University of Texas Dallas, Richardson, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Jacqueline C Alexander

    Department of Biology, Ithaca College, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Chen Qiu

    Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Marvin Wickens

    Department of Biochemistry, University of Wisconsin-Madison, Wisconsin, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Te-Wen Lo

    Department of Biology, Ithaca College, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Traci M Tanaka Hall

    Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States
    For correspondence
    hall4@niehs.nih.gov
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6166-3009
  11. Zachary T Campbell

    Department of Biological Sciences, University of Texas Dallas, Richardson, United States
    For correspondence
    zachary.campbell@utdallas.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3768-6996

Funding

National Institutes of Health (R01NS100788)

  • Zachary T Campbell

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: November 21, 2018
  2. Accepted: January 15, 2019
  3. Accepted Manuscript published: January 17, 2019 (version 1)
  4. Version of Record published: January 29, 2019 (version 2)
  5. Version of Record updated: March 5, 2019 (version 3)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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