1. Developmental Biology
  2. Structural Biology and Molecular Biophysics
Download icon

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 8
  • Views 2,035
  • Annotations
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.

Data availability

All data associated with the manuscript are present in the source data file. Data have also been deposited to PDB under the accession numbers 6NOD, 6NOH, 6NOF, and 6NOC.

The following data sets were generated

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.

Metrics

  • 2,035
    Page views
  • 271
    Downloads
  • 8
    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)

Categories and tags

Research organism

Further reading

    1. Developmental Biology
    2. Structural Biology and Molecular Biophysics

    A crystal structure of a collaborative RNA regulatory complex reveals mechanisms to refine target specificity

    Chen Qiu et al.
    Research Advance Updated

    In the Caenorhabditis elegans germline, fem-3 Binding Factor (FBF) partners with LST-1 to maintain stem cells. A crystal structure of an FBF-2/LST-1/RNA complex revealed that FBF-2 recognizes a short RNA motif different from the characteristic 9-nt FBF binding element, and compact motif recognition coincided with curvature changes in the FBF-2 scaffold. Previously, we engineered FBF-2 to favor recognition of shorter RNA motifs without curvature change (Bhat et al., 2019). In vitro selection of RNAs bound by FBF-2 suggested sequence specificity in the central region of the compact element. This bias, reflected in the crystal structure, was validated in RNA-binding assays. FBF-2 has the intrinsic ability to bind to this shorter motif. LST-1 weakens FBF-2 binding affinity for short and long motifs, which may increase target selectivity. Our findings highlight the role of FBF scaffold flexibility in RNA recognition and suggest a new mechanism by which protein partners refine target site selection.

    1. Developmental Biology
    2. Evolutionary Biology

    Single cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

    Periklis Paganos et al.
    Research Article

    Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.

    1. Developmental Biology

    Visually induced changes in cytokine production in the chick choroid

    Jody A Summers, Elizabeth Martinez
    Research Article Updated

    Postnatal ocular growth is regulated by a vision-dependent mechanism that acts to minimize refractive error through coordinated growth of the ocular tissues. Of great interest is the identification of the chemical signals that control visually guided ocular growth. Here, we provide evidence that the pro-inflammatory cytokine, interleukin-6 (IL-6), may play a pivotal role in the control of ocular growth using a chicken model of myopia. Microarray, real-time RT-qPCR, and ELISA analyses identified IL-6 upregulation in the choroids of chick eyes under two visual conditions that introduce myopic defocus and slow the rate of ocular elongation (recovery from induced myopia and compensation for positive lenses). Intraocular administration of atropine, an agent known to slow ocular elongation, also resulted in an increase in choroidal IL-6 gene expression. Nitric oxide appears to directly or indirectly upregulate choroidal IL-6 gene expression, as administration of the non-specific nitric oxide synthase inhibitor, L-NAME, inhibited choroidal IL-6 gene expression, and application of a nitric oxide donor stimulated IL-6 gene and protein expression in isolated chick choroids. Considering the pleiotropic nature of IL-6 and its involvement in many biological processes, these results suggest that IL-6 may mediate many aspects of the choroidal response in the control of ocular growth.