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

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

  • 2,205
    Page views
  • 297
    Downloads
  • 10
    Citations

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

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)

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

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

  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
  11. Zachary T Campbell
(2019)
Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
eLife 8:e43788.
https://doi.org/10.7554/eLife.43788

Further reading

    1. Chromosomes and Gene Expression
    2. Developmental Biology
    Valeria Taliani, Giulia Buonaiuto ... Monica Ballarino
    Research Article Updated

    Long noncoding RNAs (lncRNAs) are emerging as critical regulators of heart physiology and disease, although the studies unveiling their modes of action are still limited to few examples. We recently identified pCharme, a chromatin-associated lncRNA whose functional knockout in mice results in defective myogenesis and morphological remodeling of the cardiac muscle. Here, we combined Cap-Analysis of Gene Expression (CAGE), single-cell (sc)RNA sequencing, and whole-mount in situ hybridization analyses to study pCharme cardiac expression. Since the early steps of cardiomyogenesis, we found the lncRNA being specifically restricted to cardiomyocytes, where it assists the formation of specific nuclear condensates containing MATR3, as well as important RNAs for cardiac development. In line with the functional significance of these activities, pCharme ablation in mice results in a delayed maturation of cardiomyocytes, which ultimately leads to morphological alterations of the ventricular myocardium. Since congenital anomalies in myocardium are clinically relevant in humans and predispose patients to major complications, the identification of novel genes controlling cardiac morphology becomes crucial. Our study offers unique insights into a novel lncRNA-mediated regulatory mechanism promoting cardiomyocyte maturation and bears relevance to Charme locus for future theranostic applications.

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Daniel Richard, Steven Pregizer ... April M Craft
    Tools and Resources

    To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages, and sheds new light on human cartilage development and biology.