Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
- University of Texas Dallas, United States
- National Institute of Environmental Health Sciences, National Institutes of Health, United States
- Ithaca College, United States
- 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.
Article and author information
Author details
Traci M Tanaka Hall
Zachary T Campbell
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
- Timothy W Nilsen, Case Western Reserve University, United States
Version history
- Received: November 21, 2018
- Accepted: January 15, 2019
- Accepted Manuscript published: January 17, 2019 (version 1)
- Version of Record published: January 29, 2019 (version 2)
- 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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Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
eLife 8:e43788.
https://doi.org/10.7554/eLife.43788
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