Engineering a conserved RNA regulatory protein repurposes its biological function in vivo

Abstract
Data availability
Article and author information
Metrics

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

1. Bhat VD
2. McCann KL
3. Wang Y
4. Fonseca DR
5. Shukla T
6. Alexander JC
7. Qiu C
8. Wickens M
9. Lo T-W
10. Tanaka Hall TM
11. Campbell ZT
(2019) PUF-8 data from Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
Protein Data Bank, 6NOD.

http://www.rcsb.org/structure/6NOD
1. Bhat VD
2. McCann KL
3. Wang Y
4. Fonseca DR
5. Shukla T
6. Alexander JC
7. Qiu C
8. Wickens M
9. Lo T-W
10. Tanaka Hall TM
11. Campbell ZT
(2019) SS/Y data from Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
Protein Data Bank, 6NOH.

http://www.rcsb.org/structure/6NOH
1. Bhat VD
2. McCann KL
3. Wang Y
4. Fonseca DR
5. Shukla T
6. Alexander JC
7. Qiu C
8. Wickens M
9. Lo T-W
10. Tanaka Hall TM
11. Campbell ZT
(2019) AS/Y data from Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
Protein Data Bank, 6NOF.

http://www.rcsb.org/structure/6NOF
1. Bhat VD
2. McCann KL
3. Wang Y
4. Fonseca DR
5. Shukla T
6. Alexander JC
7. Qiu C
8. Wickens M
9. Lo T-W
10. Tanaka Hall TM
11. Campbell ZT
(2019) AQ/Y data from Engineering a conserved RNA regulatory protein repurposes its biological function in vivo
Protein Data Bank, 6NOC.

http://www.rcsb.org/structure/6NOC

Article and author information

Author details

Vandita D Bhat

Department of Biological Sciences, University of Texas Dallas, Richardson, United States

Competing interests
The authors declare that no competing interests exist.
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.

"This ORCID iD identifies the author of this article:" 0000-0002-7144-4851
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.
Dallas R Fonseca

Department of Biology, Ithaca College, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.
Tarjani Shukla

Department of Biological Sciences, University of Texas Dallas, Richardson, United States

Competing interests
The authors declare that no competing interests exist.
Jacqueline C Alexander

Department of Biology, Ithaca College, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.
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.
Marvin Wickens

Department of Biochemistry, University of Wisconsin-Madison, Wisconsin, United States

Competing interests
The authors declare that no competing interests exist.
Te-Wen Lo

Department of Biology, Ithaca College, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.
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.

"This ORCID iD identifies the author of this article:" 0000-0001-6166-3009
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.

"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.

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.