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

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 first 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.

Authors:
 [1]; ORCiD logo [2];  [2];  [3];  [1];  [3];  [2];  [4];  [3]; ORCiD logo [2]; ORCiD logo [1]
  1. Department of Biological Sciences, University of Texas Dallas, Richardson, United States
  2. Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States
  3. Department of Biology, Ithaca College, Ithaca, United States
  4. Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1506498
Resource Type:
Journal Article
Journal Name:
eLife
Additional Journal Information:
Journal Volume: 8; Journal Issue: 01, 2019; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Bhat, Vandita D., McCann, Kathleen L., Wang, Yeming, Fonseca, Dallas R., Shukla, Tarjani, Alexander, Jacqueline C., Qiu, Chen, Wickens, Marv, Lo, Te-Wen, Tanaka Hall, Traci M., and Campbell, Zachary T. Engineering a conserved RNA regulatory protein repurposes its biological function in vivo. United States: N. p., 2019. Web. doi:10.7554/eLife.43788.
Bhat, Vandita D., McCann, Kathleen L., Wang, Yeming, Fonseca, Dallas R., Shukla, Tarjani, Alexander, Jacqueline C., Qiu, Chen, Wickens, Marv, Lo, Te-Wen, Tanaka Hall, Traci M., & Campbell, Zachary T. Engineering a conserved RNA regulatory protein repurposes its biological function in vivo. United States. doi:10.7554/eLife.43788.
Bhat, Vandita D., McCann, Kathleen L., Wang, Yeming, Fonseca, Dallas R., Shukla, Tarjani, Alexander, Jacqueline C., Qiu, Chen, Wickens, Marv, Lo, Te-Wen, Tanaka Hall, Traci M., and Campbell, Zachary T. Thu . "Engineering a conserved RNA regulatory protein repurposes its biological function in vivo". United States. doi:10.7554/eLife.43788.
@article{osti_1506498,
title = {Engineering a conserved RNA regulatory protein repurposes its biological function in vivo},
author = {Bhat, Vandita D. and McCann, Kathleen L. and Wang, Yeming and Fonseca, Dallas R. and Shukla, Tarjani and Alexander, Jacqueline C. and Qiu, Chen and Wickens, Marv and Lo, Te-Wen and Tanaka Hall, Traci M. and Campbell, Zachary T.},
abstractNote = {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 first 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.},
doi = {10.7554/eLife.43788},
journal = {eLife},
issn = {2050-084X},
number = 01, 2019,
volume = 8,
place = {United States},
year = {2019},
month = {1}
}