Bhat, Vandita D.McCann, Kathleen L.Wang, YemingFonseca, Dallas R.Shukla, TarjaniAlexander, Jacqueline C.Qiu, ChenWickens, MarvLo, Te-WenHall, Traci M. TanakaCampbell, Zachary T.2020-09-182020-09-182019-01-172050-084Xhttps://dx.doi.org/10.7554/eLife.43788https://hdl.handle.net/10735.1/8906PUF (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.enCC0 1.0 (Public Domain Dedication)No Copyrighthttps://creativecommons.org/publicdomain/zero/1.0/MitosisLife sciencesPUF-8 protein, C elegansfem-3-binding protein, C elegansBiological Science DisciplinesarticleBhat, Vandita D., Kathleen L. McCann, Yeming Wang, Dallas R. Fonseca, et al. 2019. "Engineering a conserved RNA regulatory protein repurposes its biological function in vivo." eLife 8: art. e43788, doi: 10.7554/eLife.437888