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

Elife. 2019 Jan 17:8:e43788. doi: 10.7554/eLife.43788.

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.

Keywords: C. elegans; FBF; Motif; PUF; RNA-binding; developmental biology; elegans; molecular biophysics; pumilio; structural biology.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Caenorhabditis elegans / physiology*
  • Caenorhabditis elegans Proteins / chemistry
  • Caenorhabditis elegans Proteins / physiology*
  • Crystallography, X-Ray
  • Protein Conformation
  • Protein Engineering*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / physiology*
  • Two-Hybrid System Techniques

Substances

  • Caenorhabditis elegans Proteins
  • RNA-Binding Proteins