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
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Caenorhabditis elegans / physiology*
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Caenorhabditis elegans Proteins / chemistry
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Caenorhabditis elegans Proteins / physiology*
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Crystallography, X-Ray
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Protein Conformation
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Protein Engineering*
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / physiology*
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Two-Hybrid System Techniques
Substances
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Caenorhabditis elegans Proteins
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RNA-Binding Proteins