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A single spacer nucleotide determines the specificities of two mRNA regulatory proteins

Nature Structural & Molecular Biology volume 12, pages 945–951 (2005)Cite this article

Abstract

Regulation of messenger RNA is crucial in many contexts, including development, memory and cell growth. The 3′ untranslated region is a rich repository of regulatory elements that bind proteins and microRNAs. Here we focus on PUF proteins, an important family of mRNA regulatory proteins crucial in stem-cell proliferation, pattern formation and synaptic plasticity. We show that two Caenorhabditis elegans PUF proteins, FBF and PUF-8, differ in RNA-binding specificity. FBF requires the presence of a single 'extra' nucleotide in the middle of an eight-nucleotide site, whereas PUF-8 requires its absence. A discrete protein segment is responsible for the difference. We propose that a structural distortion in the central region of FBF imposes the requirement for the additional nucleotide and that this mode of PUF specificity may be common. We suggest that new specificities can be designed and selected using the PUF scaffold.

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Figure 1: A single nucleotide is a key determinant of the RNA-binding specificity of two PUF proteins.
Figure 2: A single nucleotide determines RNA-binding specificity in vitro.
Figure 3: RNA selection in yeast reveals that FBF requires a single 'extra' nucleotide.
Figure 4: Effect of substitutions at the 'extra' nucleotide position on binding.
Figure 5: Alignment of PUF repeats and RNA.
Figure 6: FBF RNA-binding specificity maps to a discrete, central region of the protein.
Figure 7: Model of FBF and PUF-8 interaction with RNA.

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Acknowledgements

We thank members of the Kimble and Wickens laboratories for their suggestions, advice and comments on the manuscript, and we appreciate the suggestions of J. Kimble and S. Butcher. Figures were prepared by the University of Wisconsin Biochemistry Media Center. This work was supported by US National Institutes of Health grants to M.W. L.O. was supported by a US National Institutes of Health Molecular Biology Training Grant.

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  1. Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, 53706, Wisconsin, USA

    Laura Opperman, Brad Hook, Mia DeFino, David S Bernstein & Marvin Wickens

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  1. Laura Opperman
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Correspondence to Marvin Wickens.

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Supplementary information

Supplementary Fig. 1

Alignment of RNA binding region of PUF proteins. (PDF 120 kb)

Supplementary Fig. 2

GST–FBF-2 and GST–Puf-8 proteins. (PDF 39 kb)

Supplementary Fig. 3

Junction sequences in FBF-1–PUF-8 chimeras. (PDF 43 kb)

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Opperman, L., Hook, B., DeFino, M. et al. A single spacer nucleotide determines the specificities of two mRNA regulatory proteins. Nat Struct Mol Biol 12, 945–951 (2005). https://doi.org/10.1038/nsmb1010

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