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The NMR structure of the 38 kDa U1A protein – PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein

Nature Structural Biology volume 7, pages 329–335 (2000)Cite this article

Abstract

The status of the poly(A) tail at the 3′-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3′-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.

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Figure 1: RNA sequences used in NMR studies of the U1A–PIE interaction.
Figure 2: Cooperativity of U1A binding depends on direct protein–protein interactions and is necessary for regulation of polyadenylation.
Figure 3: NMR data for the U1A–RNA complexes.
Figure 4: NOE interactions identify helix C as the site of protein–protein interactions.
Figure 5: Structure of the trimolecular U1A–PIE complex.
Figure 6: Close-up view of the intermolecular hydrophobic interactions involving Ile 93, Met 97, Gly 99, Thr 100 and Val 102.
Figure 7: The conformational change in helix C allows specific U1A–PAP interaction and selective regulation of PAP activity.

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Acknowledgements

We thank F. Allain and C. Gubser for their help at the initial stages of the project; M. Kelly (IMP Berlin) and K. Gardner (University of Toronto) for suggestions on the preparation of deuterated protein. L.V. acknowledges the support of an EU studentship and the MRC. We would also like to thank the Florence Large Scale facility for Biomolecular NMR, funded in part by the EU, for access to the 800 Mhz spectrometer.

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Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Luca Varani, David Neuhaus & Gabriele Varani

  2. EMBL, Gene Expression Program, 1 Meyerhofstrasse , Heidelberg, D-69117, Germany

    Samuel I. Gunderson & Iain W. Mattaj

  3. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, 08855, New Jersey, USA

    Samuel I. Gunderson

  4. Protein Engineering Network Centers of Excellence, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Lewis E. Kay

  5. Departments of Medical Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Gabriele Varani

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Correspondence to Gabriele Varani.

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Varani, L., Gunderson, S., Mattaj, I. et al. The NMR structure of the 38 kDa U1A protein – PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein . Nat Struct Mol Biol 7, 329–335 (2000). https://doi.org/10.1038/74101

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