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Single-molecule analysis of protein-free U2–U6 snRNAs

Nature Structural & Molecular Biology volume 16, pages 1154–1159 (2009)Cite this article

Abstract

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5 and U6) involved in substrate positioning and catalysis. It has been postulated, but never shown experimentally, that the U2–U6 complex adopts at least two conformations that reflect different activation states. We have used single-molecule fluorescence to probe the structural dynamics of a protein-free RNA complex modeling U2–U6 from yeast and mutants of highly conserved regions of U2–U6. Our data show the presence of at least three distinct conformations in equilibrium. The minimal folding pathway consists of a two-step process with an obligatory intermediate. The first step is strongly magnesium dependent, and we provide evidence suggesting that the second step corresponds to the formation of the genetically conserved helix IB. Site-specific mutations in the highly conserved AGC triad and the U80 base in U6 suggest that the observed conformational dynamics correlate with residues that have an important role in splicing.

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Figure 1: Secondary structure model of the spliceosomal snRNAs U2–U6.
Figure 2: Single-molecule FRET reveals a three-state folding pathway.
Figure 3: Mg2+ dependence of the folding rate constants for the U2–U6 complex.
Figure 4: Mutations in the AGC triad (red) show that helix IB forms only in the low FRET state.
Figure 5: smFRET histograms for the U80 mutations (U80, U80G, U80A and U80C) in 5 and 40 mM Mg2+, as indicated.

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Acknowledgements

We would like to thank S. Butcher and D. Brow for many helpful and stimulating discussions and for commenting on the manuscript. This work was supported by the NIH (R01 GM085116) and an NSF CAREER award to D.R. (MCB-0747285).

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Authors and Affiliations

  1. Department of Chemistry, Wayne State University, Detroit, Michigan, USA

    Zhuojun Guo, Krishanthi S Karunatilaka & David Rueda

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  1. Zhuojun Guo
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  2. Krishanthi S Karunatilaka
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  3. David Rueda
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Contributions

Z.G. performed experiments, analyzed data and wrote the manuscript; K.S.K. performed experiments and edited the manuscript; D.R. designed experiments and wrote the manuscript.

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Correspondence to David Rueda.

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Guo, Z., Karunatilaka, K. & Rueda, D. Single-molecule analysis of protein-free U2–U6 snRNAs. Nat Struct Mol Biol 16, 1154–1159 (2009). https://doi.org/10.1038/nsmb.1672

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