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

Nature Structural & Molecular Biology volume 16, pages 11541159 (2009) | Download Citation

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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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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).

Author information

Affiliations

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

    • Zhuojun Guo
    • , Krishanthi S Karunatilaka
    •  & 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.

Corresponding author

Correspondence to David Rueda.

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DOI

https://doi.org/10.1038/nsmb.1672

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