Abstract
The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5′ and 3′ splice sites. During splicing in vitro, we observed a multitude of generally reversible time- and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3′–splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.
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Acknowledgements
The authors wish to thank R. Lührmann and P. Fabrizio (Max Planck Institute for Biophysical Chemistry), J. Staley (Univ. Chicago) and B. Schwer (Weill Cornell Medical College) for providing splicing active yeast cell extracts at a moment, common in this field, in which we were having difficulty in making active extracts, as well as G. Whitworth, J. Staley and H. Hadjivassiliou for helpful comments on the manuscript. The oligonucleotides synthesized at Dharmacon were sometimes much longer than those they usually make, and their excellent quality was essential for this project. The work at the University of Michigan was supported by US National Institutes of Health (NIH) grant GM062357 to N.G.W. and NIH Cellular & Molecular Biology and Molecular Biophysics Training Grant fellowships to M.B. and M.A.D., respectively, as well as a Rackham Merit Fellowship to M.B. The work at the University of California, San Francisco (UCSF), was supported by an American Cancer Society Research Professor of Molecular Genetics award to C.G., by NIH grant GM021119 to C.G., by NIH postdoctoral fellowship GM077844 to C.M. and by a grant from the Agouron Institute to J.A.
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J.A. worked at the bench and led the development of the Ubc4 system at UCSF; J.A.P. and J.A. performed the microarray analysis in Supplementary Table 1; D.E.R., T.V. and C.M. participated in various phases of the biochemistry at UCSF; M.B., M.A.D., F.F. and M.W. performed the smFRET experimentation and data analysis; P.A. performed the secondary structure analysis at the University of Michigan; J.A., M.A.D., M.B., C.M., C.G. and N.G.W. wrote the manuscript.
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Supplementary Figures 1–7, Supplementary Tables 1 and 2, Supplementary Methods (PDF 7415 kb)
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Abelson, J., Blanco, M., Ditzler, M. et al. Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nat Struct Mol Biol 17, 504–512 (2010). https://doi.org/10.1038/nsmb.1767
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DOI: https://doi.org/10.1038/nsmb.1767
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