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Splicing-related catalysis by protein-free snRNAs

Nature volume 413, pages 701–707 (2001)Cite this article

Abstract

Removal of intervening sequences from eukaryotic messenger RNA precursors is carried out by the spliceosome, a complex assembly of five small nuclear RNAs (snRNAs) and a large number of proteins. Although it has been suggested that the spliceosome might be an RNA enzyme, direct evidence for this has been lacking, and the identity of the catalytic domain of the spliceosome is unknown. Here we show that a protein-free complex of two snRNAs, U2 and U6, can bind and position a small RNA containing the sequence of the intron branch site, and activate the branch adenosine to attack a catalytically critical domain of U6 in a reaction that is related to the first step of splicing. Our data provide direct evidence for the catalytic potential of spliceosomal snRNAs.

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Figure 1: Base-pairing interactions in the in vitro-assembled complex of U2–U6 and the branch oligonucleotide (Br).
Figure 2: Characterization of RNA X.
Figure 3: Mapping the site of the covalent linkage in RNA X.
Figure 4: Characterization of the new covalent bond.
Figure 5: Mutagenesis of the branch oligonucleotide and U6.
Figure 6: Requirement of the branchpoint 2′OH: a model for the linkage between U6 and Br in RNA X.

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Acknowledgements

We thank K. Ryan for advice and helpful discussions and insights, T. Nilsen for suggestions and comments on the manuscript, and A. Pyle for helpful discussions, advice and suggestions. We also thank G. Zubay, D. Herschlag, T. Tuschl, M. Konarska, G. Just, H. Lonnberg and F. Eckstein for discussions and advice on the chemistry of the reaction, and H. Robertson and D. Bartel for discussion and comments. We also thank P. Nouri for technical assistance. This work is supported by the NIH.

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  1. Department of Biological Sciences, Sherman Fairchild Center of Life Sciences, Columbia University, New York, 10027, New York, USA

    Saba Valadkhan & James L. Manley

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  1. Saba Valadkhan
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  2. James L. Manley
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Correspondence to James L. Manley.

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Valadkhan, S., Manley, J. Splicing-related catalysis by protein-free snRNAs. Nature 413, 701–707 (2001). https://doi.org/10.1038/35099500

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