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Early organization of pre-mRNA during spliceosome assembly

Nature Structural Biology volume 9, pages 576–581 (2002)Cite this article

Abstract

Intron excision from precursor mRNAs (pre-mRNAs) in eukaryotes requires juxtaposition of reactive functionalities within the substrate at the heart of the spliceosome where the two chemical steps of splicing occur. Although a series of interactions between pre-mRNAs, pre-spliceosomal and spliceosomal factors is well established, the molecular mechanisms of splicing machinery assembly, as well as the temporal basis for organization of the substrate for splicing, remain poorly understood. Here we have used a directed hydroxyl radical probe tethered to pre-mRNA substrates to map the structure of the pre-mRNA substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly/recruitment and suggest a mechanism for the formation of the final active site of the mature spliceosome.

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Figure 1: Synthesis and splicing of pre-mRNA site-specifically modified with Fe-BABE.
Figure 2: Directed hydroxyl radical probing in the spliceosome and pre-spliceosome.
Figure 3: Factor and sequence dependence of E complex formation and Fe-BABE-mediated cleavages.
Figure 4: Early organization of pre-mRNA in spliceosome assembly.

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Acknowledgements

We would like to thank C. Query, K. Wilson, M. Glover, S. Chaulk and A. Ghetu for helpful advice and discussions and D. Sigman and C. Meares for help in the early stages of this work. O.A.K. is supported by a graduate fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR). This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and by an establishment grant from AHFMR to A.M.M.

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  1. Department of Biochemistry, University of Alberta, Edmonton, T6G 2H7, Alberta, Canada

    Oliver A. Kent & Andrew M. MacMillan

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  1. Oliver A. Kent
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  2. Andrew M. MacMillan
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Correspondence to Andrew M. MacMillan.

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Kent, O., MacMillan, A. Early organization of pre-mRNA during spliceosome assembly. Nat Struct Mol Biol 9, 576–581 (2002). https://doi.org/10.1038/nsb822

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