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Comprehensive proteomic analysis of the human spliceosome

Nature volume 419, pages 182–185 (2002)Cite this article

Abstract

The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins1 and salt-stable spliceosome ‘core’ particles2,3. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography4,5 to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry6, we identify ∼145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

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Figure 1: Isolation of spliceosomes.
Figure 2: The MBP-purified spliceosomes are functional in an in vitro complementation assay.

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Acknowledgements

We thank. N. Dorman, E. Ibrahim and K. Magni for comments on the manuscript; C. Thoreen for database support; R. Das for SF3a-depleted nuclear extract; and R. Luhrmann and G. Dreyfuss for antibodies. HeLa cells were obtained from the National Cell Culture Center. This work was supported by the NIH (S.P.G. and R.R.).

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, Harvard University, Massachusetts, 02138, Cambridge, USA

    Zhaolan Zhou

  2. Department of Cell Biology, Taplin Biological Mass Spectrometry Facility, Harvard Medical School, Massachusetts, 02115, Boston, USA

    Lawrence J. Licklider, Steven P. Gygi & Robin Reed

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  1. Zhaolan Zhou
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  2. Lawrence J. Licklider
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  3. Steven P. Gygi
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  4. Robin Reed
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Correspondence to Robin Reed.

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Zhou, Z., Licklider, L., Gygi, S. et al. Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182–185 (2002). https://doi.org/10.1038/nature01031

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