1. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA
2. Department of Cell Biology, Taplin Biological Mass Spectrometry Facility, Harvard Medical School, Boston, Massachusetts 02115, USA

Correspondence to: Robin Reed² Correspondence and requests for materials should be addressed to R.R. (e-mail: Email: rreed@hms.harvard.edu).

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' particles^{2, 3}. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography^{4, 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.