Comprehensive proteomic analysis of the human spliceosome.
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 ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 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.
Zhou
Zhaolan
Z
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Licklider
Lawrence J
LJ
Gygi
Steven P
SP
Reed
Robin
R
D020816:Amino Acid Motifs; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D005816:Genetic Complementation Test; D006801:Humans; D007438:Introns / Q000235:genetics; D046911:Macromolecular Substances; D058619:Maltose-Binding Proteins; D013058:Mass Spectrometry; D021122:Protein Subunits; D020543:Proteome / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry* / Q000235:genetics
D002352:Carrier Proteins; D046911:Macromolecular Substances; D058619:Maltose-Binding Proteins; D021122:Protein Subunits; D020543:Proteome; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins