Abstract
Excision of introns from pre-mRNAs is mediated by the spliceosome, a large, dynamic complex consisting of five small ribonucleoprotein particles (snRNPs) and scores of associated proteins. Current understanding of spliceosome assembly is based largely on the procession of stable complexes that can be resolved from in vitro splicing reactions. Such ensemble experiments have suggested a highly ordered, linear assembly pathway in which initial binding of U1 snRNP to the 5′ splice site is followed by stable U2 association with the branch site and subsequent U4/U5/U6 tri-snRNP and Nineteen Complex (NTC) addition to form the fully assembled spliceosome. Previously unknown, however, were the detailed forward and reverse kinetics of each assembly step, the extent to which branched and/or dead-end assembly pathways exist, and whether or not different introns utilize the same or alternate assembly pathway(s). We are now addressing these questions by combining yeast genetic engineering, chemical biology, and multi-wavelengthfluoresence microscopy to follow assembly of single spliceosomes in real time. Because no protein purification or reconstitution is required for such Colocalization Single Molecule Spectroscopy (CoSMoS), this experimental strategy should prove widely useful for mechanistic analysis of many other macromolecular machines in environments approaching the complexity of living cells.