Abstract
Spliceosome assembly is a highly dynamic and complex process involving the addition of 5 snRNAs and >100 proteins to a pre-mRNA transcript. This process has largely been analyzed by discontinuous assays that provide limited kinetic information. By combining yeast genetics, chemical biology, and a multi-wavelength single molecule microscopy technique (Colocalization Single Molecule Spectroscopy, CoSMoS), we monitored the formation of single spliceosomes. This approach revealed that individual spliceosome subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes. No single subcomplex binding event disproportionately limits the speed of the assembly reaction. While not all pre-mRNAs acquire a functional spliceosome, commitment of pre-mRNAs to splicing increases as assembly progresses. Therefore, spliceosome assembly is not a one-way, unbranched process as some models depict. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in complex environments.
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