The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to mRNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multi-wavelength fluoresence microscopy to follow assembly of single spliceosomes in real time in whole cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes, and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

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This ED screening and diagnostic testing program found a high prevalence of hepatitis C virus antibody positivity across all groups. Challenges encountered with hepatitis C virus screening included result disclosure, confirmatory testing, and linkage to care. Our results warrant continued efforts to develop and evaluate policies for ED-based hepatitis C virus screening.