Journal
SCIENCE
Volume 331, Issue 6022, Pages 1289-1295Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.1198830
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Funding
- NIH [RO1s GM043369, GM81648, GM053007, GM54469, RC1 GM091804, GM759628]
- National Research Service Award fellowship [GM079971, K99/R00 GM086471]
- National Defense Science and Engineering Graduate fellowship
- Deutscher Akademischer Austausch Dienst fellowship
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The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence 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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