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Science. 2011 Mar 11;331(6022):1289-95. doi: 10.1126/science.1198830.

Ordered and dynamic assembly of single spliceosomes.

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  • 1Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Abstract

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.

PMID:
21393538
[PubMed - indexed for MEDLINE]
PMCID:
PMC3086749
Free PMC Article

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