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Ordered and dynamic assembly of single spliceosomes.

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.

Pubmed ID: 21393538

Authors

  • Hoskins AA
  • Friedman LJ
  • Gallagher SS
  • Crawford DJ
  • Anderson EG
  • Wombacher R
  • Ramirez N
  • Cornish VW
  • Gelles J
  • Moore MJ

Journal

Science (New York, N.Y.)

Publication Data

March 11, 2011

Associated Grants

  • Agency: NIGMS NIH HHS, Id: F32 GM079971
  • Agency: NIGMS NIH HHS, Id: F32 GM079971-03
  • Agency: NIGMS NIH HHS, Id: GM079971
  • Agency: NIGMS NIH HHS, Id: GM759628
  • Agency: NIGMS NIH HHS, Id: K99 GM086471
  • Agency: NIGMS NIH HHS, Id: K99 GM086471-02
  • Agency: NIGMS NIH HHS, Id: K99/R00 GM086471
  • Agency: NIGMS NIH HHS, Id: R01 GM043369
  • Agency: NIGMS NIH HHS, Id: R01 GM053007
  • Agency: NIGMS NIH HHS, Id: R01 GM053007
  • Agency: NIGMS NIH HHS, Id: R01 GM053007-15
  • Agency: NIGMS NIH HHS, Id: R01 GM081648
  • Agency: NIGMS NIH HHS, Id: R01 GM081648-04
  • Agency: NIGMS NIH HHS, Id: R01 GM54469
  • Agency: NIGMS NIH HHS, Id: R01 GM81648
  • Agency: NIGMS NIH HHS, Id: R37 GM043369
  • Agency: NIGMS NIH HHS, Id: R37 GM043369-21
  • Agency: NIGMS NIH HHS, Id: RC1 GM091804
  • Agency: NIGMS NIH HHS, Id: RC1 GM091804
  • Agency: NIGMS NIH HHS, Id: RC1 GM091804-02
  • Agency: NIGMS NIH HHS, Id: T32 GM007596
  • Agency: NIGMS NIH HHS, Id: T32 GM007596-30
  • Agency: Howard Hughes Medical Institute, Id:
  • Agency: Howard Hughes Medical Institute, Id:

Mesh Terms

  • Adenosine Triphosphate
  • Fluorescent Dyes
  • Introns
  • Kinetics
  • Microscopy, Fluorescence
  • Protein Binding
  • RNA Precursors
  • RNA Splicing
  • RNA, Fungal
  • Ribonucleoprotein, U1 Small Nuclear
  • Ribonucleoprotein, U2 Small Nuclear
  • Ribonucleoprotein, U4-U6 Small Nuclear
  • Ribonucleoprotein, U5 Small Nuclear
  • Ribonucleoproteins, Small Nuclear
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Spliceosomes