Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

  1. Aaron A Hoskins  Is a corresponding author
  2. Margaret L Rodgers
  3. Larry J Friedman
  4. Jeff Gelles
  5. Melissa J Moore
  1. University of Wisconsin-Madison, United States
  2. Brandeis University, United States
  3. Howard Hughes Medical Institute, University of Massachusetts Medical School, United States

Abstract

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.

Article and author information

Author details

  1. Aaron A Hoskins

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    For correspondence
    ahoskins@wisc.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Margaret L Rodgers

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Larry J Friedman

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jeff Gelles

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Melissa J Moore

    Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Jonathan P Staley, University of Chicago, United States

Publication history

  1. Received: January 3, 2016
  2. Accepted: May 26, 2016
  3. Accepted Manuscript published: May 31, 2016 (version 1)
  4. Version of Record published: June 27, 2016 (version 2)

Copyright

© 2016, Hoskins et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Aaron A Hoskins
  2. Margaret L Rodgers
  3. Larry J Friedman
  4. Jeff Gelles
  5. Melissa J Moore
(2016)
Single molecule analysis reveals reversible and irreversible steps during spliceosome activation
eLife 5:e14166.
https://doi.org/10.7554/eLife.14166

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