Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

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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.

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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.
Margaret L Rodgers

Department of Biochemistry, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.
Larry J Friedman

Department of Biochemistry, Brandeis University, Waltham, United States

Competing interests
The authors declare that no competing interests exist.
Jeff Gelles

Department of Biochemistry, Brandeis University, Waltham, United States

Competing interests
The authors declare that no competing interests exist.
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.

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