Visualizing the splicing of single pre-mRNA molecules in whole cell extract

  1. Daniel J. Crawford1,2,3,4,
  2. Aaron A. Hoskins1,3,4,
  3. Larry J. Friedman2,
  4. Jeff Gelles2, and
  5. Melissa J. Moore1,4
  1. 1Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02453, USA
  2. 2Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, USA

  1. ↵3 These authors contributed equally to this work.

Abstract

The excision of introns from nascent eukaryotic transcripts is catalyzed by the spliceosome, a highly complex and dynamic macromolecular machine composed of RNA and protein. Because of its complexity, biochemical analysis of the spliceosome has been previously limited to bulk assays in largely unfractionated cell extracts. We now report development of methodologies for studying the splicing of isolated single pre-mRNA molecules in real time. In this system, a fluorescently tagged pre-mRNA is tethered to a glass surface via its 3′-end. Splicing can be observed in Saccharomyces cerevisiae whole cell extract by monitoring loss of intron-specific fluorescence with a multi-wavelength total internal reflection fluorescence (TIRF) microscope. To prolong fluorophore lifetime, two enzyme-based O2 scavenging systems compatible with splicing were also developed. This work provides a powerful new approach for elucidating the mechanisms of spliceosome function and demonstrates the feasibility of utilizing TIRF microscopy for biochemical studies of single molecules in highly complex environments.

Keywords

Footnotes

  • ↵4 Present address: Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA.

  • Reprint requests to: Jeff Gelles, Department of Biochemistry, Brandeis University, Waltham, MA 02453, USA; e-mail: Gelles{at}brandeis.edu; fax: (781) 736-2349; or Melissa J. Moore, Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA; e-mail: Melissa.Moore{at}umassmed.edu; fax: (508) 856-1002.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.794808.

    • Received August 24, 2007.
    • Accepted October 11, 2007.
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  1. Published in Advance November 19, 2007, doi: 10.1261/rna.794808 RNA 2008. 14: 170-179 Copyright © 2008 RNA Society

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