Visualizing the Splicing of Single Pre-mRNA Molecules in Whole Cell Extract

Overview

Journal RNA

Specialty Molecular Biology

Date 2007 Nov 21

PMID 18025254

Citations 61

Authors

Daniel J Crawford

Aaron A Hoskins

Larry J Friedman

Jeff Gelles

Melissa J Moore

Affiliations

Soon will be listed here.

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.

Citing Articles

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Dynamics and Evolutionary Conservation of B Complex Protein Recruitment During Spliceosome Activation.

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