Visualizing the splicing of single pre-mRNA molecules in whole cell extract.
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.
Crawford
Daniel J
DJ
Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02453, USA.
Hoskins
Aaron A
AA
Friedman
Larry J
LJ
Gelles
Jeff
J
Moore
Melissa J
MJ
D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D012322:RNA Precursors; D012331:RNA, Fungal; D012333:RNA, Messenger