期刊
RNA
卷 14, 期 1, 页码 170-179出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.794808
关键词
RNA splicing; single molecule fluorescence; spliceosome; yeast
资金
- NIGMS NIH HHS [GM00714, R01 GM043369, R01 GM053007, R37 GM043369, K25 GM000714, GM043369, F32 GM079971, GM759628, T32 GM007596, GM081648, R01 GM081648, GM079971, GM53007] Funding Source: Medline
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM007596, R01GM043369, R37GM043369, R01GM081648, F32GM079971, R01GM053007] Funding Source: NIH RePORTER
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 O-2 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.
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