Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site
Department of Biochemistry and Molecular Pharmacology
Medical Subject Headings
Introns; RNA Splicing
Biochemistry, Biophysics, and Structural Biology | Molecular Biology
Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.
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Citation: Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):6783-8. doi: 10.1073/pnas.1219305110. Link to article on publisher's site
Crawford, Daniel J.; Hoskins, Aaron A.; Friedman, Larry J.; Gelles, Jeff; and Moore, Melissa J., "Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site" (2013). University of Massachusetts Medical School Faculty Publications. 81.