Part of the Methods in Molecular Biology™ book series (MIMB, volume 118)
Yeast Pre-mRNA Splicing Extracts
Splicing of eukaryotic precursor messenger RNAs (pre-mRNAs) excises the intron from the precursor and ligates the two exons together to produce the mature mRNA. It occurs via a two-step mechanism (Fig. 1) (reviewed in ref. 1). In the first step the 2′ hydroxyl group of an intronic adenylyl residue initiates a transesterfication reaction at the 5′ splice site. The result is the cleavage of the 5′ splice site phosphodiester bond and the formation of a new, 2′–5′ phosphodiester bond between the adenylyl residue and the 5′ end of the intron. Because the intronic adenylyl residue has both 2′–5′ and 3′–5′ phosphodiester bonds, it is commonly referred to as the branch point nucleotide. The first splicing step yields two intermediates, “free” exon 1 and the lariat intermediate, which are not covalently linked together. In the second step, the 3′ hydroxyl of the “free” exon 1 initiates a second transesterification reaction at the 3′ splice site, resulting in the ligation of the two exons together to form the mRNA and the release of the lariat intron.
KeywordsGlycerol EDTA Electrophoresis MgCl2 Fractionation
- 1.Moore, M. J., Query, C. C., and Sharp, P. A. (1993) Splicing of precursors to mRNA by the spliceosome, in The RNA World (Gesteland, R. F. and J. F. Atkins, eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 303–358.Google Scholar
© Humana Press Inc. 1999