Pre-mRNA Splicing

Traditionally, a splicing construct contains an intron flanked by two exons. However, interactions across the exons play an important role in spliceosome assembly, a phenomenon termed exon definition.6 The presence of an upstream 3 -splice site generally stimulates the function of a weak downstream 5 -splice site. Similarly, a downstream 5 -splice site induces the usage of a weak upstream 3 -splice site. In this transactivation model, the 5 -cap structure of the first exon and the polyadenylation site in the terminal exons act as 3 - and 5 -splice sites, respectively, in promoting the spliceosome assembly. This view is reinforced by the observation that less efficiently spliced introns may be stimulated by moving the 5 -cap closer to the 5 -splice site or by inserting a functional 5 -splice- or polyadenylation site in the 3 -exon. However, exon definition seems only to play a minor role for efficiently spliced introns in vitro. 

Although the splicing procedure below works well for most introns, the efficiency can generally be improved considerably by titrating the ionic conditions as discussed below. 

Bulk E. coli tRNA 5 jxg/ jl1 (RNase free from Boehringer Mannheim) 

Nuclear extract from HeLa cells prepared as described in Section 

2 mM EDTA 1 mM DTT 10% glycerol Splice stop buffer 0.5% SDS 5 mM EDTA 0.1 MNaCl 

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