Selection of Novel Exon Recognition Elements from a Pool of Random Sequences

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Abstract
A 20-nucleotide sequence close to the 3' end of the internal exon of a model two-intron, three-exon pre-mRNA (DUP184 [Z. Dominski and R. Kole, J. Biol. Chem. 269:23590-23596, 1994]) was replaced by a random 20-mer, resulting in a pool of pre-mRNAs which, like the initial DUP184 construct, were spliced in vitro by a pathway leading to predominant skipping of the internal exon. The randomized pre-mRNAs were subjected to a selection protocol, resulting in a pool enriched in pre-mRNAs that efficiently included the internal exon. Isolation and sequencing of a number of clones corresponding to the selected pre-mRNAs showed that two classes of sequences were selected from the initial pool. Most abundant among these were sequences with purine tracts similar to those in the recently identified exon-splicing enhancers while a smaller class included sequences lacking discernible purine tracts within the 20-nucleotide region. Splicing of selected pre-mRNAs showed that the purine tracts vary in their ability to promote exon inclusion and, more important, that sequences lacking purine tracts stimulate inclusion of the internal exon as efficiently as their purine-rich counterparts. The latter result indicates the existence of a novel class of exon recognition sequences or splicing enhancers.