SELECTION OF NOVEL EXON RECOGNITION ELEMENTS FROM A POOL OF RANDOM SEQUENCES

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. Kol e, 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 c onstruct, were spliced in vitro by a pathway leading to predominant sk ipping of the internal exon. The randomized pre-mRNAs were subjected t o 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 abu ndant among these were sequences with purine tracts similar to those i n the recently identified exon-splicing enhancers, while a smaller cla ss included sequences lacking discernible purine tracts within the 20- nucleotide region. Splicing of selected pre-mRNAs showed that the puri ne tracts vary in their ability to promote exon inclusion and, more im portant, that sequences lacking purine tracts stimulate inclusion of t he internal exon as efficiently as their purine-rich counterparts. The latter result indicates the existence of a novel class of exon recogn ition sequences or splicing enhancers.