Human Genomic Sequences that Inhibit Splicing 

Mol. Cell. Biol., in press, 2000

William G. Fairbrother and Lawrence A. Chasin

ABSTRACT

Mammalian genes are characterized by relatively small exons surrounded by variable lengths of intronic sequence. Sequences similar
to the splice signals that define the 5’ and 3’ boundaries of these exons are also present in abundance throughout the surrounding introns. What causes the real sites to be distinguished from the multitude of pseudo sites in pre-mRNA is unclear. Much progress has been made in defining additional sequence elements that enhance the use of particular sites. Less work has been done on sequences that repress the use of particular splice sites. To find additional examples of sequences that inhibit splicing, we searched human genomic DNA libraries for sequences that would inhibit the inclusion of a constitutively spliced exon. Genetic selection experiments suggested that such sequences were common, and we subsequently tested randomly chosen restriction fragments of about 100 bp. When inserted into the central exon of a 3-exon minigene, about 1 in 3 inhibited inclusion, revealing a high frequency of inhibitory elements in human DNA. In contrast, only 1 in 27 E. coli DNA fragments was inhibitory. Several previously identified silencing elements derived from alternatively spliced exons functioned weakly in this constitutively spliced exon. In contrast, a high affinity site for U2AF65 strongly inhibited exon inclusion. Together, our results suggest that splicing occurs in a background of repression and, as many of our inhibitors contain splice like signals, we suggest that repression of some pseudo sites may occur through an inhibitory arrangement of these sites.