Identification of splicing silencers and enhancers in sense Alus: a role for pseudoacceptors in splice site repression

Auxiliary splicing signals in introns play an important role in splice site selection, but these elements are poorly understood. We show that a subset of serine/arginine (SR)-rich proteins activate a cryptic 3' splice site in a sense Alu repeat located in intron 4 of the human LST1 gene. Utilization of this cryptic splice site is controlled by juxtaposed Alu-derived splicing silencers and enhancers between closely linked short tandem repeats TNFd and TNFe. Systematic mutagenesis of these elements showed that AG dinucleotides that were not preceded by purine residues were critical for repressing exon inclusion of a chimeric splicing reporter. Since the splice acceptor-like sequences are present in excess in exonic splicing silencers, these signals may contribute to inhibition of a large number of pseudosites in primate genomes.     

Human genomic sequences that inhibit splicing

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 pseudosites 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 three-exon minigene, about one in three inhibited inclusion, revealing a high frequency of inhibitory elements in human DNA. In contrast, only 1 in 27 Escherichia 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, since many of our inhibitors contain splice like signals, we suggest that repression of some pseudosites may occur through an inhibitory arrangement of these sites.