Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

Abstract

A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[clphenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcriptionpolymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures.The development of cell-free splicing systems has led to rapid and continuing progress in understanding biochemical mechanisms involved in intron removal and in the definition of the spliceosomal machinery. However, the exact requirements for recognition of splices sites and the rules governing splice site selection remain unclear. Analysis of consensus sequences provided the first definition of splice sites (49). Most efforts to move beyond that initial step have focused on site-directed mutagenesis studies (for reviews, see references 4 and 31). In these experiments, the effects of splice site mutations are typically assayed by using cell-free splicing extracts (in. vitro) or after transient transfection of cultured mammalian cells or injection of RNA directly into frog oocytes (in vivo).Conflicting results between these in vitro and in vivo assays for the effect of mutations on splicing have frequently been reported (2,38,39,45,83), raising the possibility that artifacts are being introduced in one system or the other. Moreover, recent experiments suggesting that the splicing machinery may be compartmentalized within t...

Dichotomous splicing signals in exon flanks

“…Exon 2+3 with its splice consensus sequences was replaced by a NotI site to serve as an insertion site for heterologous exons. Thus, the sequences surrounding the NotI insertion site in the test exon retain the original dhfr flanks, including the branch point of intron 1 at ‫42מ‬ (Chen and Chasin 1993). The experiment was designed in this way to increase the chance of revealing sequences or sequence relationships that influence splicing over and above the presence of a functional branch site; a similar consideration applies to the downstream flank; the flank deletion experiments here are testing for effects that cannot be fulfilled by any ISEs that might be present in the original dhfr flank.…”

Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes

“…4) (44). Additional constructs with variations characterized as splicing mutations in prior reports (45) were designed as sensitized reporters. Inserts were selected on the basis of their match to the cluster motif.…”

Computational definition of sequence motifs governing constitutive exon splicing

“…Thus, the induction of exon skipping was not caused by the mere introduction of a foreign sequence. We have previously shown that the insertion of much larger sequences (>100 nt) does not usually compromise splicing (Chen and Chasin 1994;Fairbrother and Chasin 2000). Interestingly, one of the two PESSs that did not inhibit splicing as a single copy, PS12, constitutes a CACACACA repeat-a sequence that has been characterized as an intronic enhancer (Hui et al 2003).…”