Exon inclusion is dependent on predictable exonic splicing enhancers - PubMed (original) (raw)

Exon inclusion is dependent on predictable exonic splicing enhancers

Xiang H-F Zhang et al. Mol Cell Biol. 2005 Aug.

Abstract

We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.

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Figures

FIG. 1.

FIG. 1.

Characteristics of PESE mutations. Site-directed mutagenesis was used to create point mutations within the indicated 6 exons positioned as the central exon in a 3-exon minigene. The sequence changes accompanying the 22 mutations created in PESEs are shown, with the mutated bases in bold. PESE scores for 8-mers overlapping the mutated sites are given as calculated previously (51) either on the basis of comparing exons with pseudo exons (P) or with the 5′UTRs of intronless genes (I). Only those overlapping 8-mers with PESE scores >2.88 based on both criteria are shown. The numbers indicate the first and last nucleotide of an octamer or cluster. The percent splicing (100 × spliced/[spliced + skipped]) is based on RT-PCR of transfected 293 cells. The boldface entries indicate a reduction in splicing to less than half that of the wild type (16 cases) or an increase in skipping of at least 10-fold (2 cases in wt1-5). The last three columns show the changes with respect to other methods of ESE prediction. The column labeled RED indicates whether the mutation also resulted in a RESCUE-ESE disruption. In the column labeled EFD, a letter or numeral indicates a disruption of an ESE predicted by ESEfinder; here the splicing factor specificity is denoted as follows: A, ASF/SF2; S, SC35; 4, SRp40; 5, SRp55. In the last column RESCUE-ESEs that do not fall within PESE sequences are noted. Mut. No., mutation number.

FIG.2.

FIG.2.

Characteristics of PESE and control mutants. The black curves in the graphs show the PESE score profile across the exon. Successive windows of 8 nt were scored as described in Zhang and Chasin (51); the average of two scoring systems (P and I) is plotted for simplicity (separate profiles for each scoring system can be seen in Fig. 4). The score for each 8-mer is plotted near the middle of the 8-mer, at position 4. Point mutations in 22 predicted ESE clusters are indicated by the red arrows. The scores for the mutant sequences are shown by the red curves where they differ from the wild type. Twenty-four control mutations were of two types, both indicated by blue arrows and curves: 12 control mutations in PESE clusters were designed to not substantially change the PESE score and 12 control mutations outside PESE or PESS clusters also had little effect on the PESE score. Splicing phenotypes are indicated by the extent of filling of the rectangles below each mutated region. The green rectangles at the left show the extent of splicing (spliced/[spliced + skipped]) for the wild-type exons. The red and blue rectangles show the extent of splicing of the experimental and control mutants, respectively. To point out the effect of mutations on splicing, the colored areas represent the extent of splicing relative to that of the wild type set equal to 1; the black regions above the red or green areas indicate the range of values from two or three replicate transfections. Note that the splicing data presented in Fig. 1 and Table 1 have not been normalized in this way. The last 40 nt of the _dhfr-2_-3 exon were not mutated and are not shown. cont., continued.

FIG. 3.

FIG. 3.

Splicing patterns in PESE mutants. A. General map of the constructs used. The dashed boxes indicate that exon flanks (∼50 nt) were included for most exons (chuk-8, _clcn7_-3, _wt1_-5, _dhfr-2_-3) and excluded for hbb-2 and _thbs4_-12 to generate a partial splicing phenotype for the wild-type exon in these cases. B. HEK 293 cells were transfected with mutant plasmids, and the resulting mRNA was amplified by RT-PCR using radioactive dATP, separated in polyacrylamide gels and subjected to phosphorimaging. The lane numbers reflect the order of the PESE disruption mutations within each exon, shown in Fig. 2. The results of one of duplicate experiment are shown. Also shown are control mutations in _chuk_-8 that do not disrupt a PESE. Control mutations in the other exons are not shown. The PhosphorImager data was converted to a tiff file and formatted using Corel PhotoPaint. C. Dependence of PhosphorImager signal intensity on PCR cycle number. D. Dependence of PhosphorImager signal intensity on the amount of RNA input. Two microliters, corresponding to the total RNA from approximately 15,000 cells, were routinely used for the RT reaction. W, wild type.

FIG. 4.

FIG. 4.

Comparison of different ESE prediction methods. A to F. The graphs show PESE scores for the six tested exons. Unlike the graphs in Fig. 2, two curves are shown here: the dark curves depict scores based on a comparison of real exons to pseudo exons and the light curve is based on a comparison to the 5′UTRs of intronless genes (51). The vertical lines under the curves denote the starting positions of individual PESEs. The dashed vertical line in panel F indicates the dhfr exons 2 and 3 joint. The rectangle directly above each graph depict the positions of ESE 6-mers predicted by the RESCUE method (15). The top rectangle in each panel depicts the positions ESEs predicted by ESEfinder (8), based on the consensuses of sequences selected for function from among random oligomers (27, 28).

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