5580 Nucleic Acids Research, 2005, Vol. 33, No. 17 Figure 5. Differences between the including and skipping isoforms of 30 random non-symmetrical alternative exons. Six different types of pairs of inclusion/ skipping isoforms were grouped into three categories (A–C). In each pair, the upper and lower isoforms represent the including and skipping mRNA, respectively. The accession numbers of all inclusion/skipping pairs that represent this category are indicated on the right (including isoform, skipping isoform). Accession numbers of PTC+ mRNAs are in bold. Open boxes indicate the same reading frame for both isoforms, and boxes with vertical lines indicate different reading frames. The boxes represent only the CDS portion of the mRNA. In category (A), skipping of the alternative exon induces a frameshift downstream from the exon. In 11 cases, the including and skipping isoforms have the same start codon (ATG). In five cases, the two isoforms use different ATGs, but the reading frame upstream from the alternative exon remains the same. In category (B), use of an alternative ATG ensures that both isoforms share the same reading frame downstream from the alternative exon. In two cases, both ATGs are upstream from the exon, and, thus, the two isoforms have a different reading frame upstream from the exon. In seven cases, the skipping isoform uses an alternative in-frame ATG downstream from the alternative exon. In the other two cases, an alternative in-frame ATG upstream from the exon is used, but an alternative splicing event downstream from the exon introduces a different stop codon at the skipping isoform. In category (C), both isoforms differ in their start and stop codons and do not share the same reading frame throughout the CDS. Table 1. Differences between symmetrical and non-symmetrical alternative exons and constitutive ones, with respect to several characteristics Characteristic Percentage of total CDS-internal exons Average exon size (nt) Percentage of exons that overlap with protein-domains Totalb Medium-sizedc Full domaind Percentage of exons with mRNA evidence of skipping Average inclusion level Average location in RefSeq mRNAs Alternative exons Symmetrical Non-Symmetrical Constitutive exons Symmetrical Non-Symmetrical 68.6% 113.3 30.3% 15% 2.3% 92.2% 0.59 0.49 31.4% 116.9a 53% 39.4% 0%a 42.7% 0.74 0.39 40% 128.4 54% 34.8% 2.6% 0.51 60% 131.2 54.3%a 36%a 0.06% 0.51a aDifference did not reach statistical significance. bPercentage of exons overlapping with protein-domains fragments or complete protein-domains. cPercentage of exons overlapping with medium-sized fragments (10–60%) of protein-domains. dPercentage of exons that possess a complete protein-domain. category B, where both isoforms share the same reading frame downstream from the alternative exon, the average location is 0.21 (median 0.19). These ﬁndings show that the location of these alternative non-symmetrical exons ensures that most of the reading frame remains unchanged in both the skipping and including isoforms. For comparison, the average normal- ized location of the 30 symmetrical exons is 0.48 (median 0.49), indicating no preference of these exons for speciﬁc locations within the CDS. DISCUSSION In this report, we found that conserved symmetrical and non- symmetrical alternative exons differ with respect to several characteristics. We showed that the tendency of symmetrical alternative exons to overlap with protein domains is signiﬁc- antly lower, compared with non-symmetrical alternative exons and all constitutive exons. This low tendency results from the bias of symmetrical alternative exons not to overlap with medium-sized fragments (10–60%) of protein domains. We also showed that non-symmetrical alternative exons tend to reside in the 50 half of the CDS and have a higher inclusion level, compared with symmetrical alternative exons. Our ﬁnd- ings are summarized in Table 1. Supplementary Table 1 shows that alternative cassette exons in mouse, rat and dog that are conserved in human possess similar characteristics. In our dataset, 68.6% of human CDS-internal alternative exons that are conserved in mouse are symmetrical. Other