Nucleic Acids Research, 2005, Vol. 33, No. 17 5577 Figure 2. Percentage of exons that overlap with protein domain coding sequence. Gray and white indicate the fraction of exons that encodes protein domain fragments or full domains and those that do not encode for any fragment of a protein domain, respectively. (A) and (B) are for alternatively spliced exons, (C) and (D) are for constitutively spliced ones. lower than that of non-symmetrical alternative exons and all constitutive exons. As shown in Figure 2, 30.3% (156/515) of symmetrical alternative exons overlap with domains, com- pared with 53% (126/236) of non-symmetrical alternat- ive exons, P < 9 · 10 10 (Fisher’s exact test). With respect to constitutive exons, 54% (8433/15 608) of symmetrical exons and 54.3% (12 850/23 645) of non-symmetrical exons overlap with coding sequences for protein domains. The differences between symmetrical and non-symmetrical constitutive exons and between non-symmetrical alternative exons and all constitutive exons are not statistically signiﬁcant (P > 0.2 in all cases). It has been previously shown that conserved alternative exons tend to be shorter than conserved constitutive exons (33). Therefore, the bias of symmetrical alternative exons against overlapping with protein domains could potentially stem from their relative short length. We found that the aver- age length of symmetrical alternative exons is indeed slightly shorter than that of non-symmetrical ones: 113.3 and 116.9 nt, respectively, but this difference is not statistically signiﬁcant (P < 0.6, t-test). Moreover, among constitutive exons, sym- metrical exons are also shorter than non-symmetrical ones: 128.4 and 131.2 nt, respectively, and this difference does reach statistical signiﬁcance (P < 0.01). Therefore, the slight difference in the average length of symmetrical and non- symmetrical alternative exons cannot account for the tendency of symmetrical alternative exons not to overlap with protein domains. Symmetrical alternative exons tend not to overlap with medium-sized fragments of protein domains To further analyze the tendency of symmetrical alternative exons not to overlap with protein domains, we compared the percentage of symmetrical and non-symmetrical alternat- ive and constitutive exons that encode different fractions of domains, in increments of 10% (Figure 3A). Among alternat- ive exons, we found that symmetrical exons are underrepres- ented among exons that overlap with domain fragments consisting of >10% and <60% of domains (which we term medium-sized fragments). Among constitutive exons, the dif- ferences between symmetrical and non-symmetrical exons reach a statistical signiﬁcance only among exons that encode complete domains (rather than only a fraction). This ﬁnding is in correlation with previous studies, which showed that most exons (constitutive and alternative) that hold complete protein domains are symmetrical (23,27). Symmetrical alternative exons also show a higher tendency to encode complete domains compared with non-symmetrical ones, but this result does not reach statistical signiﬁcance. Our analysis is summarized in Figure 3B–E. It shows that only symmetrical alternative exons tend not to encode medium-sized fragments of domains (Figure 3B compared with Figure 3C–E). Therefore, the low percentage of altern- ative symmetrical exons that encode protein domain fragments or complete domains stems from the tendency of these exons not to encode medium-sized fragments of domains. The pattern of encoding different fractions of domains is similar between non-symmetrical alternative exons and all constitu- tive exons (symmetrical or non-symmetrical). Skipping isoforms of non-symmetrical alternative exons have a low representation in GenBank Inclusion or skipping of a non-symmetrical alternative exon induces a frameshift that may result in the introduction of a PTC that induces degradation of the mRNA by the NMD mechanism [see examples in (34,35)]. Alternatively, an altern- ative exon (symmetrical or non-symmetrical) can harbor a PTC [see examples in (36,37)]. As only 3.4% of the alternative exons in our dataset encode for a stop codon, we searched for evidence of PTC generation only as result of frameshifts