5578 Nucleic Acids Research, 2005, Vol. 33, No. 17 Figure 3. Percentage of symmetrical and non-symmetrical exons that encode different fractions of protein domains. (A) The percentage of symmetrical and non- symmetrical alternative and constitutive exons that encode for different fragments of protein domains, starting with exons not encoding any domain fragment (0%), then encoding different sizes of fragments (in increments of 10%) up to encoding complete domains (the bar marked 100%). Statistically significant differences between symmetrical and non-symmetrical alternative exons and between symmetrical and non-symmetrical constitutive exons are highlighted in gray. (B–E) A histogram representation of the results shown in (A), indicating the percentage of exons that do not encode protein domains (0%), encodes more than 0% and up to 10% of protein domains (>0%, <10%), encodes medium-sized fragments (>10%, <60%), encodes fragments that consist of >60% and <100% of protein domains or holds complete domains. B and C show the distribution of alternative exons, D and E show that of constitutive exons. induced by alternative splicing of non-symmetrical exons. For that, we used BLAST to search against GenBank mRNAs for the exon-including and exon-skipping isoforms of the altern- ative exons that are CDS-internal (see Materials and Methods). We used GenBank mRNAs for this analysis because PTC+ mRNAs were found to have a low representation in RefSeq, compared with GenBank (15). Interestingly, we detected an exon-skipping mRNA for only 42.7% (101/236) of the non-symmetrical exons, compared with 92.2% (475/515) of the symmetrical exons (exons for which no skipping mRNA was detected are known to be alternative based on EST-data solely). Then, for all alternative exons for which skipping mRNAs were found, we calculated the inclusion level, which is the fraction of total transcripts of the gene that includes the exon (see Materials and Methods). As already mentioned, conserved alternative exons usually exhibit a high inclusion level (22). However, we found that the average inclusion level of non-symmetrical exons is sig- niﬁcantly higher than that of symmetrical exons: 0.74 and 0.59, respectively, P < 1 · 10 31 (t-test). The low percentage of non-symmetrical exons with mRNA evidence for their skipping isoform, along with the high inclusion level of these exons, shows that the exon-skipping isoforms of non-symmetrical alternative exons have a very low coverage among GenBank mRNAs. This may indicate that skipping of a large fraction of conserved non-symmetrical alternative exons leads to NMD activation. Non-symmetrical alternative exons tend to reside near the ends of the CDS, with a higher preference for 50 locations As NMD activation depends upon the location of the termina- tion codon relative to the last exon–exon junction, we wanted to examine whether non-symmetrical alternative exons tend to reside in speciﬁc portions of the CDS. For that, we found the location of all CDS-internal exons in their including mRNA by normalizing the CDS length between 0 and 1 (see Materials and Methods), and compared, in increments of 0.1, the per- centage of symmetrical and non-symmetrical alternative and constitutive exons at different normalized locations (Figure 4). We found that non-symmetrical exons exhibit a tendency to reside in the 50 half of the CDS, with average location of 0.39, median 0.31 (Figure 4B). There is also a slight rise in the percentage of non-symmetrical alternative exons toward the 30 end of the CDS. Symmetrical alternative exons and