studies reported varying percentages of symmetrical exons among conserved CDS-internal alternative exons, ranging from 53 to 77% (19,25,26). However, as conserved CDS- internal alternative exons show a strong bias to be symmetrical in all studies, these discrepancies probably result from differ- ences in dataset compilation and do not reﬂect meaningful contradictions between the different works. It has been previously reported that the borders of altern- ative exons tend to correlate with protein domain borders, probably to eliminate potentially deleterious splice variants that contain broken domains (39). The observed bias of sym- metrical alternative exons not to overlap with medium-sized fragments of protein domains shows that this tendency is much stronger among symmetrical alternative exons. This bias may reﬂect a negative selection on symmetrical exons that produce transcripts with broken domains. Non-symmetrical alternative exons show the same pattern of overlapping with different fractions of domains as do constitutive exons, which may indicate that non-symmetrical alternative exons are indifferent to this selection pressure. As inclusion or skipping of symmetrical exons does not alter the mRNA’s reading frame, it would be reasonable to hypothesize that, compared with non-symmetrical ones, these exons would be more prone to play the role of domain-holders (i.e. alternative exons that encode large fragments of domains and thus their skipping eliminates the domain func- tionality). We did ﬁnd that among exons that encode >60% of protein domains, symmetrical alternative exons have a slightly higher representation, but this result did not reach statistical signiﬁcance. Skipping of exons that encode >60% of protein domains is very likely to eliminate the functionality of the domains with which these exons overlap. Therefore, such exons probably act as domain-holders. However, our ﬁndings suggest that only a small fraction (<4%) of alternative exons are domain-holders. This indicates that selective removal or insertion of protein domains (at least of the type represented in Pfam and SMART databases) into different splice forms is not a major role of alternative splicing. We also found that the ratio of symmetrical and non-symmetrical alternative exons that encode such large fragments of protein domains is similar to that of symmetrical and non-symmetrical constitutive exons. This may indicate that the tendency of alternative exons to be domain-holders is not higher than expected by chance. We also found that mRNAs representing the skipping of non-symmetrical alternative exons are strongly underrepres- ented in GenBank, compared with those of symmetrical alternative exons. It has already been suggested that mRNAs subjected to NMD have a low coverage in databases because of their rapid degradation (15). Therefore, it is pos- sible that a large fraction of the exon-skipping isoforms of non- symmetrical exons underwent NMD degradation, and, thus, these isoforms are not detected. The ﬁnding that non- symmetrical alternative exons tend to reside in the 50 half of the CDS (average normalized location of half of them is lower than 0.31) can also be related to NMD activation. Reasonably, skipping of non-symmetrical exons from more upstream locations, not accompanied with use of an alternative in-frame start codon, has a higher potential to introduce a stop codon in the frameshift induced downstream from the skipped exon. Also, PTCs closer to the 50 end may trigger more robust Nucleic Acids Research, 2005, Vol. 33, No. 17 5581 NMD, as in the case of the TCR-b gene (40). Therefore, it is possible that skipping events of non-symmetrical exons are preserved in evolution for NMD activation, which may be more effective if they are skipped from locations within the 50 half of the CDS. However, there is also an increase in the representation of non-symmetrical alternative exons near the 30 end of the CDS. This increase was observed for both non-symmetrical exons with mRNA evidence for their skipping isoforms and those without such evidence. A manual inspection of the effects on the reading frame of 30 random splicing events of non- symmetrical alternative exons suggests that non-symmetrical alternative exons that do not generate PTC+ mRNAs tend to reside near the ends of the CDS, and this allows the including and skipping isoforms to use the same reading frame through- out most of the CDS. In half of such cases in our sample, the exons resided in proximity to the 50 end of the CDS and the including and skipping isoforms used a different ATG. This allowed the preservation of the reading frame downstream from the exon. Elimination of an otherwise potent PTC by means of alternative initiation has already been reported in the case of the b-globin and TPI genes (41,42). In the other cases, the exons were found to reside in proximity to the 30 end of the CDS, and, as a result, the frameshift they induced was short and did not harbor a PTC. This also allowed the preservation of most of the reading frames in both the including and skipp- ing isoforms. The low coverage of the skipping isoforms of non- symmetrical alternative exons, along with the fact that, in 2/3 of alternative splicing events that generated PTCs in our random sample, the skipping isoform was the PTC+, suggests that the most prevalent mechanism for the generation of a PTC is skipping of a non-symmetrical exon. As only 3% of alternative exons in our dataset encode for a stop codon, it is likely that creation of a stop codon by means of insertion of a cassette exon that harbors a PTC is a much less-used mechanism for NMD activation. Moreover, as our ﬁndings suggest that the including isoform of many non-symmetrical alternative exons is being translated into pro- tein and that the skipping isoforms undergoes NMD degrada- tion, it is likely that the including isoform of these exons represents the ancestral state of these exons. This supports a hypothesis we recently suggested that constitutively spliced exons can switch to alternative splicing in the course of evolution (21). The average high inclusion level of all conserved alternative exons and the tendency of these exons not to contain repetitive elements further support this idea. According to this model, the selection against transition of non-symmetrical constitutive exons to alternative splicing is weaker near the CDS ends, because skipping these exons either preserves most of the reading frames in the skipping isoform (as described above) or can be used for the generation of NMD-targeted transcripts that may play a regulatory role. Transition of symmetrical exons from constitutive to altern- ative splicing is probably under negative selection if these exons encode for medium-sized fragments of protein domains. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online.