contain PTCs (termed PTC+ mRNAs) (15). This widespread coupling of NMD and alternative splicing suggests that NMD-targeted transcripts play an important regulatory role, probably in the downregulation of gene expression (1,11,15). However, for the vast majority of alternative splicing events predicted from bioinformatic analyses, which are predomin- ately based on expressed sequence tag (EST) data, the func- tional signiﬁcance is unknown (4,16,17). Moreover, as a large fraction of ESTs in dbEST comes from cancer-related tissues, many of these alternative splicing events might represent aberrant splicing or EST artifacts (18–20). In this study, we examined a dataset of alternatively spliced cassette exons that are conserved between human and mouse. The conservation of these alternative exons in both species, which diverged from their common ancestor 75–130 million years ago, sug- gests that these exon-skipping events represent truly functional splicing events (19,21). It has been previously reported that alternative cassette exons that are conserved in several species possess some unique features that distinguish them from species-speciﬁc exons (i.e. alternative exons that exist in a human gene, but not in its mouse counterpart or vice versa). Compared with species- speciﬁc exons, conserved exons tend (i) to be shorter (19); (ii) to have a high inclusion level (i.e. the number of ESTs/ mRNAs that represent their inclusion is higher than the number of those that skip them) (22); (iii) not to contain repetitive elements (19) and (iv) to be in a length that is divisible by 3 [termed ‘symmetrical exons’, see also (23)], if they reside within the protein coding region (CDS) (19,24–26). The high percentage of symmetrical exons among conserved alternative exons within the CDS (53–77%, with respect to an expected percentage of 33% without any bias for divisibility by 3) reﬂects a functional pressure for preserving protein coding, as insertion or skipping of these exons does not alter the reading frame (21,25), whereas alternative splicing of non- symmetrical exons always induces a frameshift (Figure 1). Constitutive exons were also found to have a bias toward being symmetrical, but this bias is much more moderate ( 40% are symmetrical) (23,25,27). In this study, we examined the differences between sym- metrical and non-symmetrical conserved alternative exons with respect to several characteristics. We found that they differ (i) in their tendency to overlap with coding sequences for protein domains, (ii) in their inclusion level, (iii) in the coverage of their skipping isoforms in GenBank and (iv) in their preference for speciﬁc locations along the CDS. These ﬁndings suggest that symmetrical and non-symmetrical con- served alternative exons are under different selective pressures and may play different functional roles, such as NMD activa- tion by skipping of non-symmetrical alternative exons. MATERIALS AND METHODS Database compilation We used two databases in our analysis: one of conserved alternative exons (only cassette exons) and one of con- served constitutive exons. The conserved alternative exons consisted of 823 human exons, 664 mouse exons, 86 rat exons and 22 dog exons, along with their corresponding mRNAs from RefSeq, NCBI’s database of curated and Nucleic Acids Research, 2005, Vol. 33, No. 17 5575 Figure 1. Different impact on the reading frame of symmetrical and non- symmetrical alternatively spliced exons. (A) A gene consisting of three exons whose second exon is a symmetrical cassette exon (90 nt). Skipping and inserting of the symmetrical exon does not alter the reading frame: the sequence upstream and downstream to the alternative exon encodes for the same peptide sequence in the including and skipping isoforms. (B) A gene consisting of three exons whose second exon is a non-symmetrical cassette exon (91 nt). Skipping of the non-symmetrical exon results in a frameshift down- stream to the alternative exon. non-redundant reference sequences (http://www.ncbi.nlm. nih.gov/RefSeq/) (28). The conserved constitutive exons data- base consisted of 39 720 human–mouse homologous constitu- tive exons and their matching 6124 RefSeq mRNAs, already compiled by us (http://www.tau.ac.il/~gilast/sup_mat2.htm) (29). Compilation of the alternative exons database was performed in several stages: we started with a dataset of human–mouse homologous alternative cassette exon pairs, which was built by integrating three source databases: (i) http://www.soe.ucsc.edu/~sugnet/psb2004/altGraphXCon. html (30). (ii) http://www.tau.ac.il/~gilast/sup_mat2.htm (29). (iii) http://www.sciencedirect.com/science/MiamiMultiMedia URL/B6TCY-4B9558H-1/B6TCY-4B9558H-1-C/5183/ f9bdffb6d98512adebc2e545a0151900/ Application_1. xls (19). We removed all duplications and remained with 1285 pairs of human–mouse homologous alternative exons. As all three source databases were compiled using EST data, we blasted all exons against RefSeq, NCBI’s database of curated and non- redundant reference sequences (http://www.ncbi.nlm.nih.gov/ RefSeq/) (28), to ﬁnd their matching full-length mRNAs for further analysis. We found such RefSeq mRNAs for 823 human exons and 664 mouse exons. To ﬁnd rat conserved alternative cassette exons, we searched NCBI’s HomoloGene database (www.ncbi.nlm.