5576 Nucleic Acids Research, 2005, Vol. 33, No. 17 nih.gov/entrez/query.fcgi?db=homologene) for rat RefSeq mRNAs that are homologous to the 823 human RefSeq mRNAs in our dataset. For each pair of human and rat homo- logous mRNAs, we aligned the human alternative exon and the rat mRNA, using clustalW. This yielded 319 rat mRNAs that include a sequence with >80% identity with the human alternative exon incorporated in their human homologous mRNA. We then wanted to verify that these sequences in rat mRNAs are indeed alternative cassette exons. For that, we ran two blast searches against dbEST: (i) of the putative rat alternative exon and 50 nt on both sides (from its ﬂanking exons), representing the exon-including isoform; and (ii) of a concatenation of the ﬂanking 50 nt, representing the exon-skipping isoform. We used only blast results with E-value < 1 · 10 4. If an EST was hit on both blasts, we per- formed pairwise alignment both of the EST and the sequence representing the exon-including isoform and of the EST and the sequence representing the exon-skipping isoform, using the clustalW algorithm. According to the clustalW score, we determined whether the EST includes the exon or skips it. This left us with 86 rat exons that have EST evidence showing they incorporate an alternative cassette exon (which is con- served in human). We repeated the same procedure with dog exons and found 22 dog exons conserved in human and with evidence for both their skipping and inclusion in dog. Finding exon-skipping and exon-including isoforms Detection of exon-skipping isoforms was conducted in the same manner as the search for ESTs that provide evidence for the skipping of rat and dog alternative exons (see above). However, for this analysis, the blast was performed against human and mouse GenBank mRNAs (rather than dbEST), because we searched for full-length mRNAs where the cassette exons are skipped. GenBank mRNAs were downloaded from the UCSC genome browser (http://hgdownload.cse.ucsc.edu/ downloads.html). Inclusion level calculation We calculated the inclusion level only for exons for which at least one including and one skipping mRNA were found. The inclusion level of an exon was determined as the quotient of the number of mRNAs representing its exon-including isoform divided by the number of all mRNAs that include or skip the exon. Normalization of exon location within CDS To normalize the exon location within the CDS, we ﬁrst cal- culated N, which is the number of all possible locations for the start point of this exon in the CDS without exceeding the boundaries of the CDS (N ¼ CDS length exon length + 1). The normalized location was determined as the quotient of the actual location of the exon’s start point within the CDS divided by N. Manual analysis of the effect on the reading frame of a random sample of non-symmetrical and symmetrical alternative exons We used the mRNA/genomic alignments in the UCSC genome browser to manually compare between exon-skipping and exon-including mRNAs. We ﬁrst blasted each mRNA against the genome, at http://genome.ucsc.edu/cgi-bin/hgGateway. Then we followed the link to the mRNA information and examined its mRNA/genomic alignment to determine its CDS boundaries and exon–intron junctions. This allowed us to determine the differences between the two isoforms in the CDS level (i.e. if they use different start and stop codons, if a frameshift occurs, etc.). All the CDS annotations we used are based on SWISS-PROT, TrEMBL, mRNA and RefSeq. Statistical analysis We used two statistical tests in this work: t-test and Fisher’s exact test. t-test could be applied due to large sample size. For all statistical analyses, the level of statistical signiﬁcance was ﬁxed at 0.01. RESULTS To analyze conserved alternative splicing events, we compiled a dataset of 823 human alternative cassette exons (only exons for which the mouse homolog is alternatively spliced as well). Of these exons, 91.3% (751/823) are located within the CDS (CDS-internal exons). The rest either reside in the 50 or 30 untranslated regions (UTRs) (2.2%—18/823 and 0.4%—3/ 823, respectively) or contain the start or the stop codon (2.8%—23/823 and 3.4%—28/823, respectively). For each exon, we found a matching mRNA in NCBI’s RefSeq database (see Materials and Methods). As a control, we repeated all analyses on a dataset of 39 720 human constitutively spliced exons (only exons for which the mouse homolog is con- stitutively spliced as well) and their matching 3062 RefSeq mRNAs, which was compiled by us previously (29). Analyses were repeated with a dataset of conserved alternatively spliced and constitutively spliced mouse exons, as well as with a dataset of conserved alternatively spliced exons of rat and dog. The results of these analyses are in correlation with the results obtained from analyzing the human exons dataset (see Supplementary Data). We ﬁrst conﬁrmed that the CDS-internal alternative exons in our dataset are biased to be symmetrical. Indeed, 68.6% (515/751) were found to be symmetrical. As expected, the alternative exons that are not CDS-internal exhibit a much milder bias to be symmetrical (41.6%—30/72, are symmet- rical), as do the CDS-internal constitutive exons (39.7%— 15608/39253). Percentage of symmetrical alternative exons that overlap with protein domains is low compared with non-symmetrical ones We compared symmetrical and non-symmetrical alternative exons with respect to their potential to insert or remove protein domains. For that, we searched against Pfam and Smart databases (31,32) for protein domains in the mRNAs of the 751 alternative and 39 253 constitutive exons that are CDS-internal, and compared the percentage of symmetrical and non-symmetrical exons that overlap with domains (i.e. encode for a fragment of domain or possess the full extent of a coding sequence of a domain). We found that the percentage of symmetrical alternative exons that overlap with protein domains is signiﬁcantly