Nucleic Acids Research, 2006, Vol. 34, No. 14 3959 Figure 2. Two of the 169 human U12-type AT–AC introns are shown. These were accessed using a minimum 50 ss score of 50 and a maximum of 100. The output includes links to the GenBank accession, gene names and the intron number and length. The two highest scores for each splice-site subtype are shown. The classification of introns is explained in the Materials and Methods section. Splice-site locator. This tool can help identify potential splice sites in user-speciﬁed genes or sequences, using scores derived from the PWMs in SpliceRack. When a gene name (or sequence) is supplied along with a user-speciﬁed score threshold, the known exon–intron boundaries as well as pre- dicted splice sites are shown. In addition, a distribution of counts of predicted splice sites against scores is also shown, so that it is easy to see how the genuine splice sites compare to the predicted splice sites. A link to the sequence in the gene where the potential splice sites are located is also provided. U2-type splice sites U2-type GT–AG and GC–AG splice sites. Of all the splice sites we have classiﬁed, 99.6% (608 562) are of the U2- type in the ﬁve genomes (Table 1). GC–AG introns represent 0.82% of U2-type introns in H.sapiens, 0.86% in M.musculus, 0.8% in A.thaliana, 0.45% in D.melanogaster and 0.37% in C.elegans, indicating that more primitive organisms tend to have fewer GC–AG introns. GT–AG and GC–AG subtypes of U2-type introns are con- sidered separately. Though there is no evidence that these types are spliced by distinct mechanisms, their PWMs are signiﬁcantly different (Figure 3). The 50ss from GC–AG and GT–AG introns bind to U1 snRNP during spliceosome assembly (2). The consensus sequence for the U2-type GT– AG 50ss motif corresponds to a perfect base pairing to the U1 snRNA 50 end. Despite this, a high degree of variability seems to be tolerated at most positions of the actual 50ss. In GC–AG introns, the substitution at position +2 of the 50 ss introduces a mismatch in the U1: 50ss helix. The PWMs (Figure 3) show that the remainder of the 50ss sequence has a higher degree of match to the consensus GT–AG 50ss, apparently compensating for the mismatch at position +2 (17,23,24,50,51). Subtype switching of introns from U2-type GT–AG to U2-type GC–AG between human, rodent and chicken has been shown to be a common event (24). Non-canonical U2-type introns. Non-canonical U2-type introns with AT–AC dinucleotide intron ends have been known to occur and are functional. This is the ﬁrst genome-wide survey for U2-dependent AT–AC II introns (Table 1, Materials and Methods). Pre- viously, only two human AT–AC introns in the SCN4A and SCN5A genes were experimentally demonstrated to be spliced by the U2- but not the U12-type splicing machinery (5,51). The borders of these introns were conserved between human and puffer ﬁsh. In addition, the chicken parvalbumin gene and some xylanase genes in ﬁlamentous fungi were hypothesized to contain AT–AC II introns. Furthermore, a G to A transition at the ﬁrst intronic nucleotide can be rescued for splicing by a G to C transversion at the last nuc- leotide of U2-type introns in yeast (52,53) and human pre-mRNAs (54), although less efﬁciently. We found a total of 15 AT–AC II introns in the human, 6 in the mouse and 3 in the D.melanogaster genomes. The case for these introns is bolstered by the fact that they are pre- served in orthologous genes, such as the AT–AC II intron in the human and D.melanogaster TAF2 gene. In the human dataset, 10 out of 15 of these introns are found in members of the alpha subunit of the voltage-gated sodium channel gene family, which also have U12-type introns. Hence, the higher number of AT–AC II introns in mammals could be explained by the expansion of this gene family. All six mouse AT–AC II introns are found in members of this family, and conserved in the human genome. Interestingly, AT–AC II introns seem to be absent in C.elegans and A.thaliana. The very low abundance of these introns, and their presence in vertebrates and fungi, suggest that they constitute a relic of U2-dependent introns that have been progressively lost. We searched for other putative non-canonical U2-type splice sites as explained in the Materials and Methods section. Many of the known types (55) (see http://www.tigr. org/tdb/e2k1/ath1/Arabidopsis_nonconsensus_splice_sites. shtml) are not in our compilation as they do not occur in the RefSeq collection. We found 99 introns (33 in human, 46 in mouse, 5 in ﬂy, 11 in the worm and 4 in the plant) with an optimal U2-type 50ss and PPT, but a non-canonical 30 intron terminus. Out of these, the GT–AT and GT–TG introns in the DGCR2 and ARS2 genes, respectively, are conserved between human and mouse, suggesting that at least some of the remaining 95 might be bonaﬁde, functional splice sites. U12-type splice sites Canonical U12-type introns. We have substantially expanded the catalogue of U12-type splice sites in four of the ﬁve organisms we examined (Table 1). The PWMs for these splice sites have been derived and are consistent with pre- vious reports (6,25) (Figure 4). However, we ﬁnd a more