Table 3. Subtype switching for non-canonical U12-type introns between human and mouse H.sapiens M.musculus Subtype Human gene XPO4 AT–AG ACTR10 AT–AG NUP210 AT–AG SCN10A AT–AG CUL4B AT–AG USP21 AT–AG RAPGEFL1 AT–AG INPP5B AT–AG RASGRP4 AT–AA FLJ21106 AT–AA MSH3 AT–AA ERCC5 AT–AT IK AT–AT DEADC1 GT–AT SLC24A6 GT–AT SLC12A7 GT–GG ARAF GT–GG LZTR1 GC–AG Mouse gene 4930449E07Rik AT–AC Cacna1h AT–AC 4930590J08Rik GT–AG Gpaa1 AT–AC Zc3hdc6 AT–AC Slc9a8 AT–AC GT–AG GT–AG AT–AG GT–AG AT–AT AT–AG AT–AG AT–AG AT–AA AT–AA AT–AC AT–AC AT–AA GT–AT GT–AT GT–AG GT–GG GC–AG AT–AG AT–AG AT–AG AT–AT AT–AT AT–AT Non-canonical/canonical Non-canonical/canonical Conserved Non-canonical/canonical Non-canonical/non-canonical Conserved Conserved Conserved Conserved Conserved Non-canonical/canonical Non-canonical/canonical Non-canonical/non-canonical Conserved Conserved Non-canonical/canonical Conserved Conserved Non-canonical/canonical Non-canonical/canonical Non-canonical/canonical Non-canonical/canonical Non-canonical/canonical Non-canonical/canonical The canonical borders, AT–AC or GT–AG, for one of the species are shown in bold letters. For non-conserved introns, the differences in the nucleotides on the intron borders are underlined. There are 10 cases where the non-canonical boundaries are conserved between mouse and human, and 2 cases where one non-canonical form switched to another non-canonical form between the species. In the rest of the cases, one of the boundaries, either the mouse or human is a canonical form. U12-type AT–AC and GT–AG intron orthologs was found between human and chicken (24) Splice-site analysis by comparative genomics We used SpliceRack to analyze the species-speciﬁc pro- perties of splice sites from an evolutionary perspective. The global patterns of the splice sites were compared amongst the ﬁve species by phylogenetic approaches. Splice-site information content across species. We measured and plotted the information content at each position of the 50 and 30ss from the PWMs (Figures 5 and 6 see Materials and Methods). The information content is a measure of the degree of conservation in splice-site sequences, with lower information content corresponding to a higher degree of conservation, and vice versa (24,65–68). Information content of U2-type 50ss (Figure 5): The 50ss motifs for the two mammals are virtually identical, as described previously (24,69). The U2-type GT–AG 50ss graph ss graph shows that there is more conservation in the exonic nucleotides for H.sapiens, M.musculus and A.thaliana, com- pared to the other two simpler species, D.melanogaster and C.elegans. This is consistent with the fact that budding and ﬁssion yeast have poor conservation at the exonic 50ss posi- tions, but a very strict conservation at intronic positions (70). C.elegans and A.thaliana show weak conservation of Ts at positions downstream of +6, which is not the case for Nucleic Acids Research, 2006, Vol. 34, No. 14 3963 the other organisms (8,71). The GC–AG 50ss show higher conservation at both intronic and exonic positions when compared to the GT–AG 50ss (23,24,45). Information content of U2-type 30ss (Figure 6): The U2- type GT–AG and GC–AG 30ss show similar information con- tent, suggesting they might be functionally very similar. All species, except for C.elegans, show little conservation in the exonic or intronic portions of the 30ss, probably due to the degenerate PPT. C.elegans exhibits a shorter and more conserved PPT in the intronic portion, with a consensus sequence of TTTTCAGR, where AG is the terminus of the intron (72). The existence of this conserved 30ss motif in C.elegans is explained by a more stringent requirement for an optimal binding site for the U2AF heterodimer that is unique to this species (73). The conservation of G at position +1, conserved only in H.sapiens, M.musculus and A.thaliana, can be seen as a small decrease in information content at that position for the three species. Information content of U12-type 50ss (Figure 5): The information content distribution for U12-type GT–AG and AT–AC 50ss shows a high degree of conservation at the intronic positions that starts decaying at position +6. The conservation of T at position 1 of mammalian and plant, but not ﬂy U12-type GT–AG 50ss can be seen as a small increase in the ﬂy’s information content at that position. The information content distribution for C.elegans U12-type-like GT–AG introns is kept in the graph for comparison. Information content of U12-type 30ss (Figure 6): The information content distributions for both U12-type GT–AG and AT–AC 30ss show a very small degree of conservation in the intronic nucleotides. This observation is consistent with the notion that U12-type 30ss are slightly enriched in pyrimidines but lack a PPT. The location of a BPS near (8–14 nt) the 30 end of the U12-type intron contributes to con- servation at these positions, as seen in the slight decrease of information content on the left of these graphs. The small numbers of these types of introns makes it difﬁcult to infer general trends for D.melanogaster and A.thaliana’s AT–AC 30ss. The vestigial U12-type-like introns in the nematode also contain the conserved motif of the U2-type 30ss, consistent with the view that these introns are indeed U2-dependent. Phylogenetic trees from PWMs The Kullback–Leibler distance (40) (see Materials and Methods) between PWMs for 50 and 30ss was used to calcu- late a distance matrix between species, and phylogenetic trees were derived from these distance matrices. It is possible to use other methods to build trees, but this is the simplest one (50). Trees were generated for all the splice sites together (Figure 7), after removing the conserved dinucleotide termi- nus of the introns from consideration, as they would hide the signal from the other positions on the splice-sites. From Figure 7, it is easy to see a clear separation between U2- and U12-type 50ss motifs. The various 50 splice types separate according to splice-site type, rather than species. All the U2-type motifs cluster together, with a small separa- tion between GC–AG and GT–AG types suggesting that the GC–AG subtype is a specialized version of the U2-type