3956 Nucleic Acids Research, 2006, Vol. 34, No. 14 Figure 1. Initial steps in splice-site selection. The dark bars stand for exons, while the lines represent introns. ss stands for splice sites. AG is the 30 terminus of the intron, where Y is a pyrimidine. PPT is the poly-pyrimidine tract. BPS is the branch point sequence, U1, U2, U11 and U12 are snRNPs. U2AF65 and U2AF35 are protein splicing factors. See text for details. In U12-type introns, the roles of U1, U2, U4 and U6 snRNPs in U2-type introns are replaced by the U11, U12, U4atac and U6atac snRNPs, respectively (12–14). The over- all similarity in the predicted secondary structure between analogous U2- and U12-type snRNAs suggests that the spliceosome rearrangements during catalysis are conserved between the two spliceosomes (15,16). The 50ss, 30ss and BPS elements conform to speciﬁc consensus sequences as determined by the alignment of splice-site compilations (8,17–24). The U2-type splicing sig- nals have highly degenerate sequence motifs; many different sequences can function as U2-type splice sites. In contrast, U12-type 50ss and the BPS, which lies close to the 30 end of the intron, are highly conserved (22,25). In most U2-type cases, the PPT is located immediately upstream of the AG but there are examples in alternatively spliced exons in Droso- phila melanogaster with a longer PPT–AG distance (26), or even with the PPT placed downstream of the AG (27). Fur- thermore, the mammalian U2-type BPS can also be located very far (>100 nt) from the intron–exon junction sequence (28). U12-type introns also lack an obvious PPT at the 30ss. Historically, splice sites are ranked, based on compilations of splice sites (19,20,29,30). However, none of these ranking schemes accurately identify the bona ﬁde splice sites (31–33). In addition, alternative splicing, involving the choice of competing splice sites, is not amenable to an analysis based solely on splice sites (34). In order to identify common and distinguishing features in each splice-site type, we have collected and analyzed a com- prehensive set of naturally-occurring splice sites from the gen- omes of ﬁve model organisms: Homo sapiens, Mus musculus, D.melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. Since a well-classiﬁed dataset did not exist previ- ously, we have carefully classiﬁed the splice sites into various categories, generating a user-friendly resource, SpliceRack (http://katahdin.cshl.edu:9331/SpliceRack/). Using this large- scale splice-site database, we have revisited relevant themes in splicing, such as the frequencies of U12-type and other rare introns, and the variations in the 50 and 30ss motifs among the ﬁve organisms. We have also applied phylogenetic approaches to infer the evolutionary histories of the different splice-site subtypes. Finally, we have developed tools, avail- able on SpliceRack, to facilitate various splice-site analyses. MATERIALS AND METHODS Data collection The idea driving the collection of data was to generate a large, reliable dataset that would allow statistical analyses. For this reason, sequences from RefSeq (35) were used instead of ESTs and cDNAs and other sources of noisy data. The RefSeq collection contains sequences with different levels of conﬁdence (including categories, such as reviewed, provisional, predicted etc.). RefSeq sequence data from NCBI (from June 2005) for ﬁve reference genomes was collected, including the primate H.sapiens, the rodent M.musculus, the arthropode D.melano- gaster, the nematode C.elegans and the land plant A.thaliana. If a splice site occurred more than once in different alterna- tively spliced isoforms, it was considered only once in the database, as part of one of the variants, in order to avoid counting splice sites multiple times. The ﬂanking sequences at both exon–intron junctions were extracted, 38 nt long at the 50ss (8 nt within the exon and 30 nt within the intron), and 43 nt long at the 30ss (35 nt within the intron and 8 nt within the exon). The splice sites were identiﬁed using annotations of the genome from GenBank. C.elegans 30ss that are trans-spliced to spliced leader (SL) RNAs (36) were not included in the analysis. The data, along with lengths of introns and exons, are stored in a relational database. Position weight matrices (PWMs), information content and phylogeny Sequences at the 50 and 30 junctions for each intron were separately aligned using the intron–exon junction as the anchor. The alignment was used to calculate the frequencies of nucleotides at each position. This results in a PWM that has four rows (one for each of A, C, G and T) and the number of columns is equal to the length of the splice-site motif. We chose a 9 nt long motif for the 50ss (3 in the exon and 6 in the intron) and a 14 nt long motif for the 30ss (13 in the intron and 1 in the exon). The consensus sequence for a PWM is con- structed by choosing the nucleotide with the highest frequency of occurrence at each position. A log-odds (LOD) matrix was constructed from the PWM by the usual method of taking a logarithm to base 2 of the ratio of frequencies at each position of the matrix against a background frequency of 0.25 for each nucleotide. Using a different background will not change the results, but will change the scores and thresholds. A position on a sequence was scored by aligning it with the matrix and adding up the entries in the matrix that correspond to the nucleotides at each position on the sequence. Each position on the splice site has an associated infor- mation content that can be derived from the frequencies of the 4 nt, using information theory (37,38). For a random vari- able that can take on four values with four probabilities, pi, the information content is Spilgpi. Information content can be used to study the variability (or level of conservation) at each position, varying from 0 (one possibility is certain) to 2 (all possibilities are likely equal). Some papers use 2 þ Spilgpi as a measure of conservation, which just inverts the scale (39). A distance, d, can be derived between two PWMs, p and q, of equal length, using a measure called the Kullback–Leibler distance (40), a symmetrized form of which is deﬁned as d ¼ Si½ pilgð pi/qiÞ þ qilgðqi/piÞ ‚ 1