Published online 12 August 2006 Nucleic Acids Research, 2006, Vol. 34, No. 14 3955–3967 doi:10.1093/nar/gkl556 Comprehensive splice-site analysis using comparative genomics Nihar Sheth, Xavier Roca, Michelle L. Hastings, Ted Roeder, Adrian R. Krainer and Ravi Sachidanandam* Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA Received June 7, 2006; Revised July 13, 2006; Accepted July 17, 2006 ABSTRACT We have collected over half a million splice sites from five species—Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana—and classified them into four subtypes: U2-type GT–AG and GC–AG and U12-type GT–AG and AT–AC. We have also found new examples of rare splice-site categories, such as U12-type introns without canonical borders, and U2-dependent AT–AC introns. The splice-site sequences and several tools to explore them are available on a public website (SpliceRack). For the U12-type introns, we find several features conserved across species, as well as a clustering of these introns on genes. Using the information content of the splice-site motifs, and the phylogenetic distance between them, we identify: (i) a higher degree of conservation in the exonic portion of the U2-type splice sites in more complex organisms; (ii) conser- vation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 30 splice sites (30ss) and (iv) distinct evolutionary histories of 50 and 30ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic data- sets, provides mechanistic and evolutionary insights into pre-mRNA splicing. INTRODUCTION Pre-mRNA splicing is a nuclear process that is conserved across eukaryotes (1). This process involves the recognition of exon–intron junctions by the spliceosome, and intron exci- sion through a two-step transesteriﬁcation reaction (2,3). The spliceosome is a large and dynamic complex assembled from RNA and protein components, including four small nuclear RNAs (snRNAs) and associated proteins that make up the small nuclear ribonucleoprotein particles (snRNPs) (4). The spliceosome recognizes three conserved sequences at or near the exon–intron boundaries, namely the 50 splice site (50ss), the branch point sequence (BPS) and the 30ss (Figure 1). In the ﬁrst catalytic step, the 50 end of the intron is cleaved and covalently joined to an Adenosine (A) on the BPS. In the second catalytic step, the neighboring exons are joined and the intron is excised as a lariat. There are at least two classes of pre-mRNA introns, based on the splicing machiner- ies that catalyze the reaction: U2 snRNP-dependent introns make up the majority of all introns and are excised by spliceosomes containing the U1, U2, U4, U5 and U6 snRNPs. These introns consist of three subtypes, according to their terminal dinucleotides: GT–AG, GC–AG and AT–AC introns. U12 snRNP-dependent introns are the minor class of introns and are excised by spliceosomes containing U11, U12, U4atac, U6atac and U5 snRNPs. These introns mainly consist of two subtypes, as defined by their terminal dinucleotides: AT–AC and GT–AG introns. In addition, a small fraction of the U12-type introns exhibit other terminal dinucleotides (5–7). Whereas U2-type introns have been found in virtually all eukaryotes (1) and comprise the vast majority of the splice sites found in any organism, U12-type introns have only been identiﬁed in vertebrates, insects, jellyﬁsh and plants (8). U2-type intron splicing initially involves base pairing of U1 snRNA to the 50ss and U2 snRNA to the BPS (2) (Figure 1). The base pairing of U2 snRNA to the BPS is facilitated by the binding of the large subunit of the U2 Auxiliary Factor (U2AF65) to the poly-pyrimidine tract (PPT) located imme- diately upstream of the intron 30 terminus, and binding of the small subunit (U2AF35) to the 30 terminal AG dinucleo- tide of the intron (9,10). Following the initial recognition of the splice sites by the U1 and U2 snRNPs, the U4/U6/U5 tri-snRNP is recruited to the splice site leading to the two catalytic steps of splicing (11). *To whom correspondence should be addressed. Tel: +1 516 367 8864; Fax: +1 516 367 8389; Email: sachidan@cshl.edu Present address: Nihar Sheth, Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA 23284-2030, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.