Comprehensive splice-site analysis using comparative genomics

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) conservation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 3' splice sites (3'ss) and (iv) distinct evolutionary histories of 5' and 3'ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing.

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 3′ 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.

Figure 2

Two of the 169 human U12-type AT–AC introns are shown. These were accessed using a minimum 5′ 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.

Figure 3

PWMs for 5′ and 3′ ss for the U2-type GT–AG and GC–AG intron subtypes, for the five species. We used color-coded vertical bars stacked one over the other to represent the percentage of each nucleotide, Green (A), Blue (C), Black (G) and Red (T); similar logos have been used elsewhere (66). The bars are ordered by their percentages.

Figure 4

PWMs for 5′ and 3′ ss for the GT–AG and AT–AC U12-type intron subtypes, in the five species. For C.elegans what is shown is the vestigial U12-type GT–AG splice sites.

Figure 5

Information content in the exonic and intronic portions of 5′ ss in various organisms. (A) U2-type GT–AG introns. (B) U2-type GC–AG introns. (C) U12-type GT–AG introns. (D) U12-type AT–AC introns.

Figure 6

Information content in the exonic and intronic portions of 3′ ss in various organisms. (A) U2-type GT–AG introns. (B) U2-type GC–AG introns. (C) U12-type GT–AG introns. (D) U12-type AT–AC introns.

Figure 7

Phylogenetic trees for all splice site types. Trees were constructed in a manner similar to the trees for the individual splice site subtypes, but the terminal dinucleotides of the intron were removed from the PWM (see text for details).