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Background  Algorithmic approaches to splice site prediction have relied mainly on the consensus patterns found at the boundaries between protein coding and non-coding regions. However exonic splicing enhancers have been shown to enhance the utilization of nearby splice sites.    Results  We have developed a new computational technique to identify significantly conserved motifs involved in splice site regulation. First, 84 putative exonic splicing enhancer hexamers are identified in  Arabidopsis thaliana . Then a Gibbs sampling program called ELPH was used to locate conserved motifs represented by these hexamers in exonic regions near splice sites in confirmed genes. Oligomers containing 35 of these motifs have been shown experimentally to induce significant inclusion of  A. thaliana  exons. Second, integration of our regulatory motifs into two different splice site recognition programs significantly improved the ability of the software to correctly predict splice sites in a large database of confirmed genes. We have released GeneSplicerESE, the improved splice site recognition code, as open source software.    Conclusion  Our results show that the use of the ESE motifs consistently improves splice site prediction accuracy.

A computational survey of candidate exonic splicing enhancer motifs in the model plant Arabidopsis thaliana