3322 Nucleic Acids Research, 2010, Vol. 38, No. 10 Figure 2. Conservation of the over-abundant overlapping regulatory binding motifs. The conservation levels of the overlapping motifs were calculated between orthologous exons of human and (A) mouse, (B) rat, (C) cow, (D) dog, (E) chicken and (F) zebraﬁsh. These calculations were performed for both signiﬁcant (light-gray bars) and control (dark-gray bars) overlaps. The percent conservation level (y-axis) is indicated for both the over-abundant and the under-represented overlapping motifs (x-axis). overlap between human and mouse, for example, was calculated by multiplying the conservation level of the SRp40-binding site (40%, for example) and the conserva- tion level of the extension AG (77%, for example). The result was then multiplied by the number of times we observed this overlap in the data (128 times, for example) resulting in 39.22—the expected conservation level for the overlap TGACTCCAG. Generally, the over-abundant motifs were signiﬁcantly conserved between human and mouse (P = 2.90e 04), rat (P = 3.60e 03), cow (P = 4.88e 03) and chicken (P = 5.14e 03), but were not signiﬁcantly conserved between human and dog (P = 0.19) or human and zebraﬁsh (P = 0.89, 2 with df = 1 for all tests). Examination of the 153 under-represented motif overlaps revealed signiﬁcant, yet opposite results. These motifs were signiﬁcantly less conserved between human and mouse (P = 2.40e 17), rat (P = 8.34e 08), cow (P = 1.62e 04) and zebraﬁsh (P = 0.043), but not between human and dog (P = 0.48) or human and chicken (P = 0.13, 2 with df = 1 was used for all tests). Overall, we used diﬀerent methods to examine the conservation level of the over-abundant and under-represented motifs (see Supplementary Data for another method). The results imply that the over-abundant motif overlaps we identiﬁed are function- ally relevant. These over-abundant motifs are signiﬁcantly conserved despite millions of years of evolution since the last common ancestor of the organisms evaluated, suggest- ing that these motif overlaps play a functional role in the regulation of splicing. In contrast, under-represented motif overlaps were not signiﬁcantly conserved compared to the random data or even exhibit a signiﬁcant opposite trend. This suggests that the under-represented