3324 Nucleic Acids Research, 2010, Vol. 38, No. 10 Figure 3. Distribution of under-represented motifs along exons. The distribution of the under-represented motif overlaps along exons was calculated for exons with strong (light-gray bars) and weak (dark-gray bars) splice signals near (A) the 50 splice signal and (B) the 30 splice signal. Exon sequences were divided into four equally-sized quartiles (x-axis). The percentage of nucleotides which are part of under-represented motif overlaps, in each of the four quartiles, is indicated (y-axis). To further test the validity of the bioinformatic ﬁndings, we examined four representative over-abundant overlaps in a minigene reporting system (44). The minigene was composed of four exons, and we inserted the tested overlapping motif into the second exon. For each overlap, which is comprised of two splicing regulatory motifs (seq1 and seq2), we designed one mutation to elim- inate seq1 leaving seq2 intact, and one mutation to elim- inate seq2 leaving seq1 intact. We introduced each of the two mutations to the minigene independently to test the eﬀect of the presence of seq2 or seq1 alone. We then also introduced the two mutations simultaneously to test the eﬀect of the absence of both sequences. Two of the four overlaps we tested showed full exon skipping, a pattern that did not change regardless of the mutations we introduced (data not shown). However, the other two overlaps enabled a mode of splicing that did not occur in the presence of only one of the two regulatory sequences that comprise it, nor did it occur in the absence of the entire overlap (the overlaps’ sequence is indicated in Supplementary Table S1). The insertion of the ﬁrst overlap resulted in 80% inclusion level, while mutations that eliminated each of the regulatory sequences that form the overlap, or eliminated them both, resulted in diﬀerent splicing patterns (30, 67 and 0%, respectively; Figure 5, complex I). The insertion of the second overlap resulted in 13% inclusion level. Figure 4. Comparison between the distributions of over-abundant and under-represented motifs along exons. The distribution of the over-abundant (light gray bars) and under-represented (dark gray bars) sequence overlaps along exons was calculated for exons with (A) strong 50 splice signals and (B) weak 50 splice signals. Exon sequences were divided into four equally sized quartiles (x-axis). The percentage of nucleotides which are part of over-abundant or under-represented motif overlaps in each of the four quartiles is indicated (y-axis). Similarly, mutations that eliminated each of the regulatory sequences that form the overlap, or eliminated them both yielded diﬀerent splicing patterns (59, 71 and 0%, respec- tively; Figure 5, complex II). These experimental results are consistent with the possibility that the overlapping splicing regulatory motifs provide regulation that is diﬀer- ent from the eﬀect of each regulatory sequence alone. However, it is worth noting that further experimental analysis is required to determine this matter, as it may be that the extension itself is important, rather than the relation between two diﬀerent splicing factors. DISCUSSION To date, many layers of splicing regulation have been doc- umented. These include the consensus splice site sequences, auxiliary signals, such as exonic and intronic enhancers and silencers [reviewed in ref. (40)], chromatin structure and transcription elongation rate (45). However, despite a vast body of knowledge on elements and sequences that aﬀect splicing of particular genes, the splicing code in general is far from being fully understood and one cannot predict the mode of splicing of a given exon (or an intron) strictly from analysis of sequence. In this work we provide evidence that two exonic splicing regulatory sequences that overlap might represent