motifs are selected against, perhaps because they interfere with the splicing process. Abundance of SR overlapping binding motifs correlates with splice signal strengths We next examined whether the abundance of SR overlapping binding motifs in exons correlated with the strength of the splice sites. The splicing signals play a major role in the proper recognition of the exon/intron junctions. For example, the strength of the aﬃnity of U1 snRNA to the 50 splice site dictates both constitutive and alternative splicing, as well as regulates the inclusion/ skipping ratio in alternative splicing (40). However, the presence of a strong ESE can compensate for a weak splice site (41). Thus, if the over-abundant SR overlapping binding sites are indeed functional in splicing regulation, we would expect them to be more abundant in exons with weak splice signals. Similarly, if the under-represented SR overlapping binding sites interfere with the splicing process, we would expect them to be selected against, espe- cially in exons with weak splice signals. To test these hypotheses we divided our exon dataset into quartiles according to either their 50 splice site or their 30 splice site strength, as computed by the MaxEntScan server (42). We deﬁned strong or weak exons according to their presence in the upper or lower quartile, respectively. Next, for each of the exons, we determined the number of nucleotides that were part of a motif overlap and the number of nucleotides that were not (omitting the ﬁrst and the last three bases as these are part of the splice signals). The over-abundant overlapping motifs were signiﬁcantly more abundant in exons with weak 50 splice site splice signals than in exons with strong ones (P = 3.77e 20, 2 with df = 1). Furthermore, we found that the under-represented overlapping motifs were signiﬁcantly selected against in exons with weak 50 splice site splice signals compared to exons with strong ones (P = 2.70e 10, 2 with df = 1). Similar results were obtained when exons were classiﬁed according to their 30 splice site strengths. Namely, over-abundant motifs were found mainly in exons with weaker splice signals whereas under-represented motifs were selected against in this group of exons (over-abundant P =1.69e 34; under-represented P =1.46e 26, 2 with df =1). Negative selection acts against the under-represented sequence overlaps near the splice donor site The above analyses implied that under-represented sequences interfere with the splicing process as they are selected against. Previous studies indicated that the density of exonic splicing regulatory elements is highest near splice sites and that the relative location of these elements is crucial for their function (36,38,43). Furthermore, it appears that the eﬀect of exonic splicing regulatory sequences is more prominent near the splice donor site than the splice acceptor site (36). Thus, if the under-represented overlapping motifs we identiﬁed indeed interfere with the splicing process, we could expect they would be selected against speciﬁcally near the splice donor site where they would interfere most with splice Nucleic Acids Research, 2010, Vol. 38, No. 10 3323 site selection. Furthermore, we could expect such a phe- nomenon to be more prominent in exons with weaker splice signals, which are sub-optimally recognized to begin with, and hence depend more on auxiliary splicing factors. To reveal correlations between the strength of the splice signals and the distribution of the under-represented SR putative binding motif overlaps along the exons, we divided the sequence of each of the exons into quartiles. The ﬁrst and last quartiles were near the splice acceptor and donor site, respectively. As above, for each of the exons, we determined the number of nucleotides that were part of a motif overlap and the number of nucleotides that were not (omitting the ﬁrst and the last three bases of each exon, as these are part of the splice signals). We subdivided the exons into those with strong or weak 50 and 30 splice sites (see above). There was a signiﬁcant diﬀerence between the distributions of under-represented overlapping motifs across the entire exon for exons with strong versus weak 50 splice sites (P = 3.20e 35, 2 with df = 3; see Figure 3A). This dif- ference in the distribution was observed mainly near the splice donor site. Similarly, there was a signiﬁcant diﬀer- ence between the distributions of under-represented overlapping motifs across the entire exon for exons with strong versus weak 30 splice sites (P = 2.08e 05, 2 with df = 3; see Figure 3B). Again, this diﬀerence was found to be more prominent near the splice donor site. Diﬀerent spatial distributions of over-abundant and under-represented overlaps in exons with weak 50 splice sites The results indicated that over-abundant overlaps are functionally important and play a regulatory role in the splicing process. If the over-abundant motifs indeed par- ticipate in splicing regulation, we would expect their dis- tribution along exons to be diﬀerent from the distribution of the under-represented motifs. Namely, we expect their prevalence to be higher near the splice donor site. Our data indicated that the relative distribution of over-abundant and under-represented motifs is signiﬁcantly diﬀerent. The under-represented motifs are highly selected against and over-abundant motifs are highly preferred speciﬁcally near weak 50 splice sites (Figure 4; P = 2.56e 51, 2 with df = 1). In fact, in exons with a strong 50 splice site, there was no signiﬁcant preference for either type of motifs. These results suggest that exons with weak splice signals that rely on auxiliary factors for their splicing are more aﬀected by the presence or absence of the over-abundant and under-represented motifs; in these exons, the motifs are under tight selective pressure. Over-abundant motif overlaps facilitate a new mode of splicing in a reporting minigene system The above bioinformatic ﬁndings suggest that overlapping splicing regulatory sequences can positively or negatively interact to aﬀect splicing. Speciﬁcally, we found 72 signiﬁcantly over-abundant and conserved overlaps, suggesting they may be functional in splicing regulation.