Figure 5. The eﬀect of two over-abundant overlaps in a minigene reporting system. Each tested overlap was inserted into the second exon of a reporting minigene comprised of four exons. (A) Illustration of an overlap comprised of two spicing regulatory sequences. Seq1 is in light-gray, seq2 is in black, and the overlapping sequence is in dark-gray. For each overlap, which is comprised of two splicing regulatory motifs (seq1 and seq2), we introduced two muta- tions: (B) mutation to eliminate seq1 leaving seq2 intact, and (C) mutation to eliminate seq2 leaving seq1 intact. (D) Insertion of both mutations simultaneously. (E) Splicing patterns of each of the two overlaps (overlap I: lanes 1–4; and overlap II: lanes 5–8) is depicted. For each overlap, the splicing pattern in its presence, after the elimination of seq1, the elimination of seq2, or the elimination of both is depicted, respectively. The upper bands indicate exon inclusion, while the lower bands indicate exon skipping; the exact inclusion level is indicated above each lane. yet another level of splicing regulation. We describe a computational method, based on the natural distribution of nucleotides and splicing regulatory sequences, by which we identiﬁed overlapping putative splicing regulatory sequences. Dozens of such motif overlaps are favorable in exons and even more appear to be selected against, implying both positive and negative interplay between splicing regulatory motifs. Selection against overlap of regulatory sequences might not be surprising. Such overlapping motifs, which are presumably formed by two independently functional regulatory binding sites, may obstruct one another when in overlap resulting in improper splicing. On the other hand, we were surprised that some motifs have a speciﬁc and signiﬁcant tendency to overlap. This ﬁnding implies that there is a layer of splicing regulation provided by a competition between factors that can either assist or obstruct the splicing machinery in proper recognition of exon/intron junctions. Such a competition could depend on the strength with which each SR protein binds to its speciﬁc regulatory sequence, the expression levels of the SR proteins, their level of phosphorylation, or their spatial distribution within the cell, which could vary in diﬀerent cell types or cell stages (see also Figure 1). The outcome of such com- petition could result in the proper splicing mode (consti- tutive or alternative), or proper exon inclusion level, in each cell type or stage. It is worth noting that the complexes we identiﬁed might in fact reﬂect single longer motifs, however, our data provides evidence against such possibility (see Supplementary Data and Supplementary Figure S2). By using comparative genomics we showed that the over-abundant overlapping splicing regulatory motifs are Nucleic Acids Research, 2010, Vol. 38, No. 10 3325 highly conserved among human and six other species. This suggests an important functional role for these overlapping motifs in splicing regulation, as they are signiﬁcantly conserved despite the 450-million years of evolution since the last common vertebrate ancestor. The overlapping motifs we found to be under-represented were not conserved. In fact, in some species, these overlapping motifs were actually signiﬁcantly less conserved than expected, suggesting that selective pressure acts against them. Therefore, such overlapping motifs have been selected against and eliminated during evolution. We further showed that the prevalence of over- abundant SR overlapping binding motifs in exons corre- lates with the strength of splice signals. Over-abundant overlapping motifs were signiﬁcantly more abundant in exons with weak donor/acceptor splice signals than in exons with strong ones. This suggests that this addi- tional level of regulation introduced by the overlapping motifs is speciﬁcally important for exons with suboptimal splicing signals for which splicing regulation depends strongly on auxiliary splicing factors. The preva- lence of the under-represented overlapping motifs was negatively correlated with the strength of the splice signals. Exons with suboptimal splice signals are more prone to undergo aberrant splicing than those with consensus signals and therefore may be less permissive to the presence of sequences that obstruct the splicing process. Additional examination of the overlapping motifs revealed that over-abundant and under-represented motif overlaps exhibited diﬀerent spatial distributions. In exons with weak splice donor sites but not in those with a strong donor splice site, under-represented motifs were relatively less prevalent near the splice donor site than over-abundant motif overlaps. This implies that over-abundant and under-represented motif overlaps functionally diﬀer. We hypothesize that the over-abundant motifs we identiﬁed take part in ﬁne tuning the delicate splicing regulation and that the under-represented motifs hinder the splicing machinery and are therefore selected against. Finally, to further support the bioinformatic ﬁndings, we experimentally tested selected over-abundant overlaps in a minigene reporting system. We revealed that the overlaps enabled a mode of splicing that did not occur when either of the splicing motifs that form the overlap was present independently, nor did it occur when the entire overlap was absent. While our analyses reveal the over-abundance and conservation of overlapping sequences, both of which are indicative of functional implications, further experi- mental work is needed to determine whether there is actual binding of the SR-proteins to these speciﬁc sites. Overall, the results we present are indicative of another dimension of splicing regulation that involves overlapping splicing regulatory motifs. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online.