Nucleic Acids Research, 2010, Vol. 38, No. 10 3319 Figure 1. Possible outcomes of overlapping regulatory sequences. As an example, a potential overlap between the binding sites of SRp55 (dark gray ellipse) and SF2 (light gray ellipse) is illustrated. A speciﬁc cell type or a cell at a speciﬁc time where (A) SF2 expression is down-regulated resulting in binding of SRp55 to its binding site, (B) SRp55 expression is down-regulated resulting in binding of SF2 to its binding site, (C) both SF2 and SRp55 are expressed at signiﬁcant levels and compete with each other for binding to their overlapping regulatory sequences. Through alternative splicing more than one mRNA transcript is generated from the same mRNA precursor, giving rise to functionally diﬀerent proteins (20). This requires that exons and introns be deﬁned diﬀerently within the same sequence context (1,9,21). Thus, alterna- tive splicing is a fundamental aspect of post- transcriptional gene regulation with signiﬁcant functional implications. The regulation of splicing in eukaryotes is highly complex and often takes place through the coordinated action of multiple splicing regulatory factors. Smith and Valcarcel previously suggested that splicing is regulated by the combinatorial outcome of several regulatory sequences (22) and this hypothesis now has experimental support (23). A recent study examined the tendency of splicing factors to bind cooperatively (24); however, no extensive bioinformatic studies have examined whether overlapping regulatory sequences exhibit unique functions and provide another dimension to splicing regulation. For example, overlapping regulatory binding motifs may modulate exon selection by serving as a template for a competitive inhibition between two diﬀerent SR proteins. The outcome of such competition may depend on the strength with which each protein binds to its speciﬁc regulatory sequence or on the expression levels of the corresponding SR proteins, their level of phosphorylation, or their spatial distribution within the cell (Figure 1). This could provide another dimension for splicing regulation. This type of mechanism is known to occur during the regulation of transcription, which is aﬀected by the binding of diﬀerent transcription factors to overlapping binding motif sequences (25–28). The use of several splicing regulatory overlapping motifs in a combinatorial fashion could allow cells to express the required amount of each splice variant at the proper time. Here we describe a bioinformatic method, based on the natural distribution of nucleotides and splicing regulatory sequences, which indicates that there is a positive and negative interplay between overlapping splicing regulatory sequences. Speciﬁcally, we found 72 splicing regulatory motifs that overlapped more than expected and 153 splicing regulatory motif overlaps that were under negative selection. Examination of these motif overlaps revealed a unique spatial distribution; they tend to be found near the 50 splice sites of exons, especially in exons with weak 50 splice sites. Moreover, the over-abundant overlapping motifs are highly conserved among diﬀerent species, implying functionality. As a proof of principle, we experimentally tested the eﬀect of the signiﬁcantly over-abundant motif overlaps on the splicing pattern in a reporting minigene system. The results revealed that the overlaps facilitated 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. Overall, the results we present suggest that overlapping splicing regu- latory motifs provide another dimension of splicing regulation. MATERIAL AND METHODS Dataset compilation Exonic and intronic sequences of human (hg18), mouse (mm9), rat (rn4), dog (canFam2), chicken (gal3) and zebraﬁsh (danRer5) RefSeq genes were extracted from tables in the UCSC genome browser (http://www .genome.ucsc.edu/) (29) using GALAXY (http://main.g2 .bx.psu.edu/) (30). Orthologous exons were identiﬁed using coordinates downloaded from the UCSC genome browser. In order to obtain reliable alignments, we included only orthologous exons of the same length with identity levels higher than 75%. Construction of the SXN-derived minigenes The overlapping regulatory motifs and its mutants were cloned into SalI and BamHI sites inside the SXN reporting exon using the following oligos by annealing: 50-TCG ATG TTT GCG GCT GCT GGA ATG-30 and 50-GAT CCA TTC CAG CAG CCG CAA ACA-30; 50-TCG ATG TTA AGG GCT GCT GGA ATG-30 and 50-GAT CCA TTC CAG CAG CCC TTA ACA-30; 50-TCG ATG TTT GCG GCA AAA GGA ATG-30 and 50-GAT CCA TTC CTT TTG CCG CAA ACA-30; 50-TCG ATG TTA AGG GCA AAA GGA ATG-30 and 50-GAT CCA TTC CTT TTG CCC TTA ACA-30; 50-TCG ATG TTC GCG GAG GAG AAT G-30 and 50-GAT CCA TTC TCC TCC GCG AAC A-30; 50-TCG ATG TTC TCG GAG GAG AAT G-30 and 50-GAT CCA TTC TCC TCC GAG AAC A-30; 50-TCG ATG TTC GCG GAT CAG AAT G-30 and 50-GAT CCA TTC