3318–3327 Nucleic Acids Research, 2010, Vol. 38, No. 10 doi:10.1093/nar/gkq005 Published online 27 January 2010 Overlapping splicing regulatory motifs—combinatorial effects on splicing Amir Goren 1 , Eddo Kim1, Maayan Amit1, Keren Vaknin1, Nir Kfir1, Oren Ram1,2 and Gil Ast1,* 1Department of Human Genetics and Molecular Medicine, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel and 2Pathology and Cancer Center Massachusetts General Hospital, Boston Harvard Medical School, Boston Broad Institute, Cambridge, Massachusetts, USA Received September 1, 2009; Revised December 28, 2009; Accepted January 5, 2010 ABSTRACT Regulation of splicing in eukaryotes occurs through the coordinated action of multiple splicing factors. Exons and introns contain numerous putative binding sites for splicing regulatory proteins. Regulation of splicing is presumably achieved by the combinatorial output of the binding of splicing factors to the corresponding binding sites. Although putative regulatory sites often overlap, no extensive study has examined whether overlapping regulatory sequences provide yet another dimension to splicing regulation. Here we analyzed experimentally-identified splicing regula- tory sequences using a computational method based on the natural distribution of nucleotides and splicing regulatory sequences. We uncovered positive and negative interplay between overlapping regulatory sequences. Examination of these overlapping motifs revealed a unique spatial distri- bution, especially near splice donor sites of exons with weak splice donor sites. The positively selected overlapping splicing regulatory motifs were highly conserved among different species, implying functionality. Overall, these results suggest that overlap of two splicing regulatory binding sites is an evolutionary conserved widespread mechanism of splicing regulation. Finally, over-abundant motif overlaps were experimentally tested in a reporting minigene revealing that overlaps may facilitate a mode of splicing that did not occur in the presence of only one of the two regulatory sequences that comprise it. INTRODUCTION Splicing is a molecular mechanism by which introns are removed from an mRNA precursor and exons are ligated to form a mature mRNA (1). Most human genes that encode proteins contain multiple introns, and intronic sequence accounts for 95% of the average 28 000 nt of a transcription unit (2,3). Introns are variable in length and sequence, and thus the splicing machinery must be remarkably ﬂexible in order to correctly recognize and excise all introns. Splicing, which occurs in organisms as diverse as yeast and human, takes place within the spliceosome—a large complex comprising ﬁve small nuclear RNPs (U1, U2, U4, U5 and U6 snRNP) and as many as 150 proteins (4–7). The splicing machinery recognizes exons and introns using multiple signals, which presumably results in a network of interactions across exons and/or introns (8). Four main splice signals assist the splicing machinery in recognizing the proper exon–intron boundaries. These are the 50 and 30 splice sites, located at the 50 and 30 end of introns, and the branch site and the polypyrimidine tract, which are located upstream of the 30 splice site (1,9). In metazoans, splice sites are degenerate and are postulated to provide only half of the information required for recognition by the splicing machinery (10,11). Studies of the molecular basis of splicing revealed the existence of exonic and intronic cis-acting regulatory sequences (ESRs and ISRs, respectively) that bind trans-acting factors and inﬂuence splice-site selection. These cis-acting elements are rela- tively short, usually 4–12 nt, are classiﬁed as exonic or intronic splicing enhancers or silencers, and are required for the regulation of both constitutive and alternative splicing (12–17). Speciﬁc binding of splicing regulatory proteins (such as SR and hnRNP proteins) to these splicing regulatory elements assists in the placement of the spliceosome on the appropriate splice sites (18,19). *To whom correspondence should be addressed. Tel: +972 3 640 6893; Fax: +972 3 640 9900; Email: gilast@post.tau.ac.il The authors wish it to be known that, in their opinion, the ﬁrst three authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.