5574–5582 Nucleic Acids Research, 2005, Vol. 33, No. 17 doi:10.1093/nar/gki858 The importance of being divisible by three in alternative splicing Alon Magen and Gil Ast* Department of Human Genetics and Molecular Medicine, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel Received May 22, 2005; Revised August 10, 2005; Accepted September 7, 2005 ABSTRACT Alternative splicing events that are conserved in orthologous genes in different species are commonly viewed as reliable evidence of authentic, functionally significant alternative splicing events. Several recent bioinformatic analyses have shown that con- served alternative exons possess several features that distinguish them from alternative exons that are species-specific. One of the most striking differ- ences between conserved and species-specific alternative exons is the high percentage of exons that preserve the reading frame (exons whose length is an exact multiple of 3, termed symmetrical exons) among the conserved alternative exons. Here, we examined conserved alternative exons and found several features that differentiate between symmetrical and non-symmetrical alternative exons. We show that symmetrical alternative exons have a strong tendency not to disrupt protein domain structures, whereas the tendency of non- symmetrical alternative exons to overlap with different fractions of protein domains is similar to that of constitutive exons. Additionally, skipping isoforms of non-symmetrical alternative exons are strongly underrepresented, compared with their including isoforms, suggesting that skipping of a large fraction of non-symmetrical alternative exons produces transcripts that are degraded by the nonsense-mediated mRNA decay mechanism. Non-symmetrical alternative exons also show a tendency to reside in the 50 half of the CDS. These findings suggest that alternative splicing of symmet- rical and non-symmetrical exons is governed by different selective pressures and serves different purposes. INTRODUCTION Alternative splicing creates multiple mRNA products from a single gene. This mechanism is widespread in higher euka- ryotes: bioinformatic analyses indicate that more than half of human genes undergo alternative splicing (1,2). As the estim- ated number of human proteins ( 90 000) far exceeds the number of known protein-coding genes ( 26 000), alternative splicing was suggested as the key mechanism for bridging this numerical disparity (3–5). The ability of alternative splicing to greatly increase the protein-coding capability of a single gene is well demonstrated in the Drosophila’s Dscam gene, which has the potential capability to generate up to 38 016 distinct protein isoforms by means of combinatorial alternative splicing of multiple ‘exon cassettes’ (6). In many cases, alternative splicing creates protein isoforms, either with partially or completely different functionality from a single gene, by selectively inserting or removing protein domains encoded by alternative ‘cassette’ exons (i.e. altern- ative exons of the exon-skipping type: they are either com- pletely included or completely removed from the mRNA product). Some examples of domain architecture alternations caused by alternative splicing are the removal of signal pep- tides and transmembrane regions, which affects protein loc- alization (7); the insertion of different structural elements to ion-channel genes in different nervous system cells (8,9) and the removal of ethylene receptors in peaches, which affects the fruit’s ripening time (10). In addition to its role in increasing proteome diversity, alternative splicing is also known to produce splice forms that are not being translated into proteins, but, rather, play a regulatory role. The best-described example for such ‘unpro- ductive’ splicing is the generation of splice variants targeted for degradation by the nonsense-mediated mRNA decay mechanism (NMD) (1,11–13). NMD is an RNA surveillance function that recognizes mRNAs containing premature termi- nation codons (PTCs) and targets them for degradation, rather than translation into proteins [reviewed in (14)]. It was even suggested that as much as 35% of human alternative isoforms *To whom correspondence should be addressed. Tel: +972 3 640 6893; Fax +972 3 640 9900; Email: gilast@post.tau.ac.il The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org