Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how and when did AS evolve? Which AS events are functional? What are the evolutionary forces that shaped, and continue to shape, AS? And what determines whether an exon is spliced in a constitutive or alternative manner? In this Review, we summarize the current knowledge of AS and evolution and provide insights into some of these unresolved questions.

An increasing body of evidence indicates that transcription and splicing are coupled, and it is accepted that chromatin organization regulates transcription. Little is known about the cross-talk between chromatin structure and exon-intron architecture. By analysis of genome-wide nucleosome-positioning data sets from humans, flies and worms, we found that exons show increased nucleosome-occupancy levels with respect to introns, a finding that we link to differential GC content and nucleosome-disfavoring elements between exons and introns. Analysis of genome-wide chromatin immunoprecipitation data in humans and mice revealed four specific post-translational histone modifications enriched in exons. Our findings indicate that previously described enrichment of H3K36me3 modifications in exons reflects a more fundamental phenomenon, namely increased nucleosome occupancy along exons. Our results suggest an RNA polymerase II-mediated cross-talk between chromatin structure and exon-intron architecture, implying that exon selection may be modulated by chromatin structure.

Alternative splicing creates transcriptome diversification, possibly leading to speciation. A large fraction of the protein-coding genes of multicellular organisms are alternatively spliced, although no regulated splicing has been detected in unicellular eukaryotes such as yeasts. A comparative analysis of unicellular and multicellular eukaryotic 5' splice sites has revealed important differences - the plasticity of the 5' splice sites of multicellular eukaryotes means that these sites can be used in both constitutive and alternative splicing, and for the regulation of the inclusion/skipping ratio in alternative splicing. So, alternative splicing might have originated as a result of relaxation of the 5' splice site recognition in organisms that originally could support only constitutive splicing.

Alu repetitive elements are found in ∼1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of the Alu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon. Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain an Alu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.

Alternative splicing increases transcriptome and proteome diversification. Previous analyses aiming at comparing the rate of alternative splicing between different organisms provided contradicting results. These contradicting results were attributed to the fact that both analyses were dependent on the expressed sequence tag (EST) coverage, which varies greatly between the tested organisms. In this study we compare the level of alternative splicing among eight different organisms. By employing an EST independent approach we reveal that the percentage of genes and exons undergoing alternative splicing is higher in vertebrates compared with invertebrates. We also find that alternative exons of the skipping type are flanked by longer introns compared to constitutive ones, whereas alternative 5′ and 3′ splice sites events are generally not. In addition, although the regulation of alternative splicing and sizes of introns and exons have changed during metazoan evolution, intron retention remained the rarest type of alternative splicing, whereas exon skipping is more prevalent and exhibits a slight increase, from invertebrates to vertebrates. The difference in the level of alternative splicing suggests that alternative splicing may contribute greatly to the mammal higher level of phenotypic complexity, and that accumulation of introns confers an evolutionary advantage as it allows increasing the number of alternative splicing forms.