Evolutionary History of Exon Shuffling

Genetica (2012) 140:249–257 DOI 10.1007/s10709-012-9676-3 Evolutionary history of exon shuffling Gustavo S. França • Douglas V. Cancherini • Sandro J. de Souza Received: 2 January 2012 / Accepted: 23 August 2012 / Published online: 5 September 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Exon shuffling has been characterized as one of presence of 1-1 shuffling units in Trichoplax adhaerens and the major evolutionary forces shaping both the genome and a considerable prevalence of them in Nematostella vect- the proteome of eukaryotes. This mechanism was particu- ensis. In contrast, Monosiga brevicollis, one of the closest larly important in the creation of multidomain proteins relatives of metazoans, and Arabidopsis thaliana, showed during animal evolution, bringing a number of functional no evidence of 1-1 exon or domain shuffling above what it genetic novelties. Here, genome information from a variety would be expected by chance. Instead, exon shuffling of eukaryotic species was used to address several issues events are less abundant and predominantly mediated by related to the evolutionary history of exon shuffling. By phase 0 introns (0-0 exon shuffling) in those non-metazoan comparing all protein sequences within each species, we species. Moreover, an intermediate pattern of 1-1 and 0-0 were able to characterize exon shuffling signatures exon shuffling was observed for the placozoan T. adhae- throughout metazoans. Intron phase (the position of the rens, a primitive animal. Finally, characterization of intron regarding the codon) and exon symmetry (the pat- flanking intron phases around domain borders allowed us to tern of flanking introns for a given exon or block of adja- identify a common set of symmetric 1-1 domains that have cent exons) were features used to evaluate exon shuffling. been shuffled throughout the metazoan lineage. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the Keywords Exon shuffling Á Metazoan evolution Á predominant kind in multicellular animals. Evidence is Protein domains Á Introns provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10709-012-9676-3) contains supplementary One of the most important ways to create new genes is material, which is available to authorized users. through intron-mediated recombination, a phenomenon proposed by Gilbert more than 30 years ago and named by & G. S. França Á D. V. Cancherini Á S. J. de Souza ( ) him ‘‘exon shuffling’’ (Gilbert 1978). It is known today that Ludwig Institute for Cancer Research, Saõ Paulo Branch, Saõ Paulo 01323-903, Brazil exon shuffling is one of the most common mechanisms to e-mail: [email protected]; [email protected] create new genes and extensive reports have been published describing several mechanistic and evolutionary G. S. França aspects of exon shuffling (de Souza 2003; Eickbush 1999; Departamento de Bioquı´mica, Instituto de Quı´mica, Programa de Po´s-Graduaçaõ, Universidade de Saõ Paulo, Kaessmann et al. 2002; Liu and Grigoriev 2004; Long and Saõ Paulo 05508-900, Brazil Langley 1993; Patthy 1987; Patthy 1999). A very influential factor in intron and exon evolution is Present Address: what is called intron phase, which refers to the position of a S. J. de Souza Brain Institute, UFRN, Av. Nascimento de Castro 2155, Natal, given intron within the codon. Phase 0 introns lie between RN 59056-450, Brazil two codons, phase 1 and phase 2 introns are located after 123 250 Genetica (2012) 140:249–257 the first and second nucleotides of the codon, respectively. believed to be one of the most basal animals (Dellaporta Removal/insertion of introns or exons have very different et al. 2006;Srivastavaetal.2008), presents an interme- effects on the reading frame of a gene. Intron insertion or diary frequency of 1-1 and 0-0 shuffling units. Moreover, removal frequently occurs without any modification in the 2-2 exon shuffling seems to have had no significant con- coding sequence. However, the insertion or removal of an tribution in the building of modular proteins in any ana- exon or a block of consecutive exons in a gene may disrupt lyzed species. The availability of genome sequence for all the correct reading frame of all exons downstream from the these species allowed us to draw a map of domain shuf- insertion point if the exon or exonic block did not contain a fling occurrence along eukaryotic evolution, revealing the number of nucleotides that is an exact multiple of three. expansion of certain domains during specific periods of This latter case will take place when the phase of the two animal evolution. introns bordering the exon or exonic block is the same in both extremities. When such type of exon or exonic block is involved in an exon shuffling event, it is said that the Results and discussion shuffling unit is symmetric. Because they preserve the reading frame of genes created after exon shuffling events, Distribution of intron phase and exon symmetry they are believed to be less likely a target for purifying selection. As shown by us and others, intron phase distribution in Indeed, the excess of symmetric exons in most of the most eukaryotic species is biased toward a predominance genomes analyzed up to now suggests that exon shuffling of phase 0 introns (de Souza et al. 1998; Fedorov et al. has been a major player in shaping the genome of 1992;Longetal.1995;Nguyenetal.2006; Qiu et al. eukaryotic species (de Souza et al. 1998; Long et al. 1995). 2004). This has been a central issue in the debate about Another feature, mainly observed in metazoans, is the the existence of introns in the ancestor of eukaryotes and prevalence of shuffling units flanked by introns of phase 1 prokaryotes. To evaluate if the dataset used by us was in (1-1 exon shuffling). It has been shown that there is a large agreement with these previous observations, we calcu- excess of 1-1 exons in animal species compared to what lated the number of phase 0, 1 and 2 introns for all ana- would be expected from the total proportions of intron lyzed species. Figure 1a shows that indeed phase 0 is the phases in the corresponding genomes (Patthy 1999; Long most prevalent type of intron (42 to 57 %) in all species, et al. 1995). The same excess is also observed for protein followed by phase 1 and 2. Next, we evaluated the relative domains flanked by phase 1 introns (Kaessmann et al. excess of symmetric exons (flanked by introns of same 2002; Liu et al. 2005; Vibranovski et al. 2005). phases) and asymmetric exons (flanked by introns of In spite of all these advances, important information on distinct phases) from the expected values in a hypothetic the mechanism and functional impact of exon shuffling scenario where intron phases are randomly distributed in along evolution remains unknown. For example, was exon respect to symmetry (Fig. 1b). The main message from shuffling a frequent mechanism for the creation of new Fig. 1b is the significant excess of symmetric exons for genes in non-metazoan species? Although there are iso- almost all analyzed species (p values by Chi-square test lated examples of exon shuffling in non-metazoan species are shown in Table S1, and range from 10-2 to 10-145). (Elrouby and Bureau 2010; Long et al. 1996; Morgante We believe that the best currently available explanation et al. 2005), little is known about the overall frequency and for favored exon symmetry is that exon shuffling has mode of exon shuffling in these species. When exactly repeatedly occurred during evolution and left remnant did 1-1 exons (and domains flanked by phase 1 introns) start signals, even if sequence similarity cannot identify some to expand during evolution? Reports from Patthy’s group past shuffling events anymore. We thus speculate that the (Patthy 1999; Patthy 2003) indicate that 1-1 exon shuffling symmetric exon excesses observed by us reflect a perva- is specific to metazoans, although a more complete com- sive occurrence of shuffling events in eukaryotic gen- parative analysis is still missing. Was 0-0 and 2-2 exon omes. As seen before, higher excess was observed for 1-1 shuffling relevant in any stage of eukaryotic evolution? exons, especially for metazoans (de Souza et al. 1998; The questions posed above were addressed by gener- Long et al. 1995; Vibranovski et al. 2005). Humans, for ating a catalog of putative exon shuffling events in several example, have an excess of 11, 19 and 8 % for 0-0, 1-1 eukaryotic species, including non-metazoans and early and 2-2 symmetric exons, respectively. Interestingly, branching metazoans. We observed that 1-1 exon shuf- plants have smaller excess for symmetric exons (9, 9 and fling became the most frequent type of exon shuffling 13 % for 0-0, 1-1 and 2-2 exons, respectively). The cho- soon after the emergence of Metazoa. Furthermore, non- anoflagellate Monosiga brevicollis showed a small excess metazoan species have a predominance of 0-0 exon of symmetric exons that resulted only from an excess of shuffling. Intriguingly, Trichoplax adhaerens,whichis 0-0 exons. 123 Genetica (2012) 140:249–257 251 A 60 species. However, the number of putative exon shuffling 50 events was insufficient to perform further analyses (data not shown). This may be, in part, due to processes of both intron 40 0 gain and intron loss in extant fungi species (Stajich et al. 30 1 2 2007), obscuring the required signal of flanking introns.

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