SR proteins: a foot on the exon before the transition from intron to exon definition

Two recent publications illuminate the evolution of alternative splicing, showing that a SR (serine-arginine-rich) protein that regulates alternative splicing in multicellular organisms is also found in a unicellular organism without alternative splicing, in which it can assist in the splicing of weak introns. Moreover, insertion of SR proteins into an organism lacking such proteins can restore the splicing of weak introns. These results imply that SR proteins had already facilitated the splicing of weak introns before the evolution of alternative splicing.     

Characteristics and regulatory elements defining constitutive splicing and different modes of alternative splicing in human and mouse

Alternative splicing is a major contributor to genomic complexity, disease, and development. Previous studies have captured some of the characteristics that distinguish alternative splicing from constitutive splicing. However, most published work only focuses on skipped exons and/or a single species. Here we take advantage of the highly curated data in the MAASE database (see related paper in this issue) to analyze features that characterize different modes of splicing. Our analysis confirms previous observations about alternative splicing, including weaker splicing signals at alternative splice sites, higher sequence conservation surrounding orthologous alternative exons, shorter exon length, and more frequent reading frame maintenance in skipped exons. In addition, our study reveals potentially novel regulatory principles underlying distinct modes of alternative splicing and a role of a specific class of repeat elements (transposons) in the origin/evolution of alternative exons. These features suggest diverse regulatory mechanisms and evolutionary paths for different modes of alternative splicing.     

Evolution of Nova-dependent splicing regulation in the brain

A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.     

Alternative splicing, the gene concept, and evolution

Alternative splicing allows for the production of many gene products from a single coding sequence. I introduce the concept of alternative splicing via some examples. I then discuss some current hypotheses about the explanatory role of alternative splicing, including the claim that splicing is a significant contributor to the difference in complexity between the human genome and proteosome. Hypotheses such as these bring into question our working concepts of the gene. I examine several gene concepts introduced to cope with processes such as alternative splicing. Next I introduce some hypotheses about the evolution of mechanisms alternative splicing in higher organisms. I conclude that attention to alternative splicing reveals that we adopt an attitude that developmental theorizing must inform evolutionary theorizing and vice versa.     

Evolution of the Fgf and Fgfr gene families

Fibroblast growth factors (Fgfs) and Fgf receptors (Fgfrs) comprise a signaling system that is conserved throughout metazoan evolution. Twenty-two Fgfs and four Fgfrs have been identified in humans and mice. During evolution, the Fgf family appears to have expanded in two phases. In the first phase, during early metazoan evolution, Fgfs expanded from two or three to six genes by gene duplication. In the second phase, during the evolution of early vertebrates, the Fgf family expanded by two large-scale gen(om)e duplications. By contrast, the Fgfr family has expanded only in the second phase. However, the acquisition of alternative splicing by Fgfrs has increased their functional diversity. The mechanisms that regulate alternative splicing have been conserved since the divergences of echinoderms and vertebrates. The expansion of the Fgf and Fgfr gene families has enabled this signaling system to acquire functional diversity and, therefore, an almost ubiquitous involvement in developmental and physiological processes.     

In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments

To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor α ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results of our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution.     

Increase of functional diversity by alternative splicing

A large-scale analysis of protein isoforms arising from alternative splicing shows that alternative splicing tends to insert or delete complete protein domains more frequently than expected by chance, whereas disruption of domains and other structural modules is less frequent. If domain regions are disrupted, the functional effect, as predicted from 3D structure, is frequently equivalent to removal of the entire domain. Also, short alternative splicing events within domains, which might preserve folded structure, target functional residues more frequently than expected. Thus, it seems that positive selection has had a major role in the evolution of alternative splicing.     

Evolutionary convergence of alternative splicing in ion channels

In Drosophila melanogaster and humans, members of three different ion-channel gene families share tandem exon duplications, which are alternatively spliced. In this article, I demonstrate that the duplication events that give rise to these mutually exclusive exons are unlikely to be ancestral but have probably occurred independently in different lineages. These events provide remarkable examples of evolutionary convergence in alternative splicing. The result has important implications for the analysis of regulation of alternative splicing using comparative genomics and our understanding of molecular evolution.     

Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

Background  

Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins.     

Predominant contribution of cis‐regulatory divergence in the evolution of mouse alternative splicing

Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis‐regulatory versus trans‐acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele‐specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele‐specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis‐regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis‐regulatory changes in the evolution of mouse alternative splicing.