Alternative splicing (AS) and gene duplication (GD) both are processes that diversify the protein repertoire. Recent examples have shown that sequence changes introduced by AS may be comparable to those introduced by GD. In addition, the two processes are inversely correlated at the genomic scale: large gene families are depleted in splice variants and vice versa. All together, these data strongly suggest that both phenomena result in interchangeability between their effects. Here, we tested the extent to which this applies with respect to various protein characteristics. The amounts of AS and GD per gene are anticorrelated even when accounting for different gene functions or degrees of sequence divergence. In contrast, the two processes appear to be independent in their influence on variation in mRNA expression. Further, we conducted a detailed comparison of the effect of sequence changes in both alternative splice variants and gene duplicates on protein structure, in particular the size, location, and types of sequence substitutions and insertions/deletions. We find that, in general, alternative splicing affects protein sequence and structure in a more drastic way than gene duplication and subsequent divergence. Our results reveal an interesting paradox between the anticorrelation of AS and GD at the genomic level, and their impact at the protein level, which shows little or no equivalence in terms of effects on protein sequence, structure, and function. We discuss possible explanations that relate to the order of appearance of AS and GD in a gene family, and to the selection pressure imposed by the environment.
Alternative splicing (AS) and gene duplication (GD) are two main contributors to the diversity of the protein repertoire with enormous impact on protein sequence, structure, and function [1–5]. Interestingly, several recent studies point to a direct equivalence between AS and GD. There are some cases where alternative splice variants in one organism are similar to gene duplicates in another organism [6–9]. For example, the eukaryotic splicing factor U2AF35 has at least two functional splice variants in human, U2AF35a and U2AF35b, which differ by seven amino acids in the RNA recognition motif (Figure 1A). The fugu orthologue U2AF35-a has no splice variant; instead there is a duplicate gene U2AF35-b with changes identical to those found in the human splice variant U2AF35b [9]. Further, the changes introduced to a sequence are constrained by the need to preserve a stable and functional three-dimensional (3-D) fold [10]. Indeed, structural studies have shown that insertions and deletions between gene duplicates tend to happen at sequence locations where they are less damaging [11], such as loops at solvent-accessible locations. These restrictions will apply irrespective of the source of the changes and thus may introduce a certain degree of similarity between the sequence changes associated with GD and AS. Finally, recent studies have shown that AS and GD are inversely correlated on a genome-wide scale [12,13], i. e. , small gene families tend to have more genes with alternative splice variants than do large families. These findings together—i. e. , anecdotal examples, structural constraints, and anticorrelation at the genomic level—suggest that AS and GD are interchangeable sources of functional diversification [12]. Genes with AS would not need to produce additional variants in the form of duplicates, and vice versa. Here, we first tested the anticorrelation between AS and GD with respect to sequence divergence, function, and gene expression. Second, we studied the interchangeability hypothesis at the protein structure level and asked to what extent AS and GD introduce changes to the sequence that are equivalent in their nature and effect on structure and function. To this end, we conducted a large-scale comparison of the effects of AS and GD on human and mouse proteins (Figure 1B and 1C; the results for the analysis of the mouse data can be found in Figure S4). For the vast majority of cases, the two processes result in different protein modifications with different functional implications. This finding, while consistent with the different molecular mechanisms underlying both phenomena, contradicts the anticorrelation observed at the genomic level. We discuss some possible explanations for this paradox.
In accordance with recent findings [12,13], AS and GD are anticorrelated at the genomic level (Figure 2A). There are fewer alternatively spliced genes in large families of gene duplicates than expected in an unbiased distribution. This depletion is strongest in gene families of high sequence identity (seq. id) (>80% seq. id. , GD80, Figure 2A), and weaker but still present for more diverged families (>40% seq. id, GD40). The same trend holds true when examining orthologues: the bias is stronger amongst human–mouse or human–fly orthologues (GD80, χ2-value = 150 and χ2-value = 105, respectively; p-value ≪ 0. 001 in both cases) than amongst human–yeast orthologues (GD80, χ2-value = 84; p-value ≪ 0. 001). To test whether AS and GD are interchangeable at the structural and thus functional level, we compare sequence changes between duplicate proteins to those between alternative splice isoforms (Figure 1B–1C). There are two basic types of sequence changes in both AS and GD: substitutions, and insertions or deletions (indels) (Figure 1B). Both can be described in terms of their length, the nature of the affected amino acid residues, and their location in the structure or relative to each other in the sequence. We use these properties as changes in their values can be directly related to changes in protein structure and function [19]. We analyzed the properties strictly from a protein structure and function point of view; we do not examine the evolutionary processes (such as selection) that led to them. We focus on gene families defined using two seq. id. thresholds: 80% and 40%. The former was chosen because the anticorrelation at the genomic level is stronger (Figure 2A), and global seq. id. between alternative splice variants is >80% (see below). However, GD data at the 80% seq. id. level may display only a few sequence changes, resulting in larger than expected differences between AS and GD. To avoid this bias we have also considered more diverged families, defined by a 40% seq. id. threshold. Figure S5 contains additional analyses on GD, using families defined by common protein domains. In our analyses described below, we focus on gene families that have both alternative splice variants (AS+) and gene duplicates (GD+), i. e. , AS+/GD+, except in the case of local sequence identities, for which we also extend our study to families AS–/GD+ and AS+/GD–. First we examine substitutions, i. e. , the extent and nature of amino acid changes and the length of the substituted region. In general, substitutions amongst GD range from a small number of amino acid replacements in recent homologues to a large number of replacements in proteins as divergent as haemoglobin, for instance [10]. In AS, substitutions in one isoform as compared with another one arise by the use of mutually exclusive exons [20], although they can also be due to intron retentions accompanied by stop codons [21]. AS and GD are anticorrelated at the genomic level (Figure 2A) [12,13]. This relationship holds true for genes of different degrees of sequence divergence and of different functions, and suggests functional interchangeability between both phenomena [12]. This hypothesis is supported by known examples of equivalence [6–9] and by general, structural constraints on sequence changes [10]. In contrast, our sequence analysis shows that AS and GD followed by divergence are not directly interchangeable in their effect on protein structure and function. Indeed, they introduce very different changes with respect to indels and substitutions. The differences are summarised in Table 1. Indels caused by AS are more drastic than those observed in GD, both in type and location of affected residues. In the case of substitutions, we find that for GD they are more conservative and more broadly distributed along the whole sequence than those of AS. However, we also observe a small number of cases in which the interchangeability hypothesis may be true, and the sequence changes introduced by AS and GD are equivalent (Figure 3C, Figure 7). To explain the apparent paradox between the relationship of AS and GD at the genome and at the protein level, we speculate on alternative explanations for the depletion of AS observed in large GD families (Figure 2A) [12,13], discussing several effects that may contribute to it. Duplicated genes tend to have fewer exons than genes that have no duplicates but alternative splice variants (see Figure S8). This trend may in part be due to retrotransposition events which create duplicates which are single-exon genes that cannot have splice variants. However, when accounting for single-exon genes, the anticorrelation between AS and GD is still observed (see Kopelman et al. [12] and Figure S11). In addition, the anticorrelation may arise from a combination of the negative selection pressures introduced by dosage balance effects. In singleton genes that have evolved a splice variant, duplication may be disfavoured due to a multiple simultaneous dosage balance effect [36]: a single duplication event would produce a multitude of additional gene variants whose functions interfere with the existing, tightly regulated system of biological processes. This effect would be weaker for genes without AS, thus introducing a bias towards their duplication. This bias may be reinforced, under conditions of environmental stress [37,38], by the need to increase expression levels of a desirable function [39,40]. Further, the retention of duplicates as a potential backup system may be fostered if a gene is essential for the cell' s survival upon gene knockout [41]. In contrast, the introduction of AS in essential genes does not directly support robustness against null-mutations, and may even be slightly deleterious. Thus, essentiality of genes may contribute to the inverse relationship between AS and GD. Finally, if a gene with AS has duplicated, subsequent loss of an isoform in one of the copies may be tolerated due to the existence of an identical version of this isoform in the other copy of the gene. This explanation is supported by recent findings on the evolution of AS upon GD [13], and the fact that the depletion in AS is stronger for closely related duplicates [12,13] (Figure 2A, 80% seq. id.). Such a scenario would also be an extension of the backup role shown for paralogues [42]. A combination of these effects would, in general, result in a smaller proportion of genes with AS in gene families with more than one duplicate, in particular for recent duplicates, suggesting that the chronological order of events plays a role. Subsequent divergence of the gene duplicates may alleviate the negative impact of the dosage balance effect, allowing the evolution of AS and reducing the anticorrelation between AS and GD.
The accession numbers used in this paper are from Swiss-Prot (http: //www. ebi. ac. uk/swissprot): rat Piccolo C2A Q9JKS6), human MAPK9 (P45984), MAPK10 (P53779), and MAPK13 (O15264); and from the Protein Databank (http: //www. rcsb. org/pdb): MAPK10 (1jnk).
Alternative splicing (AS) and gene duplication (GD) followed by sequence divergence constitute two fundamental biological processes contributing to proteome variability. The former reflects the ability of many genes to express different products, while the latter results in several copies of the same gene that are similar but not identical. In spite of these obvious differences, recent computational studies as well as anecdotal experimental evidence suggested that AS and GD produce functionally interchangeable protein variants. We provide a detailed study of the differences between alternative splicing and gene duplication and discuss potential interchangeability with respect to variation in expression, protein structure, and function. In general, the contribution of these two processes to the proteome variability is substantially different, and we advance some explanations that may explain this apparent contradiction and contribute to our understanding of the evolution of complex, eukaryotic proteomes.
homo (human)
computational biology
evolutionary biology
molecular biology
mus (mouse)
The (In)dependence of Alternative Splicing and Gene Duplication