- Research article
- Open Access
Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes
© Ragg et al; licensee BioMed Central Ltd. 2009
- Received: 11 February 2009
- Accepted: 22 August 2009
- Published: 22 August 2009
Intron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes.
Here we investigated intron dynamics in the serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing.
Multiple intron acquisitions were identified in serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.
- Intron Position
- Intron Gain
- Serpin Gene
- Spliceosomal Intron
- Intron Insertion Site
Spliceosomal introns are key attributes of most eukaryotic genes. Their origin is still unclear, though descent from group II self-splicing introns seems to be likely [1, 2]. All components essential for removal of intronic sequences, a prerequisite for maturation of most transcripts and formation of functional gene products, have been identified in basal eukaryotes , indicating that the ability to cope with spliceosomal introns was fully developed in the last common ancestor of eukaryotes. In addition to their obscure provenance, spliceosomal introns present another unresolved enigma. In many taxa, intron dynamics is dominated by losses, and gains of introns were often found to be rare [reviewed in ref. ]. In recent mass analyses of various vertebrate genomes, no intron gains were detected [4, 5]. With some genes and lineages, however, intron acquisition can hardly be questioned [6–8]. Several proposals have been brought forward to explain intron birth [2, 9–11], but how these sequences were actually created, is still mysterious.
The serpins are a superfamily of proteins that cover a highly divergent spectrum of functions [12, 13]. The origin of these proteins, primarily encompassing inhibitors of serine proteases, but also including members with entirely other tasks, is not known. Serpins are found in all major branches of the tree of life, but they are rare in fungi, and their distribution in archaea and eubacteria is disjunct. In vertebrates, serpins participate in the control of blood coagulation, fibrinolysis, and other proteolytic pathways . In both vertebrates and invertebrates, serpins are also engaged in regulating the innate immune response. An arms race between proteases of pathogens and host protease inhibitors, and vice versa, was proposed to foster functional diversification of serpins [14, 15].
Here, we investigated the structures of serpin genes from lineages that pre- and postdate the split of vertebrates in order to get insight into the dynamics of their introns. The data disclose that, after establishment of major groups of vertebrate serpin genes, multiple non-canonical introns emerged in a lineage of ray-finned fishes.
Appearance of exon-intron patterns characteristic for vertebrate serpins
We first studied serpin genes and their cDNAs in two lancelet species, Branchiostoma lanceolatum (B. lanceolatum) and Branchiostoma floridae (B. floridae), representing a group of extant cephalochordates (phylum: Chordata). Experimental approaches disclosed several genes and cDNAs coding for serpins in B. lanceolatum, further superfamily members were detected by mining the genome of the closely related B. floridae . Altogether, three types of serpin genes (L1–L3) with distinctly different intron patterns were observed (Figure 1; Additional file 1). Following the previous convention , intron positions are projected onto the sequence of the human serpin α1-antitrypsin. The phases of introns are indicated by the suffixes a-c, according to their location after the first, second, or third base of the codon in question. In this reference system, the two introns of L1 genes map to positions 75c and ~176a (the exact position of this intron is ambiguous, due to alignment problems). The L2 genes contain introns at positions ~86b, 151c, 223b, 283c and 339c. The L3 genes exhibit common introns at positions 73b, 125b, ~175c and 339c, however, there are also introns that are unique to individual group members (position 280b in Bflor_Spn9; positions 224b and 278b in Bflor_Spn10). Another intron in Bflor_Spn10 is located outside the conserved serpin scaffold (gene-specific numbering of position: 29a). There are several additional serpin-like sequences in the B. floridae genome, but it is currently not discernible whether they represent intact genes (not shown); however, it is clear that serpin genes from lancelets and vertebrates differ largely with respect to their exon-intron organizations. In fact, just a single intron location (position 339c) is shared. Apparently, major changes affecting the exon-intron patterns of serpin genes have occurred since the cephalochordate/vertebrate split.
Having established lancelets as appropriate outgroup for evaluating evolution of serpins in vertebrates, we turned to lampreys, a group of basal, jawless fishes. Lampreys, in sharp contrast with lancelets, depict at least four of the six canonical groups of vertebrate serpins. A survey of cDNA and genomic sequences from Lampetra fluviatilis (L. fluviatilis; European river lamprey) and from Petromyzon marinus (P. marinus; sea lamprey) revealed representatives of groups V2, V4 and V6 (Additional file 2). We also infer that intact members of group V1 exist as indicated by the isolation of a corresponding full-length cDNA from L. fluviatilis. The associated gene contains all introns characteristic for group V1 with the exception of the 78c intron that, presumably due to its large size, remained undetected (unpublished results). Members of groups V3 and V5 were not identified.
Serpingenes with non-standard exon-intron patterns
After curtailing the emergence of major groups of vertebrate serpin genes, the dynamics of their exon-intron patterns was analyzed. The list of species investigated included: man, chicken (Gallus gallus), the clawed frog (Xenopus tropicalis; X. tropicalis), and several fishes including Danio rerio (D. rerio), Oryzias latipes (O. latipes), Gasterosteus aculeatus (G. aculeatus), Tetraodon nigroviridis (T. nigroviridis), and Takifugu rubripes (T. rubripes). Several serpin genes, though clearly members of one of the six canonical groups, were found to deflect from the standard organizations. Like their counterparts in lampreys, angiotensinogen genes of tetrapods and D. rerio exhibit the canonical gene architecture of group V2, the T. rubripes orthologue, however, contains two extra introns that split the exonic sequences at positions 77c and 233c, respectively (Figure 2; non-standard introns are marked in green). These surplus introns are also present in the orthologues of O. latipes and G. aculeatus, but they are not found in any other vertebrate serpins. In the current release of the T. nigroviridis genome, the angiotensinogen gene is not represented. EST data revealed (Additional file 2) that, in G. aculeatus, the extra introns are indeed spliced out, rejecting the argument that they are artifacts.
The HCII genes of mammals [29, 30], chicken, X. tropicalis and D. rerio uniformly depict the conserved intron pattern of group V2. Deviations from the standard structure, however, were observed in lampreys and in several fishes. The HCII orthologues of O. latipes, G. aculeatus, T. rubripes and T. nigroviridis each contain a non-standard intron mapping to position 241c (Additional file 1). EST analyses confirmed expression and correct splicing of the HCII transcript in O. latipes (Additional file 2). To our knowledge, there are no other vertebrate serpins possessing an intron at this position. A gain that occurred after the split of the D. rerio lineage from the other ray-finned fishes thus may explain appearance of this intron. Another extra intron in the HCII genes of pufferfishes maps to the inhibitor's N-terminal tail (gene-specific numbering of positions: 85b and 87b for T. rubripes and T. nigroviridis, respectively). Though also found in O. latipes and G. aculeatus, this intron here is not considered any further, due to its location outside the serpin scaffold. Examination of lamprey HCII also revealed unique introns. The 83c intron (α1-antitrypsin numbering) is embedded in a well-conserved region; its origin, however, is difficult to evaluate. The others (correctly predicted?) map to the N-terminal extension (gene-specific numbering of positions: 38b and 118c).
Database searches also disclosed members of group V2, dubbed Spn_94a, with a surplus intron at position 94a (Additional file 1). In O. latipes, G. aculeatus, T. rubripes and T. nigroviridis, these genes are flanked by a conserved set of markers (Additional file 3). The imbedded serpin genes thus are derived from a common ancestor. Position 94a was previously not known to harbor introns in vertebrate serpins. Inspection of chromosomal gene order revealed that D. rerio also contains Spn_94a; the extra intron, however, is missing, suggesting that it was gained after divergence of the D. rerio lineage. In pufferfishes, two further members of group V2 with an extra intron, located at position 215c, were identified (Spn_215c from T. rubripes and T. nigroviridis, respectively). The origin of these genes is unclear; the unique surplus intron suggests that they share a common ancestor (Additional file 1).
The intron patterns of HSP47 homologues uncover telling insight into the evolution of group V6 in fishes (Figure 3). Dan_HSP47_1, Dan_HSP47_2, Dan_HSP47_3, and HSP47 from P. marinus and tetrapods, respectively, all depict the standard intron repertoire. Gast_HSP47_1, Oryz_HSP47_1 and Fugu_HSP47_1, however, contain two additional introns (positions 36b and 102c, respectively); Gast_HSP47_2, in contrast, merely possesses the default introns. These findings suggest that the novel introns, which are restricted to HSP47_1 orthologues, were acquired by group V6 during evolution of ray-finned fishes after divergence of the D. rerio lineage, though an intron loss scenario cannot be excluded. Two intron gains or a single, coupled intron gain event are/is sufficient to explain the exon-intron patterns found in group V6; the intron removal scenario, in contrast, requires multiple intron loss events. If such losses had occurred, they must have affected several taxa, including lampreys, tetrapods and fishes. Moreover, this scenario also demands that the same two introns (positions 36b and 102c) were always deleted in parallel, while all other introns were unaffected. The parallel emergence of introns at positions 77c and 233c in angiotensinogen genes (Figure 2) provides further support for the intron gain presumption.
In contrast with several intron gains, we detected a single case of probable intron loss during evolution of vertebrate serpins. Antithrombin (AT), the only member of group V5, is a potent thrombin inhibitor in the presence of heparin. Among other characteristic features, AT orthologues are easily discernible through a highly conserved sequence centering around helix D (Additional file 1) that constitutes a major part of the heparin binding region. The AT genes of tetrapods have introns interrupting the serpin scaffold at positions 78c, 148c, 191c, 320a and 339c (Figure 1). The orthologues from fishes, however, exhibit an additional intron at position 262c (Additional file 1). This intron is also present in D. rerio and in the currently incompletely annotated AT gene of T. nigroviridis (not shown). Intron 262c is a standard attribute of group V1 that is believed to share a common ancestor with group V5 . We therefore suspect that this intron was lost in AT genes of tetrapods. We were not able to identify this gene in P. marinus; further tracing of the 262c intron was therefore not possible.
In serpins from groups V3 and V4, deviations from the standard structures were not observed (not shown). All genes from group V3 analyzed featured introns at positions ~86a-90a, 167a, 230a, 290b, 323a, 352a, and 380a. The intron pattern characteristic of group V4 (positions 67a, 123a, 192a, 238c, 307a) was also conserved.
Features of novel introns
Sequences flanking the insertion points of novel introns in vertebrate serpin genes.
Intron gain has been reported to occur very rarely in many metazoan lineages, including mammals and other vertebrates [4, 5]. To our surprise we disclosed multiple newly acquired introns by probing a vertebrate protein superfamily, the serpins, while a single intron was presumably lost. The most clear-cut example for intron gain is angiotensinogen that, in lampreys, tetrapods and D. rerio, depicts the typical exon/intron pattern of group V2. The novel introns at positions 77c and 233c, like all other non-standard introns identified, are exclusively found in a group of ray-finned fishes that emerged after the split of the D. rerio lineage. None of the novel intron positions is found in any paralogues of group V2 or in any other vertebrate serpins known. Thus, from a parsimony standpoint, the view that these introns were acquired de novo is more likely than the alternative possibility that these introns were inherited from a common ancestor. The novel introns were apparently not acquired at the expense of adjacent introns, as concomitant loss of such sequences was not observed. Since there are no reports of intron gain in serpins of other vertebrates, our findings may reflect an episode of enhanced intron acquisition that happened during radiation of ray-finned fishes. Several of the few other well-documented intron gains also occurred during diversification of these fishes [36–39].
Except for Spn_94a, the insertion points of novel introns are exact, as no deletions or insertions at the flanking sequences are evident. Many serpins tolerate indels without functional impairment, especially in loops connecting α-helices and β-strands, a noteworthy aspect that should be kept in mind in the discussion of intron gain mechanisms. Inspection of nucleotide sequences in the neighborhood of novel introns revealed compliance with the proto-splice site sequences proposed [34, 35]. A relatively high fraction of bases immediately adjacent to the splice acceptor site are pyrimidine residues, possibly biased due to the limited number of samples analyzed or due to subsequent selection processes. Alternatively, this finding may indicate that the specifications required at the 3'-side of intron insertion sites are low.
Several mechanisms have been claimed to be responsible for birth of introns [2, 41, 42]. Key to most of the proposals is duplication events operating at some stage. A popular mechanism thought to mediate intron gain involves transposons. A distinguishing feature of fish genomes is their diversity of retrotransposable elements, notably retrotransposons, some of which were active in recent times [33, 43, 44]. However, we could not detect any sequences in non-standard introns sharing similarity with known repetitive elements, participation of duplication-dependent transposons with intron gain therefore remains elusive. Preferential loss of such elements from newly acquired introns, however, cannot be excluded. In two recently described cases of intron acquisition, similarity to other genomic sequences was also not observed .
We consider that various processes might be responsible for intron birth, not necessarily related with the events responsible for their primordial emergence. Excision of introns, probably created by expansion of simple repeats or complex repetitive elements [36, 45] or generated by intronization of exon sequences , demonstrates that the spliceosome can do its job as long as the essential splice signals are present, irrespective of how the intron was created. Since we were not able to find support in favor of currently discussed mechanisms of intron gain (reviewed in [2, 9–11]), we consider that introns might also be created by other means. After the fish-specific whole genome duplication, compaction processes resulted in reduction of genome sizes in many actinopterygians [33, 46–48]. Deprivation of genomic DNA not only entailed loss of complete genes, but also affected intergenic and intronic sequences. Intron sizes, for instance, are considerably larger in D. rerio than in pufferfishes. It remains to be investigated, whether all these sequences have gone without leaving any traces behind. Loss of genomic sequences is inevitably associated with DNA breakage, requiring repair and recombination. The involvement of such processes in intron acquisition should therefore be considered; however, like for the other intron gain mechanisms suggested, conclusive evidence of a causal relationship between DNA breakage/repair and de novo intron formation is lacking as yet.
Genome-scale or local amplification of genes might conceivably favor gain and maintenance of novel introns, since unaffected copies are left in the genome. The appearance of novel introns in the serpin superfamily, however, was apparently not associated with the fish-specific genome duplication that preceded radiation of ray-finned fishes . In current phylogenetic scenarios, divergence of D. rerio, which lacks all of the non-canonical introns, antedates the split of the lineage that experienced intron gains. Retention of introns once acquired might indeed be favored by the co-existence of paralogues, relative frequencies of intron gains in single versus multi-copy gene families, however, are discussed controversially [49, 50].
By a comprehensive analysis of lineages pre- and postdating the split of vertebrates, the founding period for major groups of vertebrate serpins was ascertained. Following establishment of the canonical exon-intron patterns before or close to diversification of lampreys, a lineage of ray-finned fishes is shown to have experienced multiple intron gains. Remarkably, in two genes concomitant appearance of non-canonical introns is observed, suggesting that intron gains may even happen in parallel or in a rapidly consecutive manner. These data strongly suggest that intron acquisition occurs in at least some vertebrate taxa. The observation that all intron gain events were found in a lineage of ray-finned fishes that underwent genome compaction, leads us to assume that DNA breakage/repair processes may enable or facilitate intron acquisition. Angiotensinogen, HCII, and HSP47 were identified as ancient members of the serpin superfamily in vertebrates.
Adult lancelets (B. lanceolatum) were purchased from the Alfred-Wegener-Institut für Polar- und Meeresforschung, Helgoland, Germany. European river lampreys (L. fluviatilis) were obtained from the Bundesforschungsanstalt für Fischerei, Hamburg, Germany.
Cloning and sequencing of serpin cDNAs and genes
The isolation of poly(A)-RNA and genomic DNA, the synthesis of cDNA, PCR amplification of serpin cDNA fragments using various sets of degenerate primers, and cloning of 5'- and 3'-cDNA ends followed published procedures [20, 51]. DNA sequences (Additional file 2) have been deposited in the DDBJ/EMBL/GenBank database.
Sequence data analysis and intron annotation
Genomic data for serpins from human, chicken , X. tropicalis , D. rerio , G. aculeatus , O. latipes , T. rubripes  and T. nigroviridis  were extracted from the Ensembl genome browser, release 51 , or in the case of P. marinus , from PreEnsembl (http://www.ensembl.org). Sequences from the B. floridae genome were gathered from the JGI genome browser (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html). EST and cDNA data mining included searches in the NCBI trace archive (http://www.ncbi.nlm.nih.gov/) and in the UCSC genome browser , applying the BLAST algorithm. Some gene models were refined using EST data (Additional file 2).
All intron positions predicted by gene models were examined visually, corrected and amended manually, if necessary. Whenever cDNA or EST sequences were available, intron positions were checked by means of GENEWISE . Protein sequences were aligned with CLUSTALW  with some manual improvements. Intron positions were projected onto the sequence of mature human α1-antitrypsin as described . All intron locations allude to the reference protein, unless stated otherwise. Only introns mapping to the conserved serpin scaffold (i.e. positions 33 to 394 of human α1-antitrypsin) were considered.
Sequences of non-canonical introns were searched for repetitive elements with the RepeatMasker package (version 3.2.6; (http://www.repeatmasker.org)) and with RepBase Censor (http://www.girinst.org/censor/index.php)  using default settings.
Phylogenetic analysis was performed using the Neighbor-Joining method  conducted in MEGA4 . All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). There were a total of 340 positions in the final dataset.
We thank Professor Dr S. Ehrlich, BFA für Fischerei, Hamburg, for providing lampreys. This work was supported by the Deutsche Forschungsgemeinschaft, Graduate Program 'Bioinformatics' at Bielefeld University.
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