The recent origins of spliceosomal introns revisited

Does the intron/exon structure of eukaryotic genes belie their ancient assembly by exon-shuffling or have introns been inserted into preformed genes during eukaryotic evolution? These are the central questions in the ongoing ‘introns-early’ versus ‘introns-late’ controversy. The phylogenetic distribution of spliceosomal introns continues to strongly favor the intronslate theory. The introns-early theory, however, has claimed support from intron phase and protein structure correlations.     

Genomic organization of the human multidrug resistance (MDR1) gene and origin of P-glycoproteins

The MDR1 gene, responsible for multidrug resistance in human cells, encodes a broad specificity efflux pump (P-glycoprotein). P-glycoprotein consists of two similar halves, each half including a hydrophobic transmembrane region and a nucleotide-binding domain. On the basis of sequence homology between the N-terminal and C-terminal halves of P-glycoprotein, we have previously suggested that this gene arose by duplication of a primordial gene. We have now determined the complete intron/exon structure of the MDR1 gene by direct sequencing of cosmid clones and enzymatic amplification of genomic DNA segments. The MDR1 gene includes 28 introns, 26 of which interrupt the protein-coding sequence. Although both halves of the protein-coding sequence are composed of approximately the same number of exons, only two intron pairs, both within the nucleotide-binding domains, are located at conserved positions in the two halves of the protein. The other introns occur at different locations in the two halves of the protein and in most cases interrupt the coding sequence at different positions relative to the open reading frame. These results suggest that the P-glycoprotein arose by fusion of genes for two related but independently evolved proteins rather than by internal duplication.     

Organization and evolution of the rat tyrosine hydroxylase gene

This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out of 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylase genes are members of a gene family which has a common evolutionary origin. We predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family.     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

Arthropod and mollusk defensins--evolution by exon-shuffling

Arthropod and mollusk defensins are secreted antibacterial proteins that exhibit similarity in sequence, mode of action and structure and are expressed ubiquitously. Comparison of the gene organization of a newly cloned scorpion defensin gene, with that of other arthropods and the mussel, revealed that all exons and introns, aside from the exon encoding the mature protein, differ widely in number, size and sequence. This variability suggests that the exon encoding the mature defensin has undergone exon-shuffling and integrated downstream of unrelated leader sequences during evolution. Unlike other exon-shuffling events, in which modules are added into existing proteins, arthropod and mollusk defensins represent the first instance of exon-shuffling of autonomous modules.     

Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications for the origin and the diversification of IF proteins.

The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results.     

Constraints on the evolution of plastid introns: the group II intron in the gene encoding tRNA-Val(UAC).

The evolution of the group II intron in the plastid gene encoding tRNA(Val)UAC (trnV) from seven plant taxa was studied by aligning secondary and other structural features. Levels of evolutionary divergence between six angiosperms and a liverwort, Marchantia polymorpha, were compared for the six domains commonly demonstrated for group II introns and were shown to be statistically heterogeneous. Evolutionary rates varied substantially among various domains and other features. Domain II showed the highest evolutionary rate, approaching the synonymous substitution rate reported for cpDNA-encoded genes, while domain VI and the helix and loop region bearing EBS1 evolved at rates similar to those for nonsynonymous substitutions of a number of cpDNA-encoded genes. The minimum free-energy structure of domain I varied among the seven taxa, suggesting that possible protein-RNA or tertiary interactions are important for intron processing.     

The Exon/Intron Organization and Chromosomal Mapping of the Mouse ADAMTS-1 Gene Encoding an ADAM Family Protein with TSP Motifs

ADAM is a recently discovered gene family that encodes proteins with a disintegrin and metalloproteinase. ADAMTS-1 is a gene encoding a new member protein of the ADAM family with the thrombospondin (TSP) type I motif, the expression of which is associated with inflammatory processes. In the present study, we have characterized the exon/intron organization of the mouse ADAMTS-1 gene. The ADAMTS-1 gene is composed of nine exons, all of which are present within the 9.2-kb genomic region. Among the nine exons, exons 1, 5, and 6 encode a proprotein domain, a disintegrin-like domain, and a TSP type I motif, respectively, of the ADAMTS-1 protein, suggesting that there is a correlation between exon/intron organization and functional domains. In addition, the exon/ intron organization of the ADAMTS-1 gene is very different from that of the metalloproteinase-like/disintegrin-like/cysteine-rich protein gene (MDC) (ADAM11), suggesting that the genomic structure of ADAM family genes is not necessarily conserved. Furthermore, fluorescencein situhybridization revealed that the ADAMTS-1 gene is located in region C3–C5 of chromosome 16, to which none of the previously identified ADAM genes have been mapped.     

Deoxyribonuclease II: structure and chromosomal localization of the murine gene, and comparison with the genomic structure of the human and three C. elegans homologs

Deoxyribonuclease II (DNase II) has been implicated in diverse functions including degradation of foreign DNA, genomic instability, and in mediating the DNA digestion associated with apoptosis. The production of a mouse deleted for DNase II would clearly help to discriminate these functions. We have cloned and sequenced the mouse gene encoding DNase II. It was found to have a similar intron/exon structure to the human gene, although introns 3 and 5 are considerably shorter. The gene is located on mouse chromosome 8. The order of genes at this locus is mGCDH, mEKLF, mDNase II, mSAST, which is the same order that these genes are found on human chromosome 19. The GenBank database contains incorrect expressed sequence tags (ESTs) for the 3' end of the mouse mRNA. Furthermore, the gene structure of two of the three homologs in C. elegans is also incorrectly predicted in the database. We have established the correct intron/exon structure for these genes and show the conserved sequence and structure of the C. elegans, murine and human genes.     

A primary survey on bryophyte species reveals two novel classes of nucleotide-binding site (NBS) genes

Due to their potential roles in pathogen defense, genes encoding nucleotide-binding site (NBS) domain have been particularly surveyed in many angiosperm genomes. Two typical classes were found: one is the TIR-NBS-LRR (TNL) class and the other is the CC-NBS-LRR (CNL) class. It is seldom known, however, what kind of NBS-encoding genes are mainly present in other plant groups, especially the most ancient groups of land plants, that is, bryophytes. To fill this gap of knowledge, in this study, we mainly focused on two bryophyte species: the moss Physcomitrella patens and the liverwort Marchantia polymorpha, to survey their NBS-encoding genes. Surprisingly, two novel classes of NBS-encoding genes were discovered. The first novel class is identified from the P. patens genome and a typical member of this class has a protein kinase (PK) domain at the N-terminus and a LRR domain at the C-terminus, forming a complete structure of PK-NBS-LRR (PNL), reminiscent of TNL and CNL classes in angiosperms. The second class is found from the liverwort genome and a typical member of this class possesses an α/β-hydrolase domain at the N-terminus and also a LRR domain at the C-terminus (Hydrolase-NBS-LRR, HNL). Analysis on intron positions and phases also confirmed the novelty of HNL and PNL classes, as reflected by their specific intron locations or phase characteristics. Phylogenetic analysis covering all four classes of NBS-encoding genes revealed a closer relationship among the HNL, PNL and TNL classes, suggesting the CNL class having a more divergent status from the others. The presence of specific introns highlights the chimerical structures of HNL, PNL and TNL genes, and implies their possible origin via exon-shuffling during the quick lineage separation processes of early land plants.     

Evolutionary conservation of intron position in a subfamily of genes encoding carbohydrate-recognition domains

The structure of the gene encoding a chicken liver receptor, the chicken hepatic lectin, which mediates endocytosis of glycoproteins has been established. The coding sequence is divided into six exons separated by five introns. The first three exons correspond to separate functional domains of the receptor polypeptide (cytoplasmic tail, transmembrane sequence, and extracellular neck region), while the final three exons encode the Ca(2+)-dependent carbohydrate-recognition domain. These results, as well as computer-assisted multiple sequence comparisons, establish this receptor as the evolutionary homolog of the mammalian asialoglycoprotein receptors. It is interesting that the chicken receptor falls into a subfamily of proteins along with the mammalian asialoglycoprotein receptors, since the saccharide-binding specificity of the chicken receptor resembles more closely that of a different set of calcium-dependent animal lectins, which includes the mannose-binding proteins. The portions of the genes encoding the carbohydrate-recognition domains of these proteins lack introns. The results suggest that divergence of intron-containing and intron-lacking carbohydrate-recognition domains preceded shuffling events in which other functional domains were associated with the carbohydrate-recognition domains. This was followed by further divergence, generating a variety of saccharide-binding specificities.     

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.     

Nucleotide sequence of the Neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae and Escherichia coli

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.     

Cryptic Exons as a Source of Increased Diversity of Ewing Tumor-Associated EWS–FLI1 Chimeric Products

In the Ewing family of tumors (EFT), the EWS gene is rearranged with members of the ets oncogene family. Variability in genomic breakpoint locations is the source of significant heterogeneity in fusion product structure. As a consequence of variably included exon sequences from the two partner genes, a variable amount of additional peptide sequence is inserted in between the minimal transforming domains. Some of this molecular diversity has recently been correlated with disparate clinical outcome. Here we report on cryptic exons found in the chimeric RNA of three EFT with different EWS-FLI1 fusions. In two tumors, the emergence of a cryptic exon from FLI1 intron 5 in the chimeric RNA was apparently unrelated to the genomic rearrangement that occurred in FLI1 introns 4 and 5, respectively. In one case, a novel exon was generated through the creation of an artificial splice acceptor site in FLI1 intron 6 by the genomic rearrangement that occurred in EWS intron 8. These results further extend the spectrum of molecular diversity in EFT.     

Evolutionary history of exon shuffling

Exon shuffling has been characterized as one of the major evolutionary forces shaping both the genome and the proteome of eukaryotes. This mechanism was particularly important in the creation of multidomain proteins during animal evolution, bringing a number of functional genetic novelties. Here, genome information from a variety of eukaryotic species was used to address several issues related to the evolutionary history of exon shuffling. By comparing all protein sequences within each species, we were able to characterize exon shuffling signatures throughout metazoans. Intron phase (the position of the intron regarding the codon) and exon symmetry (the pattern of flanking introns for a given exon or block of adjacent exons) were features used to evaluate exon shuffling. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the predominant kind in multicellular animals. Evidence is provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable presence of 1-1 shuffling units in Trichoplax adhaerens and a considerable prevalence of them in Nematostella vectensis. In contrast, Monosiga brevicollis, one of the closest relatives of metazoans, and Arabidopsis thaliana, showed no evidence of 1-1 exon or domain shuffling above what it would be expected by chance. Instead, exon shuffling events are less abundant and predominantly mediated by phase 0 introns (0-0 exon shuffling) in those non-metazoan species. Moreover, an intermediate pattern of 1-1 and 0-0 exon shuffling was observed for the placozoan T. adhaerens, a primitive animal. Finally, characterization of flanking intron phases around domain borders allowed us to identify a common set of symmetric 1-1 domains that have been shuffled throughout the metazoan lineage.     

The analysis of genomic structures in the L1 family of cell adhesion molecules provides no evidence for exon shuffling events after the separation of arthropod and chordate lineages

Members of the L1 family of neural cell adhesion molecules consist of multiple extracellular immunoglobulin and fibronectin type III domains that mediate the adhesive properties of this group of transmembrane proteins. In vertebrate genomes, these protein domains are separated by introns, and it has been suggested that L1-type genes might have been subject to exon-shuffling events during evolution. However, comparison of the human L1-CAM and the chicken neurofascin gene with the genomic structure of their Drosophila homologue, neuroglian, indicates that no major rearrangement of protein domains has taken place subsequent to the split of the arthropod and chordate phyla. The Drosophila neuroglian gene appears to have lost most of the introns that have been conserved in the human L1-CAM and the chicken neurofascin gene. Nevertheless, exon shuffling or the generation of new exons by mutational changes might have been responsible for the generation of additional, alternatively spliced exons in L1-type genes.     

Group II introns deleted for multiple substructures retain self-splicing activity.

Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5 gamma of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.