Patterns of intron gain and conservation in eukaryotic genes

Background:  

The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions.     

Prevalence of intron gain over intron loss in the evolution of paralogous gene families

The mechanisms and evolutionary dynamics of intron insertion and loss in eukaryotic genes remain poorly understood. Reconstruction of parsimonious scenarios of gene structure evolution in paralogous gene families in animals and plants revealed numerous gains and losses of introns. In all analyzed lineages, the number of acquired new introns was substantially greater than the number of lost ancestral introns. This trend held even for lineages in which vertical evolution of genes involved more intron losses than gains, suggesting that gene duplication boosts intron insertion. However, dating gene duplications and the associated intron gains and losses based on the molecular clock assumption showed that very few, if any, introns were gained during the last ~100 million years of animal and plant evolution, in agreement with previous conclusions reached through analysis of orthologous gene sets. These results are generally compatible with the emerging notion of intensive insertion and loss of introns during transitional epochs in contrast to the relative quiet of the intervening evolutionary spans.     

Intron gain and loss in the evolution of the conserved eukaryotic recombination machinery

Intron conservation, intron gain or loss and putative intron sliding events were determined for a set of three genes (SPO11, MRE11 and DMC1) involved in basic aspects of recombination in eukaryotes. These are ancient genes and present in nearly all of the major kingdoms. MRE11 is of bacterial origin and can be found in all kingdoms. DMC1 is a specialized homolog of the bacterial RecA protein, whereas the SPO11 gene is of archaebacterial origin. Only unique homologs of SPO11 are found in animals and fungi whereas three distantly related SPO11 copies are present in plant genomes. A comparison of the respective intron positions and phases of all genes was performed, demonstrating that a quarter of the intron positions were perfectly conserved over more than 1000000000 years. Regarding the remaining three quarters of the introns we found insertions to be about three times more frequent than deletions. Aligning the introns of the three different SPO11 homologs of Arabidopsis thaliana we propose a conclusive model of their evolution. We postulate that at least one duplication event occurred shortly after the divergence of plants from animals and fungi and that a respective homolog has been retained in a protist group, the apicomplexa.     

The biology of intron gain and loss

Intron density in eukaryote genomes varies by more than three orders of magnitude, so there must have been extensive intron gain and/or intron loss during evolution. A favored and partial explanation for this range of intron densities has been that introns have accumulated stochastically in large eukaryote genomes during their evolution from an intron-poor ancestor. However, recent studies have shown that some eukaryotes lost many introns, whereas others accumulated and/or gained many introns. In this article, we discuss the growing evidence that these differences are subject to selection acting on introns depending on the biology of the organism and the gene involved.     

Remarkable sequence conservation of the last intron in the PKD1 gene

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.     

Patterns of intron loss and gain in plants: intron loss-dominated evolution and genome-wide comparison of O. sativa and A. thaliana

Numerous previous studies have elucidated 2 surprising patterns of spliceosomal intron evolution in diverse eukaryotes over the past roughly 100 Myr. First, rates of recent intron gain in a wide variety of eukaryotic lineages have been surprisingly low, far too low to explain modern intron densities. Second, intron losses have outnumbered intron gains over a variety of lineages. For several reasons, land plants might be expected to have comparatively high rates of intron gain and thus to represent a possible exception to this pattern. However, we report several studies that indicate low rates of intron gain and an excess of intron losses over intron gains in a variety of plant lineages. We estimate that intron losses have outnumbered intron gains in recent evolution in Arabidopsis thaliana (roughly 12.6 times more losses than gains), Oryza sativa (9.8 times), the green alga Chlamydomonas reinhardtii (5.1 times), and the Bigelowiella natans nucleomorph, an enslaved green algal nucleus (2.8 times). We estimate that during recent evolution, A. thaliana and O. sativa have experienced very low rates of intron gain of around one gain per gene per 2.6-8.0 billion years. In addition, we compared 8,258 pairs of putatively orthologous A. thaliana-O. sativa genes. We found that 5.3% of introns in conserved coding regions are species-specific. Observed species-specific A. thaliana and O. sativa introns tend to be exact and to lie adjacent to each other along the gene, in a pattern suggesting mRNA-mediated intron loss. Our results underscore that low intron gain rates and intron number reduction are common features of recent eukaryotic evolution. This pattern implies that rates of intron creation were higher during earlier periods of evolution and further focuses attention on the causes of initial intron proliferation.     

A very high fraction of unique intron positions in the intron-rich diatom Thalassiosira pseudonana indicates widespread intron gain

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.     

双翅目昆虫与脊椎动物内含子丢失和获得的比较分析

内含子插入和丢失的进化动力及机制尚存有许多疑问。我们拟通过对真核生物的604个同源基因的蛋白高度保守区域内含子-外显子的结构研究, 对人Homo sapiens、大鼠Rattus norvegicus、小鼠Mus musculus、黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae和拟南芥Arabidopsis thaliana中的12 585个内含子、3 074个保守内含子进行分析, 推断出不同系统中内含子进化趋势。结果显示在进化中双翅目昆虫丢失了约850多个内含子, 脊椎动物获得了1 600多个内含子, 而双翅目昆虫获得的内含子及脊椎动物丢失的内含子则较少。在内含子分布上, 除酵母有明显5′末端倾向性外, 双翅目昆虫也显示出内含子分布倾向于基因的5′端, 而在脊椎动物及拟南芥中则没有这种分布的倾向性。这可能是由于双翅目昆虫丢失的内含子大多位于基因的3′端造成的。通过对现在脊椎动物内含子分布及获得的内含子的插入相的研究, 发现内含子的获得可能在一定程度上导致了现存基因的内含子中插入相0的内含子最多这一倾向。     

Generation of a database containing discordant intron positions in eukaryotic genes (MIDB)

MOTIVATION: Intron sliding is the relocation of intron-exon boundaries over short distances and is often also referred to as intron slippage or intron migration or intron drift. We have generated a database containing discordant intron positions in homologous genes (MIDB--Mismatched Intron DataBase). Discordant intron positions are those that are either closely located in homologous genes (within a window of 10 nucleotides) or an intron position that is present in one gene but not in any of its homologs. The MIDB database aims at systematically collecting information about mismatched introns in the genes from GenBank and organizing it into a form useful for understanding the genomics and dynamics of introns thereby helping understand the evolution of genes. RESULTS: Intron displacement or sliding is critically important for explaining the present distribution of introns among orthologous and paralogous genes. MIDB allows examining of intron movements and allows mapping of intron positions from homologous proteins onto a single sequence. The database is of potential use for molecular biologists in general and for researchers who are interested in gene evolution and eukaryotic gene structure. Partial analysis of this database allowed us to identify a few putative cases of intron sliding. AVAILABILITY: http://intron.bic.nus.edu.sg/midb/midb.html     

On the incidence of intron loss and gain in paralogous gene families

Understanding gene duplication and gene structure evolution are fundamental goals of molecular evolutionary biology. A previous study by Babenko et al. (2004. Prevalence of intron gain over intron loss in the evolution of paralogous gene families. Nucleic Acids Res. 32:3724-3733) employed Dollo parsimony to infer spliceosomal intron losses and gains in paralogous gene families and concluded that there was a general excess of gains over losses. This result contrasts with patterns in orthologous genes, in which most lineages show an excess of intron losses over gains, suggesting the possibility of fundamentally different modes of intron evolution between orthologous and paralogous genes. We further studied the data and found a low level of intron position conservation with outgroups, and this led to problems with using Dollo parsimony to analyze the data. Statistical reanalysis of the data suggests, instead, that intron losses have outnumbered intron gains in paralogous gene families.     

High rate of recent intron gain and loss in simultaneously duplicated Arabidopsis genes

We examined the gene structure of a set of 2563 Arabidopsis thaliana paralogous pairs that were duplicated simultaneously 20-60 MYA by tetraploidy. Out of a total of 23,164 introns in these genes, we found that 10,004 pairs have been conserved and 578 introns have been inserted or deleted in the time since the duplication event. This intron insertion/deletion rate of 2.7 x 10(-3) to 9.1 x 10(-4) per site per million years is high in comparison to previous studies. At least 56 introns were gained and 39 lost based on parsimony analysis of the phylogenetic distribution of these introns. We found weak evidence that genes undergoing intron gain and loss are biased with respect to gene ontology terms. Gene pairs that experienced at least 2 intron insertions or deletions show evidence of enrichment for membrane location and transport and transporter activity function. We do not find any relationship of intron flux to expression level or G + C content of the gene. Detection of a bias in the location of intron gains and losses within a gene depends on the method of measurement: an intragene method indicates that events (specifically intron losses) are biased toward the 3' end of the gene. Despite the relatively recent acquisition of these introns, we found only one case where we could identify the mechanism of intron origin--the TOUCH3 gene has experienced 2 tandem, partial, internal gene duplications that duplicated a preexisting intron and also created a novel, alternatively spliced intron that makes use of a duplicated pair of cryptic splice sites.     

Poxvirus genome evolution by gene gain and loss

The poxviruses (Poxviridae) are a family of viruses with double-stranded DNA genomes and substantial numbers (often >200) of genes per genome. We studied the patterns of gene gain and loss over the evolutionary history of 17 poxvirus complete genomes. A phylogeny based on gene family presence/absence showed good agreement with families based on concatenated amino acid sequences of conserved single-copy genes. Gene duplications in poxviruses were often lineage specific, and the most extensively duplicated viral gene families were found in only a few of the genomes analyzed. A total of 34 gene families were found to include a member in at least one of the poxvirus genomes analyzed and at least one animal genome; in 16 (47%) of these families, there was evidence of recent horizontal gene transfer (HGT) from host to virus. Gene families with evidence of HGT included several involved in host immune defense mechanisms (the MHC class I, interleukin-10, interleukin-24, interleukin-18, the interferon gamma receptor, and tumor necrosis factor receptor II) and others (glutaredoxin and glutathione peroxidase) involved in resistance of cells to oxidative stress. Thus "capture" of host genes by HGT has been a recurrent feature of poxvirus evolution and has played an important role in adapting the virus to survive host antiviral defense mechanisms.     

EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies

Recent innovations in next-generation sequencing have lowered the cost of genome projects. Nevertheless, sequencing entire genomes for all representatives in a study remains expensive and unnecessary for most studies in ecology, evolution and conservation. It is still more cost-effective and efficient to target and sequence single-copy nuclear gene markers for such studies. Many tools have been developed for identifying nuclear markers, but most of these have focused on particular taxonomic groups. We have built a searchable database, EvolMarkers, for developing single-copy coding sequence (CDS) and exon-primed-intron-crossing (EPIC) markers that is designed to work across a broad range of phylogenetic divergences. The database is made up of single-copy CDS derived from BLAST searches of a variety of metazoan genomes. Users can search the database for different types of markers (CDS or EPIC) that are common to different sets of input species with different divergence characteristics. EvolMarkers can be applied to any taxonomic group for which genome data are available for two or more species. We included 82 genomes in the first version of EvolMarkers and have found the methods to be effective across Placozoa, Cnidaria, Arthropod, Nematoda, Annelida, Mollusca, Echinodermata, Hemichordata, Chordata and plants. We demonstrate the effectiveness of searching for CDS markers within annelids and show how to find potentially useful intronic markers within the lizard Anolis.     

Extraordinary conservation, gene loss, and positive selection in the evolution of an ancient neurotoxin

The recent determination of the genetic basis for the biosynthesis of the neurotoxin, saxitoxin, produced by cyanobacteria, has revealed a highly complex sequence of reactions, involving over 30 biosynthetic steps encoded by up to 26 genes clustered at one genomic locus, sxt. Insights into evolutionary-ecological processes have been found through the study of such secondary metabolites because they consist of a measurable phenotype with clear ecological consequences, synthesized by known genes in a small number of species. However, the processes involved in and timing of the divergence of prokaryotic secondary metabolites have been difficult to determine due to their antiquity and the possible frequency of horizontal gene transfer and homologous recombination. Through analyses of gene synteny, phylogenies of individual genes, and analyses of recombination and selection, we identified the evolutionary processes of this cluster in five species of cyanobacteria. Here, we provide evidence that the sxt cluster appears to have been largely vertically inherited and was therefore likely present early in the divergence of the Nostocales, at least 2,100 Ma, the earliest reliably dated appearance of a secondary metabolite. The sxt cluster has been extraordinarily conserved through stabilizing selection. Genes have been lost and rearranged, have undergone intra- and interspecific recombination, and have been subject to duplication followed by positive selection along the duplicated lineage, with likely consequences for the toxin analogues produced. Several hypotheses exist as to the ecophysiological role of saxitoxin: as a method of chemical defense, cellular nitrogen storage, DNA metabolism, or chemical signaling. The antiquity of this gene cluster indicates that potassium channels, not sodium channels, may have been the original targets of this compound. The extraordinary conservation of the machinery for saxitoxin synthesis, under radically changing environmental conditions, shows that it has continued to play an important adaptive role in some cyanobacteria.     

Patterns and implications of gene gain and loss in the evolution of Prochlorococcus

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.     

Widespread conservation of genetic redundancy during a billion years of eukaryotic evolution

Genetic redundancy means that two genes can perform the same function. Using a comprehensive phylogenetic analysis, we show here in both Saccharomyces cerevisiae and Caenorhabditis elegans that genetic redundancy is not just a transient consequence of gene duplication, but is often an evolutionary stable state. In multiple examples, genes have retained redundant functions since the divergence of the animal, plant and fungi kingdoms over a billion years ago. The stable conservation of genetic redundancy contrasts with the more rapid evolution of genetic interactions between unrelated genes and can be explained by theoretical models including a 'piggyback' mechanism in which overlapping redundant functions are co-selected with nonredundant ones.