Lateral transfer of introns in the cryptophyte plastid genome

Cryptophytes are unicellular eukaryotic algae that acquired photosynthesis secondarily through the uptake and retention of a red-algal endosymbiont. The plastid genome of the cryptophyte Rhodomonas salina CCMP1319 was recently sequenced and found to contain a genetic element similar to a group II intron. Here, we explore the distribution, structure and function of group II introns in the plastid genomes of distantly and closely related cryptophytes. The predicted secondary structures of six introns contained in three different genes were examined and found to be generally similar to group II introns but unusually large in size (including the largest known noncoding intron). Phylogenetic analysis suggests that the cryptophyte group II introns were acquired via lateral gene transfer (LGT) from a euglenid-like species. Unexpectedly, the six introns occupy five distinct genomic locations, suggesting multiple LGT events or recent transposition (or both). Combined with structural considerations, RT–PCR experiments suggest that the transferred introns are degenerate ‘twintrons’ (i.e. nested group II/group III introns) in which the internal intron has lost its splicing capability, resulting in an amalgamation with the outer intron.     

Novel Introner-Like Elements in fungi Are Involved in Parallel Gains of Spliceosomal Introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom.     

Analysis of Fungal Genomes Reveals Commonalities of Intron Gain or Loss and Functions in Intron-Poor Species

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.     

The exon/intron organization of the globin gene of Scapharca inaequivalvis homodimeric hemoglobin: unusual intron homology with other bivalve mollusc globin genes

In this study, we have investigated the positions of introns in the globin gene of Scapharca inaequivalvis homodimeric hemoglobin. We found the three exon/two intron organization typical of vertebrate globin genes, with the two introns in highly conserved positions, as it occurs in the A and B globin genes of the tetrameric hemoglobin from the same organism, confirming the absence of the so-called `central intron' found in the globin genes of plants and of some invertebrates. We identified two homodimeric globin genes (3207 and 2723 bp) that differ only with respect to the size of the first intron. Sequence analysis of the two first introns (1668 and 1364 bp) has revealed that they are highly homologous, except for a 569- and 296-bp insertion in each intron I. Interestingly, the two first introns contain regions with an unusually high identity (∼80%) with regions of the first intron of the congeneric clam Anadara trapezia and the related clam Barbatia reveana globin genes, suggesting that these uncoding regions may have played a regulatory role that has subsequently been lost during the course of the evolution.     

Conservation versus parallel gains in intron evolution

Orthologous genes from distant eukaryotic species, e.g. animals and plants, share up to 25–30% intron positions. However, the relative contributions of evolutionary conservation and parallel gain of new introns into this pattern remain unknown. Here, the extent of independent insertion of introns in the same sites (parallel gain) in orthologous genes from phylogenetically distant eukaryotes is assessed within the framework of the protosplice site model. It is shown that protosplice sites are no more conserved during evolution of eukaryotic gene sequences than random sites. Simulation of intron insertion into protosplice sites with the observed protosplice site frequencies and intron densities shows that parallel gain can account but for a small fraction (5–10%) of shared intron positions in distantly related species. Thus, the presence of numerous introns in the same positions in orthologous genes from distant eukaryotes, such as animals, fungi and plants, appears to reflect mostly bona fide evolutionary conservation.     

Evolutionary dynamics of introns and their open reading frames in the U7 region of the mitochondrial rnl gene in species of Ceratocystis

The mtDNA rnl-U7 region has been examined for the presence of introns in selected species of the genus Ceratocystis. Comparative sequence analysis identified group I and group II introns encoding single and double motif LAGLIDADG open reading frames (ORFs) at the following positions L1671, L1787, and L1923. In addition downstream of the rnl-U7 region group I introns were detected at positions L1971 and L2231, and a group II intron at L2059. A GIY-YIG type ORF was located within one mL1923 LAGLIDADG type ORF and a degenerated GIY-YIG ORF fused to a nad2 gene fragment was found in association with the mL1971 group I intron. The diversity of composite elements that appear to be sporadically distributed among closely related species of Ceratocystis illustrates the potential for homing endonucleases and their associated introns to invade new sites. Phylogenetic analysis showed that single motif LADGLIDADG ORFs related to the mL1923 ORFs have invaded the L1787 group II intron and the L1671 group I intron. Phylogenetic analysis of intron encoded single and double motif LAGLIDADG ORFs also showed that these ORFs transferred four times from group I into group II B1 type introns.     

Retroduplication and loss of parental genes is a mechanism for the generation of intronless genes in Ciona intestinalis and Ciona savignyi

Tunicates, the sister clade of vertebrates, have miniature genomes and numerous intronless genes compared to other animals. It is still unclear how the tunicates acquired such a large number of intronless genes. Here, we analyzed sequences and intron–exon organizations of homologous genes from two closely related tunicates, Ciona intestinalis and Ciona savignyi. We found seven cases in which ancestral introns of a gene were completely lost in a species after their divergence. In four cases, both the intronless copy and the intron-containing copy were present in the genome, indicating that the intronless copy was generated by retroduplication. In the other three cases, the intron-containing copy was absent, implying it was lost after retroduplication. This result suggests that retroduplication and loss of parental genes is a major mechanism for the accumulation of intronless genes in tunicates.     

Insights into the history of a bacterial group II intron remnant from the genomes of the nitrogen-fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

Group II introns are self-splicing catalytic RNAs that act as mobile retroelements. In bacteria, they are thought to be tolerated to some extent because they self-splice and home preferentially to sites outside of functional genes, generally within intergenic regions or in other mobile genetic elements, by mechanisms including the divergence of DNA target specificity to prevent target site saturation. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti and was first described in the GR4 strain. Like other bacterial group II introns, RmInt1 tends to evolve toward an inactive form by fragmentation, with loss of the 3′ terminus. We identified genomic evidence of a fragmented intron closely related to RmInt1 buried in the genome of the extant S. meliloti/S. medicae species. By studying this intron, we obtained evidence for the occurrence of intron insertion before the divergence of ancient rhizobial species. This fragmented group II intron has thus existed for a long time and has provided sequence variation, on which selection can act, contributing to diverse genetic rearrangements, and to generate pan-genome divergence after strain differentiation. The data presented here suggest that fragmented group II introns within intergenic regions closed to functionally important neighboring genes may have been microevolutionary forces driving adaptive evolution of these rhizobial species.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

GROUP I INTRON VERSUS ITS SEQUENCES IN PHYLOGENY OF CETRARIOID LICHENS

Abstract: Phylogenetic trees based on group I intron sequences and on internal transcribed spacer (ITS) sequences of mycobiont ribosomal genes were calculated and compared. Eight cetrarioid and four non-cetrarioid species of the Parmeliaceae were compared. The phylogeny based on group I intron sequences is partly congruent with the ITS sequence phylogeny. Group I intron sequences are presumably less informative for infragenic studies. The introns have a length of 214–233 nucleotides, and differ at up to 33% of the bases between species. All introns analysed are located between the positions 1516 and 1517 of the fungal 18S ribosomal RNA gene. Cetrarioid lichens form a non-homogeneous group within theParmeliaceae according to both group I intron and ITS sequences.     

Fungal origin by horizontal transfer of a plant mitochondrial group I intron in the chimeric coxI gene of Peperomia

We present phylogenetic evidence that a group I intron in an angiosperm mitochondrial gene arose recently by horizontal transfer from a fungal donor species. A 1,716-bp fragment of the mitochondrial coxI gene from the angiosperm Peperomia polybotrya was amplified via the polymerase chain reaction and sequenced. Comparison to other coxI genes revealed a 966-bp group I intron, which, based on homology with the related yeast coxI intron aI4, potentially encodes a 279-amino-acid site-specific DNA endonuclease. This intron, which is believed to function as a ribozyme during its own splicing, is not present in any of 19 coxI genes examined from other diverse vascular plant species. Phylogenetic analysis of intron origin was carried out using three different tree-generating algorithms, and on a variety of nucleotide and amino acid data sets from the intron and its flanking exon sequences. These analyses show that the Peperomia coxI gene intron and exon sequences are of fundamentally different evolutionary origin. The Peperomia intron is more closely related to several fungal mitochondrial introns, two of which are located at identical positions in coxI, than to identically located coxI introns from the land plant Marchantia and the green alga Prototheca. Conversely, the exon sequence of this gene is, as expected, most closely related to other angiosperm coxI genes. These results, together with evidence suggestive of co-conversion of exonic markers immediately flanking the intron insertion site, lead us to conclude that the Peperomia coxI intron probably arose by horizontal transfer from a fungal donor, using the double-strand-break repair pathway. The donor species may have been one of the symbiotic mycorrhizal fungi that live in close obligate association with most plants. Correspondence to: J.C. Vaughn     

Group I introns interrupt the chloroplast psaB and psbC and the mitochondrial rrnL gene in Chlamydomonas.

The polymerase chain reaction was used to identify novel IAI subgroup introns in cpDNA-enriched preparations from the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii. These experiments along with sequence analysis disclosed the presence, in both green algae, of a single IA1 intron in the psaB gene and of two group I introns (IA2 and IA1) in the psbC gene. In addition, two group I introns (IA1 and IB4) were found in the peptidyltransferase region of the mitochondrial large subunit rRNA gene at the same positions as previously reported Chlamydomonas chloroplast introns. The 188 bp segment preceding the first mitochondrial intron revealed extensive sequence similarity to the distantly spaced rRNA-coding modules L7 and L8 in the Chlamydomonas reinhardtii mitochondrial DNA, indicating that these two modules have undergone rearrangements in Chlamydomonas. The IA1 introns in psaB and psbC were found to be related in sequence to the first intron in the C. moewusii chloroplast psbA gene. The similarity between the former introns extends to the immediate 5' flanking exon sequence, suggesting that group I intron transposition occurred from one of the two genes to the other through reverse splicing.     

Compartmentalized isozyme genes and the origin of introns

Summary Both the mouse cytosolic malate dehydrogenase gene and its mitochondrial counterpart contain eight introns, of which two are present at identical positions between the isozyme genes. The probability that the two intron positions coincide by chance between the two genes has been shown to be significantly small (=1.3×10^–3), suggesting that the conservation of the intron positions has a biological significance. On the basis of a rooted phylogenetic tree inferred from a comparison of these isozymes and lactate dehydrogenases, we have shown that the origins of the conserved introns are very old, possibly going back to a date before the divergence of eubacteria, archaebacteria, and eukaryotes. In the aspartate aminotransferase isozyme genes, five of the introns are at identical places. The origins of the five conserved introns, however, are not obvious at present. It remains possible that some or all of the conserved introns have evolved after the divergence of eubacteria and eukaryotes.     

Hypervariable and Highly Divergent Intron–Exon Organizations in the Chordate Oikopleura dioica

Oikopleura dioica is a pelagic tunicate with a very small genome and a very short life cycle. In order to investigate the intron–exon organizations in Oikopleura, we have isolated and characterized ribosomal protein EF-1, Hox, and -tubulin genes. Their intron positions have been compared with those of the same genes from various invertebrates and vertebrates, including four species with entirely sequenced genomes. Oikopleura genes, like Caenorhabditis genes, have introns at a large number of nonconserved positions, which must originate from late insertions or intron sliding of ancient insertions. Both species exhibit hypervariable intron–exon organization within their -tubulin gene family. This is due to localization of most nonconserved intron positions in single members of this gene family. The hypervariability and divergence of intron positions in Oikopleura and Caenorhabditis may be related to the predominance of short introns, the processing of which is not very dependent upon the exonic environment compared to large introns. Also, both species have an undermethylated genome, and the control of methylation-induced point mutations imposes a control on exon size, at least in vertebrate genes. That introns placed at such variable positions in Oikopleura or C. elegans may serve a specific purpose is not easy to infer from our current knowledge and hypotheses on intron functions. We propose that new introns are retained in species with very short life cycles, because illegitimate exchanges including gene conversion are repressed. We also speculate that introns placed at gene-specific positions may contribute to suppressing these exchanges and thereby favor their own persistence.Supplementary material () is available for this article.     

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.     

Divergent Histories of rDNA Group I Introns in the Lichen Family Physciaceae

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. Reviewing Editor: Dr. W. Ford Doolittle     

The recent transfer of a homing endonuclease gene

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.     

Separate origins of group I introns in two mitochondrial genes of the katablepharid Leucocryptos marina

Mitochondria are descendants of the endosymbiotic α-proteobacterium most likely engulfed by the ancestral eukaryotic cells, and the proto-mitochondrial genome should have been severely streamlined in terms of both genome size and gene repertoire. In addition, mitochondrial (mt) sequence data indicated that frequent intron gain/loss events contributed to shaping the modern mt genome organizations, resulting in the homologous introns being shared between two distantly related mt genomes. Unfortunately, the bulk of mt sequence data currently available are of phylogenetically restricted lineages, i.e., metazoans, fungi, and land plants, and are insufficient to elucidate the entire picture of intron evolution in mt genomes. In this work, we sequenced a 12 kbp-fragment of the mt genome of the katablepharid Leucocryptos marina. Among nine protein-coding genes included in the mt genome fragment, the genes encoding cytochrome b and cytochrome c oxidase subunit I (cob and cox1) were interrupted by group I introns. We further identified that the cob and cox1 introns host open reading frames for homing endonucleases (HEs) belonging to distantly related superfamilies. Phylogenetic analyses recovered an affinity between the HE in the Leucocryptos cob intron and two green algal HEs, and that between the HE in the Leucocryptos cox1 intron and a fungal HE, suggesting that the Leucocryptos cob and cox1 introns possess distinct evolutionary origins. Although the current intron (and intronic HE) data are insufficient to infer how the homologous introns were distributed to distantly related mt genomes, the results presented here successfully expanded the evolutionary dynamism of group I introns in mt genomes.     

Evolutionary dynamics of spliceosomal intron revealed by <Emphasis Type="Italic">in silico</Emphasis> analyses of the P-Type ATPase superfamily genes

It has been long debated whether spliceosomal introns originated in the common ancestor of eukaryotes and prokaryotes. In this study, we tested the possibility that extant introns were inherited from the common ancestor of eukaryotes and prokaryotes using in silico simulation. We first identified 21 intron positions that are shared among different families of the P-Type ATPase superfamily, some of which are known to have diverged before the separation of prokaryotes and eukaryotes. Theoretical estimates of the expected number of intron positions shared by different genes suggest that the introns at those 21 positions were inserted independently. There seems to be no intron that arose from before the diversification of the P-Type ATPase superfamily. Namely, the present introns were inserted after the separation of eukaryotes and prokaryotes.