Introns and their splicing are tightly coupled with the subsequent mRNA maturation steps, especially nucleocytoplasmic export. A remarkable fraction of vertebrate introns have a minimal size of about 100 bp, while majority of introns expand to several kilobases even megabases in length.We carried out analyses on the evolution and function of minimal introns (50–150 bp) in human and mouse genomes. We found that minimal introns are conserved in terms of both length and sequence. They are preferentially located toward 3′ end of mRNA and non-randomly distributed among chromosomes. Both the evolutionary conservation and non-random distribution are indicative of biological relevance. We showed that genes with minimal introns have higher abundance, larger size, and tend to be universally expressed as compared to genes with only large introns and intron-less genes. Genes with minimal introns replicate earlier and preferentially reside in the vicinities of open chromatin, suggesting their unique nuclear position and potential relevance to the regulation of gene expression and transcript export.Based on these observations, we proposed a nuclear-export routing model, where minimal introns play a regulatory role in selectively exporting the highly abundant and large housekeeping genes that reside at the surface of chromatin territories, and thus preventing entanglement with other genes located at the interior locations. Gene expression program, rather than a simple assembly line to process mRNAs, is a complex network systematically coordinating many cellular pathways including transcription initiation-elongation-termination, RNA processing, transcription-coupled DNA repair, nuclear export of mRNAs, translation and RNA/protein degradation An intriguing example of the coupling among the expression machineries is the observation that RNA splicing influences many subsequent steps of mRNA metabolism such as nucleocytoplasmic export We previously reported a conspicuous feature of vertebrate introns that a remarkable fraction of introns have a lineage-specific minimal size (∼100 bp), which were termed as “minimal introns” In order to understand their functions, we analyzed genes with minimal introns and found some unique characteristics associated with the presence of minimal introns. In general, genes with minimal introns have higher abundance and larger size, and tend to be universally expressed as compared to genes with only large introns and intron-less genes. The presence of minimal intron is also correlated with the replication timing and chromatin structure of the gene locus, implying specific nuclear positions of these genes. Based on these observations, we proposed a nuclear export routing model where minimal introns play regulatory role to selectively export some highly abundant and large housekeeping genes that reside at the surface of chromatin territory, thus preventing the entanglement with other genes located at the interior locations. Although this model is largely descriptive and hypothetical, it provides a necessary framework to design experiments to test the exact role of minimal introns in post-transcriptional regulation.A remarkable fraction of vertebrate introns have a minimal size (∼100 bp) peaking at the low end of the size distribution, while majority of introns expand to several kilobases even megabases in length . Over laWe analyzed 18,468 human and 18,889 mouse RefSeq loci, containing 175,723 and 165,351 introns, respectively. We defined introns with length of 50–150 bp as minimal introns and those with length of >150 bp as large introns . In totaP<2.2×10−16). We concluded that minimal introns are evolutionarily conserved in terms of both length and sequence.In order to understand the intron evolution between human and mouse, we first analyzed the length differences of human-mouse orthologous introns as a function of intron length. We found that most of large introns fluctuate in length, with a median length difference of 452 bp. In contrast, the lengths of minimal introns are highly conserved, with a median length difference of 8 bp . At presith intron in a locus with N introns with a position value of i/N, and observed that minimal introns preferentially reside toward the 3′ end within the sequential arrangement of exon-intron structure  pathway tructure . It's intructure .We observed that minimal introns are non-randomly distributed among chromosomes. Using the fraction of minimal intron within a chromosome to index its enrichment, some chromosomes  are significantly enriched by minimal introns whereas others  are significantly deficient .r = 0.8217, P = 8.5×10−7), GC-rich , replicate earlier  and have more open chromatin .It has been reported that chromosome features such as gene density, GC content, replication time, compactness and nuclear position are all correlated—open chromatins tend to locate at the nuclear interior, replicate early, and have higher gene density and GC content as opposite to close chromatins—although the causal relationship among them remains to be elucidated P = 3.7×10−196), but chromosome 18 is among the most minimal intron deficient . Chromosome 19 replicates early and has open chromatin structure  as compared to chromosome 18 . In human lymphocyte nuclei, which exhibit an spherical shape, chromosome 19 is consistently localized toward the nuclear center without any detectable attachment to the nuclear envelope, whereas chromosome 18 is positioned close to the nuclear border Two striking example are chromosome 19 and 18 . AlthougThe evolutionary conservation, 3′-positional preference and non-random chromosomal distribution all indicate that minimal introns may have special biological functions. We previously showed that minimal introns are not randomly distributed among genes P<2.2×10−16; In order to look further into the features of minimal introns-containing genes, we separated genes into three classes: (1) genes with minimal introns, (2) genes with only large introns and (3) genes without introns. We observed that intron-containing genes have significantly higher mRNA concentration and are significantly larger than genes without introns, consistent with the experimental observation that splicing can enhance mRNA nuclear export P<2.2×10−16) than that of other spliced genes with only large introns (with a mean S∶G1 ratio of 1.58), which is again significantly earlier  than that of intron-less genes  than that of other spliced genes with only large introns (with a mean open∶input ratio of 1.39), which is again significantly more open  than that of intron-less genes  is significantly earlier  genes with minimal introns, (2) genes with only large introns and (3) genes without introns, and found that the presence of minimal introns is associated with gene's length, expression level, replication timing and chromatin structure. Based on these observatons, we proposed a nucleocytoplasmic export routing model . Genes wThere are several pieces of evidence supporting our model. Nuclear pore complex (NPC) contains 9-nm aqueous channels, through which small water-soluble molecules are moved back-and-forth through passive diffusion; the channel of NPC expands to about 25 nm when exporting macromolecules, such as protein and mature mRNP, in an energy-dependent way How minimal introns specifically regulate the export of these highly abundant and giant mRNAs? In lower eukaryotes, introns are generally small and could be directly recognized by the splicing machinery—the intron definition model; but in higher eukaryotes, such as vertebrates where most of introns have been expanded, the splicing machinery would use exons as the unit of recognition to facilitate the identification of exons among the intronic oceans—the exon definition model The current mammalian nuclear architecture model, i.e., chromosome territory - interchromatin compartment (CT-IC) model, depicts that chromosomes in the nucleus are organized as chromosome territories (CTs), which are sponge-like structure built up from condensed high-order chromatin fibers. CTs occupy spatially limited volume and are non-randomly positioned within the nucleus Interchromatin compartment (IC) is a contiguous network of DNA-free space, starting at nuclear pores and expanding between CTs and into their interior. IC can form lacunas with diameters of up to several micrometers containing nuclear bodies such as speckle, Cajal and PML bodies in situ hybridization (FISH) and establishing mutated genes where minimal introns are artificially lengthened and/or disrupted would provide very important evidence for their function. Although the results presented in this study are largely descriptive and correlative and the model are hypothetical, these observations provided a necessary framework to design experiments to test the exact role of minimal introns in post-transcriptional regulation.So far, we still only have a very fragmented view of the molecular mechanism associated minimal intron. As the EJC composition can influence almost every stage of RNA metabolism, including export, localization, translation and NMD pathway G. gallus, X. tropicalis and D. rerio are processed in the same way. Intron positions are derived from RefSeq alignments. Human EST sequences and their genomic alignments were retrieved from UCSC annotation database , and clustered into RefSeq loci as previously described We aligned 25,127 human and 21,153 mouse RefSeq transcripts  onto their reference genomes  using BLAT program We retrieved 13,382 human-mouse orthologs from NCBI HomoloGene (Build 61), and aligned amino acid sequences by using CLUSTALW2 Information on the replication time and chromatin structure of human genome were obtained from previous studies Table S1List of 6,327 human genes with minimal introns.(1.30 MB XLS)Click here for additional data file.Figure S1Gene ontology annotation of human genes with minimal introns.(0.12 MB PDF)Click here for additional data file.


In this essay, I argue that unraveling the full genetic architecture  and molecular landscape of host–pathogen interactions can only be achieved by accounting for their genetic specificity. Indeed, the outcome of host–pathogen interactions often depends on the specific pairing of host and pathogen genotypes y is the phenotypic value of the individual, μ is the mean value of the population, g is the genetic contribution to the deviation from the mean , and ε is the environmental (non-genetic) deviation. By extending this model to a quantitative trait resulting from the interaction between a host and a pathogen, the model becomes:gH is the host genotypic value, gP is the pathogen genotypic value, and gHP is the genotypic value due to the specific G×G interaction. This simple model ignores interactions between genes and environment (G×E and G×G×E effects), which occur when genotypic values vary across environments. The genetic component of phenotypic variance in a host–pathogen interaction can thus be partitioned into three distinct terms: variance due to the additive effect of the host genotype, variance due to the additive effect of the pathogen genotype, and variance due to the specific interaction between the two genomes. Whereas the first two terms can be characterized by considering either the host or the pathogen genetic variation alone, exploring the genetic basis of host–pathogen specificity requires that genetic variations in both the host and the pathogen are considered simultaneously.Quantitative genetics is the area of genetics dealing with the inheritance of traits showing continuous phenotypic variation i and j of a single organism, we can define αi the additive effect of locus i, αj the additive effect of locus j, and βij the interaction effect between loci i and j to decompose the genotypic value into:i and j are two homologous alleles of the same diploid locus the interaction effect is generally referred to as “dominance”. By defining Σα as the sum of all additive effects and Σβ as the sum of all interaction effects (both within and between loci), the previous equation can be generalized to any trait determined by multiple (n>2) loci:HP = ΣαH+ΣαP (sum of additive effects of host and pathogen loci) and ΣβHP = ΣβH+ΣβP+gHP , the equation becomes:In the case of a trait determined by two haploid loci In some instances, the specificity of host–pathogen associations can be explained to a large extent by major genes of hosts and pathogens, as in the gene-for-gene model of plant–pathogen compatibility It is striking that, to date, quantitative genetic studies of host–pathogen systems have neglected the specific component of the interaction. Dissecting the genetic architecture of complex infection traits has traditionally relied on QTL mapping strategies From a fundamental standpoint, improved knowledge of the genetic architecture of host–pathogen specificity has important implications for our understanding of the ecology and evolution of host–pathogen associations. The genetic specificity of host–pathogen interactions is thought to promote the maintenance of host and pathogen genetic diversity via frequency-dependent coevolutionary cycles From a more applied perspective, exploring the genetic basis of host–pathogen specificity will provide important insights into the mechanisms of disease emergence. Pathogens with a broad host range  are those most likely to emerge or re-emerge following ecological changes


Many multicellular eukaryotes have two types of spliceosomes for the removal of introns from messenger RNA precursors. The major (U2) spliceosome processes the vast majority of introns, referred to as U2-type introns, while the minor (U12) spliceosome removes a small fraction (less than 0.5%) of introns, referred to as U12-type introns. U12-type introns have distinct sequence elements and usually occur together in genes with U2-type introns. A phylogenetic distribution of U12-type introns shows that the minor splicing pathway appeared very early in eukaryotic evolution and has been lost repeatedly.Drosophila melanogaster, we found one case of U2 to U12-type conversion, apparently mediated by the activation of cryptic U12 splice sites early in the dipteran lineage. Overall, loss of U12-type introns is more common than conversion to U2-type and the U12 to U2 conversion occurs more frequently among introns of the GT-AG subtype than among introns of the AT-AC subtype. We also found support for natural U12-type introns with non-canonical terminal dinucleotides  that have not been previously reported.We have investigated the evolution of U12-type introns among eighteen metazoan genomes by analyzing orthologous U12-type intron clusters. Examination of gain, loss, and type switching shows that intron type is remarkably conserved among vertebrates. Among 180 intron clusters, only eight show intron loss in any vertebrate species and only five show conversion between the U12 and the U2-type. Although there are only nineteen U12-type introns in Although complete loss of the U12-type spliceosome has occurred repeatedly, U12 introns are extremely stable in some taxa, including eutheria. Loss of U12 introns or the genes containing them is more common than conversion to the U2-type. The degeneracy of U12-type terminal dinucleotides among natural U12-type introns is higher than previously thought. In most eukaryotic protein-coding genes, the sequences found in mature messenger RNAs (exons) are discontinuous, and are separated by intervening sequences known as introns. The removal of introns, in a process known as pre-mRNA splicing, is performed by the spliceosome, which consists of the U1, U2, U4, U5 and U6 snRNPs , and more than 200 non-snRNP proteins .The terminal dinucleotides GT and AG at the 5' and 3' end of introns, respectively, are nearly universal -6. Howevet al. [SCN4A gene, which has AT-AC termini, requires a major-type spliceosome [et al. [Introns in the minor class were originally called AT-AC introns for their unusual terminal dinucleotides. However, Dietrich et al.  demonstriceosome . Since t [et al.  coined tet al. [In the first study that investigated the evolution of U12-type introns, Burge et al.  proposedC. elegans and yeast. The question whether these two seemingly parallel splicing machineries descended from the same ancestor is intriguing but remains unsolved. Burge et al. [at al. analyzed protosplice sites in the human and Arabidopsis thaliana genomes and concluded that U2-type introns predate the U12-type [The wide spread of the U12-type spliceosome in eukaryotes and its independent loss in distantly related species was recently studied by computational scanning for spliceosomal snRNA genes in 149 eukaryotic genomes [e et al.  proposede et al. . In thisU12-type . This fiHomo sapiens [Arabidopsis thaliana [Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris), 2) vertebrates , 3) chordates , 4) Homo sapiens vs. Drosophila melanogaster), and 5) Homo sapiens vs. Arabidopsis thaliana.Since the publication of Burge and colleagues' evolutionary study of U12-type introns, many complete genomes have become available. U12-type introns have been computationally identified and characterized in several genomes, including  sapiens  and Arabthaliana . Here wethaliana  and U12Dthaliana . More U1D. melanogaster genome appeared to be the first known case of U2 to U12-type conversion. In general, loss of U12-type introns is more common than their conversion to U2-type and U12 to U2 conversion occurs more frequently among U12 introns of the GT-AG subtype. We also found support for natural U12-type introns with non-canonical terminal dinucleotides  that have not been previously reported.Our results reveal that U12-type introns are remarkably conserved among vertebrates. On the other hand, there has been a massive loss of U12-type introns in multiple invertebrate lineages, suggesting that the rate of U12-type intron loss varies significantly among taxa. Interestingly, one of only nineteen U12-type introns that were detected in the Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Canis familiaris, Bos taurus, Monodelphis domestica, Gallus gallus, Xenopus laevis, Fugu rubripes, Tetraodon nigroviridis, Danio rerio, Ciona intestinalis, Apis mellifera, Drosophila melanogaster, Anopheles gambiae, Caenorhabditis elegans, and Arabidopsis thaliana. We utilized orthologous U12-type intron clusters and related data available in the U12DB [To study the evolution of U12-type introns, we investigated conservation and changes of the intron state among U12-type introns and their orthologs in twenty sequenced eukaryotic genomes: he U12DB . MembersDrosophila (see below) and Arabidopsis [Pairwise comparisons to the human genome (prior to the detailed investigation described below) as a reference provide an overview of the entire dataset (see Table bidopsis .Ciona), or phylogenetically distant species (such as Arabidopsis). 3) Ancient paralogs resulting from gene duplication before speciation are not distinguished from recent paralogs.In addition to possible inaccuracy in genome assembly or annotation, the way U12DB was constructed also poses the following limitations for the present study. 1) The number of reported U12-type introns for non-reference genomes may be underestimated, since these genomes have not been fully searched. 2) Identification of the orthologous intron relies on correct assignment of orthologous genes since only sequences of the members of the same ortholog group were searched for flanking exonic sequences. Errors in orthologous gene identification can lead to overestimate of gain and loss of introns. This is particularly a challenge for genomes that have less than ideal genome assembly (such as We addressed some of these issues by 1) comparing genomes at various levels, starting with more closely related species and gradually adding more distantly related lineages; 2) inferring evolutionary events  from intron clusters where all orthologous genes of compared species are present; 3) manually curating when necessary and manageable. This includes identifying homologous genes by BLAST search (tblastn), tree construction of homologous protein sequences, inspection of pairwise protein sequence alignments, and annotation of intron splice sites.Drosophila melanogaster genome is exceptionally complete and well-annotated, it has undergone extensive loss of U12 introns relative to other insects (see below). A thorough analysis of U12 intron evolution in insects using different methods is in progress, but not as part of the present study .Although a U12 intron that was not present in any of the four reference genomes could be missed (and therefore not listed in any of the full set of 20 genomes), we note that the human genome, which is a U12DB reference, is also the most complete. It is unlikely that the human genome has lost more than one or two introns present in other vertebrates, because the rate of intron loss in vertebrates is low overall (see below). Furthermore, we note that almost all of the U12 introns that have been retained in fruit flies are also retained in humans and the others are missing from all chordates (see below). Thus, we feel that use of the human genome as a reference allows an accurate description of all U12 introns in vertebrates, with only a few exceptions. The one possibility we cannot rule out is high rates of U12 intron gain in some non-human vertebrate clade, but we consider this unlikely because we have observed only a single case of U12 intron gain in any species. The situation in insects is quite different. Although the Syt16) contains one "U12", one "U2", and two "ambiguous" introns. After manual inspection of the seventeen intron clusters having putative U12-type intron losses ("U12" to "absent"), only one case appears to be a true intron loss by deletion, in the gene encoding the mouse elongation factor 1 (Elof1). The other "absent" cases (twenty-four introns in seventeen clusters) are artefacts. Interestingly, in fourteen cases non-orthologs were compared. In the other three cases, non-cognate transcripts were compared, or genes that are present were not annotated, or were partially annotated, in the comparison genome. Thus, a case-by-case analysis reveals only two cases in which a U12 intron changes state, one loss (the second intron of the Elof gene in mouse) and one switch to the U2 type (the second intron of the rat Syt16 gene).We analyzed four eutherian genomes that are of relatively good quality to gain insight into the evolution of U12-type introns through closely related species and to reduce the effect of poor data quality. According to the initial automated results, there are 442 intron clusters where the human, mouse, rat, and dog orthologous genes are all present and at least one of member intron has a "U12" status  were represented as a puffer lineage. As more species were added to the analysis, the number of shared genes decreased. There are 180 orthologous intron clusters where orthologous genes for all vertebrates are available and there is at least one "U12" intron present in a cluster. This is considerably less than the 442 clusters in the eutherian analysis. U12-type introns are conserved in all eight lineages for 156 of the 180 intron cases. This number (156) reflects an increase from 144 after manual inspection. As in the eutherian comparison, several apparent cases of change were found to be due to ambiguous orthology or incompleteness in the genome assemblies and/or gene annotation. Furthermore, 11 of the remaining 24 cases are ambiguous, and may also represent U12 introns without change. We were able to confirm intron losses in eight clusters and U12-U2 changes in five cases. Intron state compositions of these 180 intron clusters are listed in Additional file We then added Ciona intestinalis reveals only 77 gene clusters available for analysis. The Ciona intron states in these seventy-seven clusters are twenty-five "U12", one "ambiguous", seventeen "U2", and thirty-four "absent". This low conservation is likely to be exaggerated by an under-detection of tunicate U12-type introns. As mentioned earlier, the relatively poor quality of the draft genome assembly [Ciona genomes. Nonetheless, the low number of genes compared and the high proportion of absent cases agree with the findings that the Ciona genome has undergone excessive loss of ancestral genes [Ciona.Comparison of the human and assembly  and lessal genes  and intral genes  that werD. melanogaster U12-type introns. We were able to identify orthologous U12-type introns in the human genome for sixteen of the nineteen introns. The remaining three cases are an intron loss in early vertebrates  . Fish orthologs have an additional subtype switch from AT-AC to GT-AG in the Syntaxin-6 gene. We also examined orthologs of D. melanogaster U12-type introns in twelve drosophila and one mosquito genome whenever data was available. Interestingly, we found a case of two independent switches from a U12-type to a U2 intron in the ZDHHC8 gene. This intron is of the U12-type in vertebrates and most dipterian species, but of the U2-type in D. ananassae and Anopheles gambiae.Fifteen orthologous mammalian introns have the same terminal dinucleotides as their fruitfly orthologs, the only exception is the one with GC-AG in A. thaliana, only two of them are reported by U12DB to have orthologous U12 introns in humans. These two introns are in genes encoding exosome component 5 (EXOSC5) and BRCA1/BRCA2-containing complex, subunit 3 (BRCC3). Most Arabidopsis U12 introns are in genes that are reported to lack human orthologs. Twenty-five A. thaliana U12-type introns are in genes with human orthologs but no orthologous intron, and another twenty-eight have orthologous genes (but not introns) in non-human animals. If these numbers are correct, the number of A. thaliana U12-containing genes that have human orthologous genes is underrepresented compared to the number of A. thaliana genes that have human orthologous genes . Howeve [et al.  reportedElof1 in the mouse genome) and one potential U12-U2 transition (synaptotagmin XIV in rats). Among the eight vertebrates, in 156 (86.7%) of the 180 clusters, U12-type introns are conserved in all eight lineages. There are intron losses in eight clusters and U12-U2 conversion in five clusters. Eleven clusters contain ambiguous introns. No gain of a U12-intron was observed. These results indicate that U12-type introns have been tremendously stable in vertebrates during the last 550 million years after the divergence of Ciona. There were only a few losses or U12-U2 conversions. Conservation between Ciona and vertebrates is certainly underestimated mainly due to the relatively poor quality of the Ciona genome draft, which leads to under detection of U12-type introns and difficulties in ortholog prediction. The conservation of U12-type introns between humans and fruitflies is very low (7.5% of 200 introns) since there are only nineteen U12-type introns in D. melanogaster. Fifteen of these have orthologs in humans; one is specific to diptera, and two are lost in humans .The above analyses on various subsets of genomes were based on relatively conservative datasets; intron clusters were not considered for those genes where orthologous gene information is missing for any of the species compared. In summary, introns are classified as U12-type in all four eutheria for 423 (95.7%) of the 442 clusters (unique intron positions) where four eutherian orthologous genes are present. Seventeen clusters contain ambiguous introns, indicating potential conversion, but as many as 440 of the 442 clusters may be U12-type in all genomes considered. There is only one unambiguous case of U12 intron deletion  in the U12DB. Since only fourty-seven intron clusters contain non-chordate U12-type introns, obviously, the modern-day U12-type introns identified in extant animals are mostly limited to vertebrates. To learn how these orthologous vertebrate U12-type introns (those that are present in the human and at least one non-primate genomes) relate to other lineages, we investigated their conservation pattern from a broader and more inclusive perspective.Platynereis dumerilii), C. elegans, insects, ciona, and humans, Raible et al. [D. melanogaster genome appears to have undergone extensive intron loss [D. melanogaster, 1.04/0.02 = ~50 in C. elegans, 0.77/0.04 = ~20 in Ciona, and 0.35/0.21 = ~2 in humans [We were able to find invertebrate orthologous genes for 267 vertebrate genes that contain a U12 intron. In 194 cases the orthologous intron was missing from the invertebrate gene. Although the data does not provide direct evidence whether these are intron gains in vertebrates or losses in non-chordates, the following observations suggest that U12-type intron loss is more frequent than U12-type intron gain. First, our four eutherian and eight vertebrate analyses did not uncover a single case of U12-type intron insertion. Second, as mentioned above, the minor-type splicing system independently went extinct in some organisms via intron deletion and/or conversion to U2-type, indicating that the loss rate of U12-type introns is greater than gain rate and that the entire system has been lost repeatedly . Third, e et al.  reportedron loss ,27-29. An humans  argued tn humans  and highn humans ,31. TherOf the 549 clusters with a U12-type intron present in humans and at least one non-primate vertebrate, invertebrate orthologous genes are absent in 254 of them. Thus, not only are the majority of U12-type introns found in metazoans present in vertebrates but also the genes that harbour them are often vertebrate-specific. While some of the 254 cases of vertebrate-specific genes might be due to extensive gene losses in invertebrates ,32, otheSCN and CACN1 genes have two U12-type introns [SCN genes are located in four chromosomal regions that, interestingly, also harbour HOX genes. This suggests that the large-scale segmental duplications and subsequent tandem gene duplications led to the expansion of SCN genes in mammals [Mammals have eleven sodium and ten calcium channel alpha subunit genes while fruitflies have two and three, respectively -37. Most introns ,39, howeet al. [A unique aspect of the evolution of U12-type introns is the conversion from a U12- to a U2-type which was first proposed by Burge et al. . Both inet al. , inverteet al. . Such muCCDC16, the vertebrate ortholog of the D. melanogaster U12-containing gene CG11839, has undergone three independent retropositions followed by a loss of intron-containing alleles, one in early mammals and two in distinct fish lineages. Only thirteen of the 160 D. melanogaster orthologs of the vertebrate U12-containing genes are intronless. However, retrotransposition followed by intron gain is more likely at such a long phylogenetic distance, so we cannot rule out a larger role of retrotransposition in U12 intron removal.We have observed several cases of U12 intron loss that can be attributed to retrotransposition. In vertebrates, where U12-type introns are extremely conserved, gene retroposition appears to be responsible for three out of the eight losses of U12-type introns (the affected genes are intronless). Interestingly, et al. [Of the nearly one thousand U12-type intron clusters in the U12DB, eighty-seven clusters have both U12- and U2-type introns in orthologous position. These eighty-seven non-redundant U12-type introns can be presumed to have undergone type-conversion at least once during the course of evolution. Eight of them also have both of the U12 subtypes. Of the remaining seventy-nine clusters, seventy-four are of the GT-AG subtype and five are of the AT-AC. The ratio of GT-AG to AT-AC subtypes in various genomes catalogued in the U12DB ranges between 3:1 and 1:1. Comparing this ratio to the total number of clusters of each subtype, 565:204 (GT-AG:AT-AC), indicates that the underrepresentation of the AT-AC subtype among clusters with conversions is statistically significant . Moreover, all eighty-seven U2-type introns involved in conversion have GT-AG termini. This suggests that GT-AG U12-type introns are much more likely to be converted to a U2-type. There are thirty-six clusters with U12-introns with both subtypes indicating subtype switching. Thus, it seems likely that AT-AC intron conversion to the U2-type is preceded by a subtype switching from AT-AC to GT-AG, as previously proposed by Burge et al. .Although most type conversions from an AT-AC U12-type are likely to involve prior subtype switching, we think that the existing AT-AC U2-type introns are the outcome of a much more rare U12 to U2 conversion pathway. These AT-AC U2-type introns in the sodium channel genes all have a G at position +5, a feature that has been shown to be important for recognition of the 5' splice site by the U2-spliceosome [Trappc9, Nibp), three GG-AG introns in the macaque and cow SLC12A4 genes and the rat SLC25A30 gene, and one GA-AG in the zebrafish actr10 gene. In all of these cases, orthologous introns in other vertebrates have either AT-AC or GT-AG consensus dinucleotide termini. Naturally occurring U12-type introns with these three sets of termini  have never been reported before. In a mutational study of terminal dinucleotide in U12-type introns, all sixteen possible combinations of first and last nucleotides were tested in the context of NU-AN [U-AN; denoted as +2 at the 5' end and the -2 at the 3' end, respectively) are highly conserved according to database search and mutant analyses; the authors also tested the functionality of twelve combinations (excluding G2) of nucleotides at these two positions in the context of AN-NC. The results suggested that the nucleotides at +2 and -2 do not interact in the same way as those at +1 and -1. Therefore, no experimental splicing data appear to challenge the functionality of GG-AG and GA-AG termini.Given that the 5' end dinucleotides of U12-type introns seem to be under less constraint than are U2-type introns, we investigated non-canonical 5' dinucleotides present in U12-type introns. In the U12DB, nineteen U12-type introns do not have canonical donor sites . Manual examination revealed that six are annotation errors (four have AT-AC termini and two have GT-AG). Six of the remaining thirteen are unambiguous U12 non-canonical 5' dinucleotides. There are two CT-ACs in the mouse and rat genes encoding trafficking protein particle complex 9 . This indicates that U12-type 5' termini are more degenerate than previously thought. At most, one case of U12-type intron gain was found.We investigated conservation and changes of the intron state among orthologous U12-type introns identified in eighteen metazoan genomes. From our study it is clear that intron loss contributes much more than conversion of U12- to U2-type introns to the evolution of U12-type introns. Conversion occurred more frequently in the GT-AG subtype of U12 introns than in the "classic" AT-AC subtype. Intron subtype switch has been widely regarded as a unidirectional process ,21. HoweDaphnia [The presence of orthologous U12-type introns among vertebrates has been highly conserved in the past 550 million years. The conservation is especially marked among eutheria. Of the 442 unique intron positions, only one unambiguous loss in mice and one U12 to U2 conversion in rats were observed. The non-chordates investigated have considerably fewer U12-type introns than vertebrates. Interestingly, the lineage with the fewest U12 introns, diptera, is the only one that acquired a new U12-type intron. Although there is no orthologous intron from outgroups in most of the nearly 200 cases of intron loss several facts support intron loss view. Nearly half of vertebrate U12-containing genes are absent in non-chordates. This is likely a combination of gene losses in the non-chordates and neofunctionalization or subfunctionalization of genes arising from large-scale segmental duplication or whole genome duplication that occurred in early vertebrates, as is exemplified by the multiple-U12-containing genes that encode the ion channel alpha subunit. Thus, while our summary clearly shows that all changes occur only slowly, it appears that the loss of U12 introns greatly outstrips the gain of U12 introns. We find only a single clear-cut case of U12 intron gain. We note the recent exciting discovery of high rates of intron gain in the crustacean ftp://genome.imim.es/pub/software/u12/u12db_v1_0.sql.gz. U12DB, a database with a web interface, provides information for U12-type intron identified in twenty sequenced eukaryotic genomes and clusters of U12-type introns and orthologous introns. In U12DB, orthology between two introns was determined by sequence similarity of their flanking exonic sequences. Each orthologous U12-type intron cluster represents a unique intron position with respect to the protein sequence that is derived from the hosting genes. At least one of the member introns is of the U12-type. Therefore, a set of orthologous genes can share more than one U12-type intron cluster.We downloaded the MySQL version of the entire U12DB version 1.0 published by Alioto ftp://genStatuses of the introns in a cluster were encoded with "1" - U12-type, "2"-ambiguous, which is denoted as "U12/U2" in the U12DB, "3" - U2-type, and "4" - intron absent. Intron states of all intron clusters were converted into a matrix of intron status versus species. Each orthologous intron cluster is represented in the matrix as one line of intron states for all species analyzed. If gene orthology information is lacking for a certain species, the state of this species is coded by a "5" - missing data. On the other hand, if multiple genes from one species are present in one cluster, intron states of the same species were ranked in the order of U12-type, ambiguous, U2-type introns, and intron absent. The top ranking was retained to represent the species. Patterns of intron states were used to infer conservation of the U12-type intron among taxa at varying phylogenetic distances. We conducted manual inspections extensively to correct potentially false cases of intron absence and missing orthologs in the U12DB. We relied on a variety of sources for validation of automated results when necessary. These sources included Entrez Gene , evidencCL carried out the acquisition, analysis, and interpretation of data, and drafted the manuscript. SM participated in analysis and interpretation of data, and writing. AJ conceived of the study and participated in interpretation of data. WM coordinated and designed the study, participated in analysis and interpretation of data, and writing. All authors read and approved the final manuscript.Here we present several examples of the evolutionary fates of specific U12-type introns. These examples illustrate the modes of evolutionary changes for U12 introns.Click here for file


Ostreococcus lucimarinus has one of the highest gene densities known in eukaryotes, yet it contains many introns. Phylogenetic studies suggest this unusually compact genome (13.2 Mb) is an evolutionarily derived state among prasinophytes. The presence of introns in the highly reduced O. lucimarinus genome appears to be in opposition to simple explanations of genome evolution based on unidirectional tendencies, either neutral or selective. Therefore, patterns of intron retention in this species can potentially provide insights into the forces governing intron evolution.The genome of the pico-eukaryotic  prasinophyte green alga O. lucimarinus using expressed sequence tags (ESTs) to annotate the current genome assembly. ESTs were assembled into unigene clusters that were mapped back to the O. lucimarinus Build 2.0 assembly using BLAST and the level of gene expression was inferred from the number of ESTs in each cluster. We find a positive correlation between expression levels and both intron number  and intron density .Here we studied intron features and levels of expression in In a species with a genome that has been recently subjected to a great reduction of non-coding DNA, these results imply the existence of selective/functional roles for introns that are principally detectable in highly expressed genes. In these cases, introns are likely maintained by balancing the selective forces favoring their maintenance with other mutational and/or selective forces acting on genome size. Ostreococcus encompasses a group of globally distributed photosynthetic, unicellular green algae in the anciently diverged class Prasinophyta Ostreococcus species also contain the most compact  genomes Saccharomyces cerevisiae (12.1 Mbp) that contains ∼6000 open reading frames, with one gene every 2.0 kb, O. tauri and O. lucimarinus have genomes sizes of 12.6 and 13.2 Mbp with a gene occurring every 1.6 and 1.7 kb, respectively O. tauri, which is the smallest among eukaryotes Ostreococcus genomes because introns are present in an estimated 20–25% of transcripts, compared to less than 4% of intron-containing genes in the budding yeast Ostreococcus species display the highest ratio of intragenic to intergenic noncoding DNA detected thus far.The genus Ostreococcus genomes which are characterized by a high degree of synteny for a core set of 18 chromosomes. Apart from this conservation, several chromosomes appear to have unique characteristics when compared both to other chromosomes and when compared across Ostreococcus spp. Chromosome 21 appears to be a unique feature of the O. lucimarinus genome resulting from a fusion of duplicated regions within Chromosomes 9 and 13 This extreme level of genome compression has lead to both a downsizing of gene families and in some cases to the creation of gene fusion proteins Ostreococcus genomes, trends common to both suggest that similar mechanisms have led to the optimization of cell and genome size O. lucimarinus. Our results reveal significant correlations between the levels of expression and the number of introns present in assembled EST clusters, strongly suggesting non-neutral processes. We present and discuss several possible selective mechanisms acting on intronic DNA focusing on explanations for intron retention in compact genomes.Despite these differences both within and between the O. lucimarinus that are available at the Joint Genome Institute (http://jgi.doe.gov/) were downloaded and assembled into unigene clusters using the TGI clustering package (http://www.tigr.org/software). Single contig clusters were mapped backed to the genome assembly of this species using blastn. Individual clusters which mapped to multiple loci and/or arose from cluster assemblies producing multiple contigs were omitted to reduce the mapping of clusters potentially containing EST reads of duplicate genes and closely related members of gene families.EST reads of Ostreococcus spp. due to the known presence of overlapping genes on opposite DNA strands in these species Assembled clusters were verified as originating from a single locus by examining the clustered output for congruency within the directionality of forward and reverse EST reads. Clusters that contained forward or reverse reads in both the sense and antisense orientation of an assembled contig were examined to identify potential over-clustering within overlapping 3′ ends of transcripts. Over-clustering is not unexpected in http://www.jmp.com/). Given the unique features of Chromosome 2 with respect to increased intron presence, we redid the analyses excluding this chromosome. Differences in the level of gene expression for Chromosome 2 were also compared against all other chromosomes to assess potential differences in gene expression and their influence on the correlations of expression and intron presence. Functional annotation of the 265 intron-containing clusters was performed using Blast2GO (www.blast2go.de).Tentative introns were identified as instances where complete and uninterrupted queries were not recovered with a single high-scoring pair (HSP). Intron presence was confirmed only in cases where non-overlapping HSPs, with an identity of 99% or greater, could be used to recover >97% of the query length. In cases where blastn was not capable of unambiguously aligning the terminal edges of exons, absolute intron size was calculated using the genomic distance between aligned neighboring HSPs. Adjustments to absolute intron size were performed by centering HSPs along their respective neighboring exons and stipulating the single inclusion of each query nucleotide in either one or the other HSP. Absolute intron size was therefore either decreased or increased in situations where terminal exon nucleotides were omitted or accounted for multiple times respectively in the local alignments. Statistical analyses of expression and intron composition were done using the JMP statistical software package  database in an orientation indicative of paired loci overlapping on opposite strands. Three of the intronless clusters were discarded from further analysis due to an extensive amount of overlap and the inability to properly bin the EST reads as belonging to one of the working models. Corrections of the starting clusters in this manner resulted in a final data set of 1,670 clusters for downstream analysis.Investigation of potentially over-clustered ESTs due to overlapping transcriptional units on opposite strands that span small intergenic regions identified 8.9% (148/1647) of these cases See . Of thesO. lucimarinus Build 2.0 with an average of 22.4 ± 3.4% of annotated genes per chromosome within our mapped unigene clustered dataset  suggesting that although different in intron composition, the levels of relative expression for the genes on Chromosome 2 are comparable to the remainder of the genome.Although our limited EST data address only ∼22% of the annotated genes , the coverage of these transcripts is largely unbiased in their chromosomal distribution. Furthermore, our analyses indicate a similar average intron size (188 bp) to that described for the complete genome assembly that utilized both experimental and bioinformatic methods to calculate this number (187 bp). This suggests the EST data utilized here do not deviate significantly from characteristics of the complete genome and therefore represent an accurate picture of the overall pattern of intron evolution in p<0.0005); an equivalent result is observed after excluding clusters mapped to Chromosome 2  and significantly reduced expression for intron-less genes when compared to intron-containing genes . These latter results are not surprising given the limited contribution of ribosomal proteins to the EST library  returned a positive correlation between intron presence and increased expression  and corresponding longest open reading frame  in all analyses. The tendency for EST libraries to be biased towards longer transcripts has been previously reported p<0.0001). In addition, no statistical differences were found between intron-containing or intron-less unigene clusters in terms of either the assembled cluster lengths or longest open reading frames , whereas intron number and assembled cluster length was consistently found to be non-significant in all analyses. The removal of ribosomal proteins from the analysis depicts an equivalent trend, with intron density increasing with expression .Lastly, the trend of intron presence and increased expression does not appear to be an artifact of longer transcripts simply containing more introns because a measure of intron density (number of introns/length ORF) is also positively associated with expression  to be biased towards deletions (deletion bias) that progressively eliminate nonessential portions of the genome. This observation offers a quandary in terms of intron retention in the reduced genomes of 2 in plants Several biological arguments have previously been made to justify extreme directional shifts in genome size as a byproduct of selection working towards larger and smaller cell volumes. Examples of cell size and corresponding shifts in genome size have been proposed for gamete dispersal, duration of meiotic division, and responses to COO. lucimarinus when compared to other non-coding regions of the genome. Interestingly, we find a positive correlation of intron presence and increased expression, which suggests a selective advantage  for introns in highly expressed genes that is not present  in genes with reduced expression. This finding is not unique to the miniaturized genome of Ostreococcus because it has previously been reported in yeast. In S. cerevisiae, a few intron-containing genes produce nearly a third of the total mRNA transcripts, with a large fraction of these intron-containing genes corresponding to ribosomal proteins Ostreococcus represents a more extreme case in this respect because its genome is not only more gene dense but also clearly shows a much higher proportion of genes with introns. Furthermore, the reported relationships between expression and intron features are also observed after removing ribosomal proteins from the analyses in Ostreococcus. Indeed, several functional categories were represented by intron-containing genes with diverse roles in metabolism, responses to light, and chloroplast function  in highly expressed genes include their role in transcriptional elongation, nonsense-mediated mRNA decay (NMD), amelioration of linkage effects between neighboring sites under selection, and mRNA transport Arabidopsis nucleolus extracts have identified putative orthologs to exon-junction complex (EJC) proteins previously shown to increase the export of spliced mRNAs in animals Arabidopsis EJC proteins identified in O. lucimarinus protein models for the existence of these sequences in the current assembly  converged on a single Ostreococcus model. Reciprocal BLAST analyses performed on these best candidate gene models using the nr protein data available at NCBI identified most gene models as their corresponding homologs through the conservation of protein domains. Of particular interest is the tentative identification of an ALY/REF homolog because this component has previously been characterized as an EJC interacting protein that is responsible for the enhanced export efficiency of spliced mRNAs Ostreococcus nuclear pore complexes are never found to be facing the exterior membrane of the cell and are always directed towards the cell interior facing the chloroplast Recent proteomic analyses of assembly . SeveralArabidopsis genomes have revealed that highly expressed genes not only contain more introns but the average intron length and primary transcript length also increase with increasing expression Although it is to be expected that not all introns currently existing in the genome are of functional significance, the correlations described here are consistent with other findings that correlate intron features and increased levels of gene expression in plants. Analyses of rice and Drosophila melanogaster, Caenorhabditis elegans, and humans suggest that intron size decreases with expression, opposite to the trend observed in plants DrosophilaC. elegans exhibits the opposite trend Analyses done on animal models however have reported a more diverse pattern when investigating relationships between expression levels and intron size and density. Studies in O. lucimarinus genome (16% vs. 20%) Ostreococcus and other pico-eukaryotes.Finally, it should be also noted that differences exist between the absolute numbers of intron containing transcripts reported here and those previously reported for the O. lucimarinus. A note of caution is that current EST data for this species are rather limited and future experiments are needed to fully corroborate our results, using more complete expression data from Ostreococcus spp. This work would allow us to further explore the potential contribution that introns make in reduced genomes of free-living picoeukaryotes.Although our study is consistent with previously documented intron composition trends seen in other plant genomes, it clearly underscores the existence of selective  roles of introns in genes that are highly expressed in Table S1BLAST results generated using nuclear encoded ribosomal proteins within the clustered EST data(0.05 MB DOC)Click here for additional data file.Table S2BLAST2GO annotated intron clusters(0.14 MB DOC)Click here for additional data file.


Arabidopsis thaliana, Caenorhabditis elegans, and Drosophila melanogaster) which show that the first intron in the 5′ UTR is - on average - significantly longer than all downstream introns within a gene. A partial explanation for increased first intron length in A. thaliana is suggested by the increased frequency of certain motifs that are present in first introns. The phenomenon of longer first introns can potentially be used to improve gene prediction software and also to detect errors in existing gene annotations.While many properties of eukaryotic gene structure are well characterized, differences in the form and function of introns that occur at different positions within a transcript are less well understood. In particular, the dynamics of intron length variation with respect to intron position has received relatively little attention. This study analyzes all available data on intron lengths in GenBank and finds a significant trend of increased length in first introns throughout a wide range of species. This trend was found to be even stronger when using high-confidence gene annotation data for three model organisms ( The discovery of RNA splicing in the late 1970s paved the way for our understanding of the exon-intron structure of eukaryotic genes Drosophila melanogasterArabidopsis thalianaThe few studies that have combined intron data from multiple species have found that the first intron is - on average - longer than later introns A. thalianaC. elegansA. thaliana and in rice One reason for distinguishing between introns that occur at different positions in a gene, is that introns from the 5′ proximal region of a gene (‘early’ introns) have been shown to have important functional properties, often relating to gene expression. The ability of an intron to enhance gene expression has been termed intron-mediated enhancement (IME) Drosophila species have also shown that first introns contain more conserved elements than later introns There are other reasons to suggest that early introns might be functionally different from those that occur later on in the gene. First introns in humans (in comparison to later introns) contain fewer SNPs A. thaliana, this increased length can partly be accounted for by the presence of an IME motif that increases in frequency in early introns. Finally, we also show that because first introns tend to be longer, this information can be used to detect errors in existing gene annotations.In this study, we have looked at the issue of first intron length in several different ways. Firstly, and most importantly, we used information from GenBank to look at intron length data from a much larger number of species than has been previously studied. Secondly, by also studying high-quality gene annotations from three model organisms, it has been possible to systematically evaluate differences in intron length at different positions within a gene, ensuring that UTR introns are evaluated separately from CDS introns. By finding that a trend for increased length occurs in the vast majority of eukaryotic species, we establish that this is a general feature of eukaryotic gene structure. This phenomenon exhibits itself most strongly in the first intron of the 5′ UTR . Additionally, we find that in P<0.05) longer than the average of the non-first introns , suggesting that this increased length is typical for all eukaryotic species.Using data from GenBank, intron lengths were calculated for all available species with sufficient numbers of full-length CDSs (at least 500). For each species, the average length of all first introns was compared to the average length of all other introns (‘non-first’ introns). For 30 of 36 species with sufficient data, the average length of the first intron was seen to be significantly , or as many as 20 introns .The trend for longer first introns was examined in more detail by partitioning the data for ‘non-first’ introns into separate classes, i.e. when 1st and 2nd intron lengths are compared in CDSs that have exactly the same number of introns. In total, 118 comparisons were made for species that have specific numbers of introns (excluding single intron CDSs). Of these, the majority (78%) showed a significant difference between the lengths of the 1st and 2nd intron . Analysis of these introns confirmed the prior observation of longer first introns and also revealed that while the first intron of the coding region remains significantly longer than downstream coding introns, it is the first intron of the 5′ UTR, if present, that is the longest intron within a transcript . These annotations were all supported by cDNA evidence and revealed introns in the 5′ UTR of many coding transcripts  are arranged in operons C. elegans intron (344 nt), with the very first intron in the operon transcript being notably shorter than the average.An unusual feature of the A. thaliana introns that is postulated to be involved in the process of intron mediated enhancement (IME) of gene expression A. thaliana; the aforementioned IME motif accounts for a greater proportion of early introns than later introns  were revealed to be annotation errors. In some cases the problem with the annotation was obvious as there was clear supporting evidence that the first intron should be deleted from the annotation  was the only one that was found to have notably shorter first introns. This is likely to be caused by a small sample size (N = 645 CDSs) which also features several genes which are overrepresented due to being sequenced multiple times from different strains .While there are many individual examples of genes with short first introns, the dominant trend appears to be that, on average, it is the first intron that is the longest intron in a gene. Utilizing data from GenBank, we show that the first intron is the longest intron for the majority 32 of 36) of species  and 2. W of 36 ofAny limitations in the GenBank data can be overcome by focusing on high-quality annotations in which only one isoform is represented from each locus, and in which introns from 5′ UTRs can be clearly distinguished from introns in the CDS. Such data  confirmsC. elegans genes that occur in operons. First introns in operons are surprisingly shorter than expected and the shortest introns are those that occur first in the operon transcript. It is unclear why introns in operons differ in this way, and this suggests that the processes that contribute to increasing first intron length might act differently in operons . Operons are known to differ in other ways, containing genes that are known to be biased towards certain essential functions such as transcription and translation C. elegans operons, we believe that this work underlines the generality of this phenomenon across phylogenetically diverse species.One notable exception to the pattern of increased first intron length was found in the subset of C. elegans suggest that this is a justifiable method for finding errors in annotations , and can include models to reflect different classes of intron length e.g. . Howeverotations . As it iA. thaliana and rice C. elegans or D. melanogaster (unpublished data), suggesting that it might be a motif common to plants. In our analysis, we find that while this 11 nt motif is present at a background level in all A. thaliana introns, it occurs far more frequently in early introns.A simple hypothesis to explain the increased length of first introns is that these introns contain functional elements that are present in addition to what might be regarded as ‘normal’ intron structure. There is evidence that this might be the case for why the first introns of mouse and rat are longer D. melanogaster Adh gene contains a hairpin structure that appears important for normal splicing activity of that gene Given the numerous reports that have shown that first introns tend to be under more selective constraint see , it seemA perl script was written to extract data from release 164 of the GenBank database For each species, the mean length of introns at different positions within the CDS was calculated in two different ways. First, CDSs were separated into different classes - based on how many introns they contained - and mean intron length was calculated at each intron position within each class (with a requirement of at least 100 introns in each position). Secondly, all introns were classified as either ‘first intron’ or ‘non-first intron’. Additional filtering restricted this data to species with at least 500 CDSs in GenBank, and excluded any single-intron CDSs. Mean intron lengths were compared by use of a Z-test.A. thaliana, C. elegans, and D. melanogaster were extracted from their respective model organism databases TAIR, WormBase and FlyBase Details of the genome annotations of Used TAIR release 7Only retained transcripts with annotated 5′ and 3′ UTRsRemoved genes that are 95% identical to others over 400 ntRemoved genes with introns <60 bp or >1000 bp, or CDS lengths <50 amino acidsFinal data set: 13,163 transcripts containing 59,260 intronsUsed FlyBase release r5.7Only retained transcripts that had a FlyBase evidence score of at least 8Final data set: 7,486 transcripts containing 19,772 intronsUsed WormBase release WS190Only kept transcripts deriving from genes with a WormBase ‘Confirmed’ designationFinal data set: 6,232 transcripts containing 30,916 intronsAverage intron lengths were calculated for each intron position, pooling data from transcripts with different numbers of introns. I.e. the 2nd intron of a gene with just two introns was included with the 2nd intron of a gene with ten introns. A small proportion of 5′ UTRs contained two or more introns, but only the first 5′ UTR intron was considered in this analysis.A. thaliana introns in the high-confidence data set to a previously discovered A. thaliana sequence motif believed to be involved in IME We compared the sequences of


Previous studies have indicated that the wide variation in intron density (the number of introns per gene) among different eukaryotes largely reflects varying degrees of intron loss during evolution. The most popular model, which suggests that organisms lose introns through a mechanism in which reverse-transcribed cDNA recombines with the genomic DNA, concerns only one mutational force.Using exons as the units of splicing-site recognition, exon definition constrains the length of exons. An intron-loss event results in fusion of flanking exons and thus a larger exon. The large size of the newborn exon may cause splicing errors, i.e., exon skipping, if the splicing of pre-mRNAs is initiated by exon definition. By contrast, if the splicing of pre-mRNAs is initiated by intron definition, intron loss does not matter. Exon definition may thus be a selective force against intron loss. An organism with a high frequency of exon definition is expected to experience a low rate of intron loss throughout evolution and have a high density of spliceosomal introns.The majority of spliceosomal introns in vertebrates may be maintained during evolution not because of potential functions, but because of their splicing mechanism . Further research is required to determine whether exon definition is a negative force in maintaining the high intron density of vertebrates.This article was reviewed by Dr. Scott W. Roy (nominated by Dr. John Logsdon), Dr. Eugene V. Koonin, and Dr. Igor B. Rogozin (nominated by Dr. Mikhail Gelfand). For the full reviews, please go to the Reviewers' comments section. Encepalitozoon cuniculi [Plasmodium species, which lack known transposable elements, have much lower rates of intron loss as compared with other eukaryotic lineages, such as plants, fungi, and nematodes [Plasmodium species are comparable with those in vertebrates [Spliceosomal introns are sequences that interrupt nuclear genes and will be removed from the corresponding RNA transcripts by spliceosomes. They are found in all eukaryotic groups, but their density, i.e., the average number of introns per gene, varies dramatically among eukaryotes from 8.4 in humans to 0.0075 in the microsporidian cuniculi -4. Whilecuniculi . Howevercuniculi ,15-18. Icuniculi ,15,19. Icuniculi . Plasmodematodes . Howevertebrates , the gentebrates ,23. Anottebrates . By analtebrates  found thtebrates ,27. It sThe second model of intron loss is genomic deletion via unequal recombination between two copies of genomic DNA -30. To mIn vitro, expanding the small internal exons of vertebrate genes to 300 nt inhibits the assembly of spliceosomes [in vivo analysis of a gene with long introns showed that exon size turned out to be problematic when it was expanded to 432 nt, and exon skipping became predominant when the exon was expanded to 526 nt [Although most of the key components of spliceosomes are present in all eukaryotic organisms , there aceosomes . Similaro 526 nt .Loss of an intron from a gene results in fusion of two flanking exons. Multi-species comparisons of exon-intron structures have shown that adjacent introns tend to be lost in concert ,20,40 an2 is improperly recognized, and splicing fails  are synthesized. Abnormal proteins will be produced if the mRNA molecules are not degraded by mRNA surveillance mechanisms. Thus, we are inclined to suggest that intron loss notably affects fitness and is under strong negative selection.1. In an organism in which exon definition is predominant and introns are very large, intron loss produces a large exon (> 526 nt), which causes exon skipping because exon A) and normal mRNA products (transcript B) will be synthesized. Intron loss partially affects fitness and thus is under weak negative selection.2. In an organism in which exon definition is predominant and introns are very large, intron loss produces a middle-sized exon (432–526 nt); both truncated mRNA products (transcript a and b) that flank the newly formed large exon (exon 2) happen to be small, the splicing sites of intron a as well as b will be recognized by means of intron definition [B) will therefore be synthesized. Loss of those three internal introns will not affect the splicing of nearby introns. Thus, intron loss does not reduce fitness.3. Accumulating evidence indicates that both exon definition and intron definition are effective splicing-site-recognition mechanisms in many eukaryotes ,41,42, afinition ,39 and nB) will be produced.4. In lower eukaryotes, in which intron definition is predominant, large exon size does not affect splicing; normal mRNA molecules . Finally, it seems very unlikely to me that arguments relying on vague selective differences that are expected to vary considerably across exons within a genome  could explain the nearly complete lack of intron loss in, for instance, mammals or Theileria. That is, whereas I think Dr. Niu's argument could indeed explain a fewfold reduction in intron loss, I cannot see how it could completely stem intron loss  as seen in some lineages.I am unconvinced by this argument. The comparison here is largely between vertebrates – the only lineage known to extensively utilize exon definition – and everything else. Insofar as this is true, this result only restates something that is already known – that vertebrates are unlike most sampled eukaryotes both in their very high intron density and utilization of exon definition. Given the large number of other vertebrate genomic and organismal characteristics that differ from many eukaryotic lineages – large genomes and introns, high content of transposable elements, high degree of alternative splicing, large body size, multicellularity, small population size, etc. – all of which can (and have) been argued to make a difference in both of the traits compared by Dr. Niu, I am unconvinced that this is good evidence for a causal relationship. In addition, since (apparently) simultaneous loss of adjacent introns creates much longer exons than single intron losses, in the context of Dr. Niu's argument it is surprising that such multiple losses seem to account for a relatively large fraction of overall intron losses in many lineages in which such losses are rare , I have revised the manuscript and clarified that this hypothesis is applied primarily to chordates.Dr. Niu highlights yet another chicken-and-egg relationship between characteristics of vertebrate genomes: high intron density is likely to favor emergence of exon definition, but the reverse may also be true. Dr. Niu adds another strand to the complex web of cause and effect in genome evolution, and his hypothesis is worthy of consideration for future research.I agree with this comment.This is an elegant hypothesis that strives to explain, with one unifying idea, two rather puzzling observations: i) the extremely low rate of intron loss in the course of evolution of some groups of animals, particularly, vertebrates, and ii) the typical small size and narrow size distribution of exons in those same organisms. Of course, the two trends are likely to be related as introns loss results in fusion of exons and the formation of a new, long exon, so if intron loss rate is low, there will be fewer long exons. However, the connection needs not be deterministic because exons could expand by other mechanisms as well. Dend-Ke Niu's hypothesis is that exon expansion, by any mechanism, be intron loss, duplication or insertion, is deleterious because splicing in the respective organisms occurs, mostly, via the exon definition.I find this to be an interesting, credible hypothesis with considerable explanatory power. This being said, there are several issues that deserve some criticism or at least awareness. First, I would suggest that the paper should make it clearer that the hypothesis applies, primarily, to chordates, as intron loss is pretty extensive in other animals like nematodes or arthropods. This does not really detract from the hypothesis but there is no need to claim more generality than justified.I agree with this comment and have revised the manuscript accordingly. Second, it should be noticed that that test of the hypothesis illustrated in Figure 2 is weak, and indeed, rather ambiguous because the ratio of the   numbers of exon skipping to intron retention events is a very indirect proxy of exon definition, and intron density is not intron loss either. With regard to the latter, I think that it is possible to use estimates of actual intron loss rates from: Carmel et al. Genome Res. 2007 Jul; 17(7):1034-44, to at least diminish the uncertainty associated with the use of intron density. This is, in a sense, not a crucial issue because the paper is a   hypothesis, and the decisive test in any case remains to be done.I thank Dr. Koonin for pointing out this flaw. The related text and figure have been removed from the main article. However, to aid readers' understanding of the reviewing process for this paper, I included them as an online supplementary file  to aid readers' understanding of the reviewing process for this paper.I would like to emphasize that the above criticisms do not invalidate Dend-Ke Niu's hypothesis which, on the whole, I find interesting, stimulating, and plausible.The author suggested a hypothesis that exon definition is a potential negative force against intron losses. This is an interesting hypothesis, however I have certain reservations regarding this paper.N. vectensis and Arabidopsis are among the most intron-rich species that contain a lot of ancient introns. However, splicing in N. vectensis is largely consistent with the exon definition  whereas splicing in Arabidopsis is more consistent with the intron definition (CE fraction = 0.17). This result immediately suggests that the hypothesis is not able to explain the extreme cases of high intron density.1) 2) Correlation coefficient is a measure of the linear dependence between two random variables. It is obvious that these variables are not random. Many species  are closely related. Thus the correlation coefficient might simply reflect the non-randomness of the analyzed variables. Removal of closely related species may substantially change results of the correlation analysis .3) Even if the correlation is significant, this does not mean that there is a true connection between two variables. Pearson correlation coefficient (0.49) is not exciting. There might be some other variables  which would produce a stronger correlation and explain larger fraction of variation in the data.I agree with these comments and thank Dr. Rogozin for pointing out these flaws. The relevant text and figure have been deleted from the main text and are now included as an online supplementary file  to aid readers' understanding of the reviewing process for this paper.Text and figure removed from the main text according to reviewers' comments. Presentation of those contents is intended to aid the readers' understanding of the reviewing process for this paper.Click here for file


MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition.It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the  MET oncogene, not only in primary bladder tumors but also in premalignant urothelium across entire bladders with tumors. Our study has important implications for tumor biology, cancer detection, and treatment, and it also answers the long-standing question of whether hypomethylation of retrotransposons induces ectopic gene expression and influences disease susceptibility in humans, a phenomenon first described in agouti mice.A surprisingly large portion of our transcriptome originates within repetitive elements, most commonly LINE-1s. However, the mechanism of activation has not been definitively shown. We directly demonstrate for the first time the causal relationship between DNA hypomethylation and transcriptional activation of LINE-1 promoters. Hypomethylation of specific LINE-1 promoters can alter the transcriptome, including activating an alternate transcript of the  Ectopic induction of the agouti gene results in altered coat color, obesity, and an increased incidence of tumors Aberrant DNA methylation is involved in the initiation and progression of carcinogenesis and includes both hypermethylation of CpG islands at gene promoters and global hypomethylation. While a small portion of hypomethylation occurs at gene promoters, resulting in overexpression of certain oncogenes ArabidopsisA recent study has revealed that more than 30% of transcription start sites in the human genome are located within repetitive elements, with just over 7% in L1s MET oncogene is active across entire bladders with cancer. The clinical implication of our finding is that surgical excision of the tumor would leave behind large areas of the bladder that remain epigenetically altered and express a potential oncogene. We also provide evidence that an active L1 acquires H2A.Z and nucleosome free regions upstream of TSSs, which has only been described previously at single copy genes, and undergoes chromatin remodeling from an inactive tetranucleosomal structure to an active dinucleosomal structure.Until recently it has not been possible to study the promoters of individual L1s since the sequences are too similar to design primers for one particular locus MET oncogene (L1-MET) MET transcripts by sequencing EST clones obtained from a bladder carcinoma cell line (GenBank accession no. BF208095) and placenta (BX334980). Both transcripts have start sites located in the L1 promoter, share the same reading frame as MET  (−5) but not as dramatic as L1-MET hypomethylation and that the methylation pattern can be quite different between global L1s and a specific L1, such as in the cell lines LD137, T24, and RT4 (To examine the methylation status at a specific L1 we designed bisulfite-specific PCR primers with one located in the L1 promoter and the other in the surrounding intronic region of the host gene . The L1-4×10−10) . We also4×10−10) . We foun and RT4 . This reMET anti-sense promoter contains its own exons 1 and 2, referred to as L1-MET exon 1 and L1-MET exon 2  and two non-tumorigenic urothelial cell lines, UROtsa MET was also not expressed in normal tissues except for placenta (data not shown). Therefore L1-MET hypomethylation correlated with the expression of the alternate transcript , allowing the promoter to be methylated without affecting the plasmid backbone. We created two plasmids, differing only the orientation of the L1-MET promoter, allowing us to measure either the L1 transcriptional activity or the L1-MET activity transcriptional activity  has previously been used to obtain single molecule resolution of nucleosome positioning at unmethylated CpG island promoters MET promoter in the UROtsa immortalized urothelial cell line was completely occupied by nucleosomes, revealing that the methylated L1-MET promoter exists in a tetranucleosomal structure .The endogenously methylated L1-tructure . GpCpG sf either . We werethylated . HoweverMLH1 bidirectional promoter has demonstrated that while each transcription start site loses the nucleosome directly upstream when active (−1 nucleosome), the nucleosome directly downstream is always maintained (+1 nucleosome) MET and global L1s (MET (data not shown) and nucleosome eviction at the L1-MET promoter   curves for L1-MET revealed an extraordinary degree of both sensitivity and specificity for detecting bladder tumors (AUC of 0.97) and premalignant tissue (AUCs of 0.89)  . Hypomet1-RAB3IP  [35]. Thof 0.89) . Since apatients , a noninMET was not correlated with hypomethylation of the L1-MET promoter, since the expression of MET is regulated by its endogenous promoter and not by the specific L1 promoter  mice revealed that hypomethylation of a retrotransposon could induce ectopic expression of a gene and influence disease susceptibility et al. The consequences of global hypomethylation at repetitive elements in cancer has long been the subject of speculation regarding the generation of genomic instability and potential activation of oncogenes. Ever since studies on viable yellow agouti  according to the manufacturer's instruction. See The non-tumorigenic human urothelial cell lines UROtsa and NK2426 and the normal fibroblast cell line LD419 have been described previously Tumor tissue samples were collected from the patients undergoing cystectomy or TURBT for bladder cancer. Normal bladder epithelium was obtained from 12 patients undergoing radical prostatectomy for prostate cancer (aged from 50 to 80) and 7 autopsy patients aged from 34 to 82, 5 of which were from non-cancer related deaths and 2 from deaths due to cancers other than bladder). All of these collections took place at Norris Cancer Hospital in IRB-approved protocols with patients' consent. Hematoxylin and eosin (H&E) sections marked with the location of the adjacent urothelium or tumor were used to guide in microdissection. DNA was bisulfite treated as previously described Methylation-sensitive single nucleotide primer extention (MS-SNuPE) was performed as previously described Expression was determined by quantitative RT–PCR as described previously MET and L1 promoters were cloned into the pCpGL luciferase vector MET promoter cloned was 555 bp, with 535 bp within the L1 and 20 bp within the MET gene (ch7:116364010–564). These experiments were performed as described previously The L1-ChIP was performed as described previously SssI. DNA was isolated, bisulfite converted, and PCR fragments were cloned for sequencing of individual molecules. In order to examine endogenously methylated promoters and increase the resolution of this method, chromatin from 250,000 cells was treated with the enzyme M. CviPI, which methylates GpC sites M-SPA was performed as described previously MNase digestion and sucrose density gradient centrifugation were performed as described previously Significant differences in methylation and expression levels in normal, corresponding normal, and tumor tissues were determined using a Mann-Whitney test.Figure S1Specific L1s with alternate transcripts located in intron of genes. Black boxes represent exons of the host gene while red boxes represent a specific L1. The black arrow represents the transcriptional start site of the host gene while the red arrow represents the alternate transcriptional start site within the potentially active L1 promoter. GenBank accession numbers for representative alternate transcripts are followed by the number in parentheses of similar transcripts transcribed from the individual L1. All L1s are antisense to their host genes, yielding alternate transcripts that are sense with their host genes.(0.56 MB TIF)Click here for additional data file.Figure S2MET. (A) The functional domains of MET include the signal peptide (SP), sema domain at the N-terminus, the PSI domain, IPT repeats, the transmembrane domain (TM), and the kinase domain at the C-terminus. The structure of truncated MET proteins 1 and 2 are shown, encoded by transcripts derived from placenta (GenBank accession no. BX334980) and a bladder carcinoma cell line (BF208095), respectively. (B) The two L1-MET transcripts, truncated L1-MET-1 (T-MET-1) and truncated L1-MET-2 (T-MET-2), were cloned into a pMEV expression vector with 2 HA tags fused at the N-terminal. Hela cells were transfected with either the empty pMEV vector, pMEV T-MET-1, or pMEV T-MET-2 and protein was extracted after 48 hours. The expression of truncated MET-1 (90 kDa) and truncated MET-2 (60 kDa) was detected by western blot using an HA antibody. (C) Results of 5′RACE reveal the start site for L1-MET within the L1 element. The transcriptional start site of L1-MET was confirmed by 5′RACE in the T24 cell line which expressed L1-MET. The underlined sequence is located inside of the LINE-1. (D) RT–PCR analysis of reactivation of L1-MET by 1 or 3 µM of 5-Aza-CdR treatment for 24 hours (day 3 after treatment). β-actin expression level was used as a control.The truncated MET protein encoded by L1-(1.22 MB TIF)Click here for additional data file.Figure S3ACVR1C correlates in cell lines. (A) Map of alternate transcripts from L1-ACVR1C. Exons are represented by black boxes while the specific L1s are represented by red boxes. The lower tick marks represent each CpG site. The left bent arrow indicates transcriptional start sites and ATGs indicate translational start sites. Green arrows indicate the primers used to amplify the pyrosequencing product and the black arrow in between indicates the location of the pyrosequencing primer for L1-ACVR1C. (B) L1-ACVR1C methylation (red bars) and L1 methylation (black bars) was analyzed by pyrosequencing in 6 normal tissues, one normal bladder fibroblast cell line (LD419), one non-tumorigenic urothelial cell lines (UROtsa), and 10 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1-ACVR1C from two technical duplicates. (C) Expression of L1- ACVR1C was measured using real-time RT PCR in one normal bladder fibroblast cell line, one normal urothelial cell line, and 10 bladder carcinoma cell lines. Values are also the average from two technical duplicates. Red bars indicate the methylation status of L1-ACVR1C, which is also represented in (B), and green bars represent the level of expression relative to GAPDH.Methylation and expression of L1-(0.86 MB TIF)Click here for additional data file.Figure S4RAB3IP correlates in cell lines. (A) Map of alternate transcripts from L1-RAB3IP. Exons are represented by black boxes while the specific L1s are represented by red boxes. The lower tick marks represent each CpG site. The left bent arrow indicates transcriptional start sites and ATGs indicate translational start sites. Green arrows indicate the primers used to amplify the pyrosequencing product and the black arrow in between indicates the location of the pyrosequencing primer for L1-RAB3IP. (B) L1-RAB3IP methylation (red bars) and L1 methylation (black bars) was analyzed by pyrosequencing in 6 normal tissues, one normal bladder fibroblast cell line (LD419), one non-tumorigenic urothelial cell lines (UROtsa), and 10 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1-RAB3IP from two technical duplicates. (C) Expression of L1-RAB3IP was measured using real-time RT–PCR in one normal bladder fibroblast cell line, one normal urothelial cell line, and 10 bladder carcinoma cell lines. Values are also the average from two technical duplicates. Red bars indicate the methylation status of L1-RAB3IP, which is also represented in (B), and green bars represent the level of expression relative to GAPDH.Methylation and expression of L1-(0.88 MB TIF)Click here for additional data file.Figure S5Chromatin remodeling occurs at active L1 promoters. (A) DNA methylation at specific and global L1s (with p16 as a control) was determined by pyrosequencing in the immortalized urothelial cell line UROtsa and bladder carcinoma cell line T24. The specific L1s had less methylation in the cancer cell line. Chromatin immunoprecipitation was performed using antibodies for (B) H3K4me3; (C) acetylated H3; and (D) H2A.Z. The values of the ChIP assay are the average of three experiments with technical duplicates. Error bars represent the standard deviation. The presence of active histone marks was associated with absence of DNA methylation at the specific L1s in the cancer cell line.(0.67 MB TIF)Click here for additional data file.Figure S6MET promoter. Nucleosome positioning in an active fully unmethylated L1-MET promoter in T24 bladder carcinoma cells reveals a dinucleosomal structure, as determined by both M. SssI, a CpG methyltransferase and M. CviPI accessibility.Chromatin remodeling occurs an active L1-(1.29 MB TIF)Click here for additional data file.Figure S7ACVR1C was analyzed by pyrosequencing in normal tissues (N), corresponding normal tissues (CN), and bladder tumors (T). Values are an average of two CpG sites. (B) Expression of the alternate transcript from L1-ACVR1C and (C) the host gene ACVR1C, and the control gene GAPDH was measured by real-time RT–PCR. *** represents p<0.001, ** represents p<0.01, and * represents p<0.05. (D) Methylation status of L1-RAB3IP was analyzed by pyrosequencing in normal tissues (N), corresponding normal tissues (CN), and bladder tumors (T). Values are an average of two CpG sites. (E) Expression of the alternate transcript from L1-RAB3IP and F. the host gene RAB3IP, and the control gene GAPDH was measured by real-time RT–PCR. *** represents p<0.001, ** represents p<0.01, and * represents p<0.05 as determined by the Mann-Whitney test. While there are no error bars for the clinical sample analysis due to the extremely limited amount of sample DNA. the results show a consistent trend.Methylation and expression status of specific L1s correlates in bladder tissues. Horizontal lines represent the mean. (A) Methylation status of L1-(0.58 MB TIF)Click here for additional data file.Figure S8MET methylation distinguish between normal bladder tissue (N) and corresponding normal bladder tissues (CN), N and bladder tumors (T), and CN and T. (B) ROC curves using L1-ACVR1C methylation, and (C) ROC curves using L1-RAB3IP methylation. *** represents p<0.001 and * represents p<0.05.ROC curves for specific L1s. (A) ROC curves using L1-(0.65 MB TIF)Click here for additional data file.Figure S9MET. Bladder tissues (N) from age-matched patients without bladder cancer (n = 10) and urine (N) from age-matched healthy volunteers (n = 10) showed low levels of L1-MET hypomethylation. However, urine (n = 20) from patients with TCC showed high levels of L1-MET hypomethylation, which was specific to the bladder since it was not detected in their white blood cells (WBC) (n = 20). Unmethylated levels (Y axis) indicate the Percent of fully Unmethylated Reference (PUR) values.Detection of L1-MET hypomethylation in urine sediments of patients with bladder cancer. Bisulfite-specific primers and a probe were designed for the MethyLight assay that amplified only completely unmethylated strands of L1-(0.30 MB TIF)Click here for additional data file.Figure S10ACVR1C and (C) L1-RAB3IP was measured by pyrosequencing. The green line represents the mean methylation of 12 normal samples from cancer-free patients. While there are no error bars for the clinical sample analysis due to the extremely limited amount of sample DNA. the results show a consistent trend.Methylation of specific L1s across the bladder. (A) Tissue samples were taken from five patients of their tumors  and at increasing distances from the tumor (0.5 to 2 cm) in the surrounding normal-appearing tissue in multiple directions . Additionally, distant normal-appearing samples were taken at least 5 cm from the tumor . (B) Methylation at L1-(1.62 MB TIF)Click here for additional data file.Figure S11MET. Biphasic distribution of L1-MET methylation status in corresponding tissue from a patient with bladder cancer is revealed by plotting the number of DNA strands by the percent of CpG sites methylated.Bisulfite sequencing of L1-(0.18 MB TIF)Click here for additional data file.Figure S12Ms-SNuPE and pyrosequencing yield similar methylation results. While both Ms-SNuPE and Pyrosequencing are quantitative assays, Pyrosequencing is much more high throughput. Therefore, we developed a Pyrosequencing assay for the rest studies. (A) We measured 4 CpG sites by Pyrosequencing assay in contrast with the 2 CpG sites by Ms-SNuPE. (B) We randomly chose 66 samples previously analyzed by Ms-SNuPE to perform Pyrosequencing on and the results are very similar from both assays (R = 0.91).(0.42 MB TIF)Click here for additional data file.Table S1Primer sequences.(0.08 MB DOC)Click here for additional data file.


Nephtys), where the complete sequence revealed a 1819 bp group II intron inside the cox1 gene. We infer that this intron is the result of a recent horizontal gene transfer event from a viral or bacterial vector into the mitochondrial genome of Nephtys sp. Our findings hold implications for understanding mechanisms, constraints, and selective pressures that account for patterns of animal mitochondrial genome evolutionGroup II introns are ribozymes, removing themselves from their primary transcripts, as well as mobile genetic elements, transposing via an RNA intermediate, and are thought to be the ancestors of spliceosomal introns. Although common in bacteria and most eukaryotic organelles, they have never been reported in any bilaterian animal genome, organellar or nuclear. Here we report the first group II intron found in the mitochondrial genome of a bilaterian worm. This location is especially surprising, since animal mitochondrial genomes are generally distinct from those of plants, fungi, and protists by being small and compact, and so are viewed as being highly streamlined, perhaps as a result of strong selective pressures for fast replication while establishing germ plasm during early development. This intron is found in the mtDNA of an annelid worm, (an undescribed species of  With more than 900 complete animal mtDNA sequences available, only some members of the basal groups, sponges and cnidarians (which have group I introns) and the placozoan Nephtys sp., a carnivorous polychaete inhabiting the intertidal and subtidal zones. This genome is typical of animal mtDNAs in possessing 37 genes on a single circular molecule with few and short non-coding regions cox1 contains a group II intron  conserved GUGYG and AY nucleotides at the 5′ and 3′ intron boundaries, respectively; (2) conserved sequence of domain V, which is the catalytic core of the intron's ribozymic activity; (3) presence of an ORF for a contiguous reverse transcriptase (RT) gene and a partial maturase gene; (4) potential secondary structure with six helical domains radiating from a central core consistent with the highly conserved secondary structure of group II introns  into the alignment of Zimmerly et al. mparison . HoweverNephtys sp. and T. adhaerens  have only 29% identity, indicating that these introns diverged long ago, presumably long-predating the divergence of these animal groups. Although both the T. adhaerens and Nephtys ORFs are found in the cox1 mitochondrial gene, their positions differ by 108 nucleotides. For these reasons it seems very likely that this intron has integrated into these two mtDNAs in separate events, particularly because the alternative would require the hypothesis of many parallel losses in related lineages.The amino acid sequences of the intronic ORFs of Wolbachia) invade the female reproductive tissues of their host (i.e. Drosophila) and live intracellularly inside of the eggs, leading to inheritance of the microbial population in subsequent generations NephtysNephtys sp. harbors endosymbionts is yet unknown.Thus, this group II intron is most likely the result of recent horizontal gene transfer, presumably from a bacterial or a viral intermediary. This would require that the transferred genetic material was specifically sequestered by the germline in order to be inherited. Interestingly, some bacterial lineages  may illuminate the extent and patterns of intron gain as well as provide further genome-level data for better understanding the forces shaping mitochondrial genome evolution.4, dried, and resuspended in 100 µl of water. This was sheared into ∼1.5 kb fragments using a Hydroshear device (GeneMachines), then the fragment ends were repaired using Klenow fragment and T4 polymerase. The product was size-selected using an agarose gel and ligated into pUC18 vector. These clones were introduced into E. coli cells by electroporation. This was plated and grown overnight at 37°. Colonies were picked and processed to generate reads from each end of randomly selected clones.Total genomic DNA was extracted from frozen tissue using a Qiagen DNeasy kit according to supplier's instructions. The mtDNA was amplified by long PCR (using the Takara polymerase kit) in three overlapping pieces (∼8 kb each) using specific and universal primers  [29]. Eacox1 mRNA was amplified by PCR using primers matching the mtDNA sequence, and this product was isolated, cloned, and sequenced as above.Total RNA was extracted from tissues samples using Quiazol reagent (Quiagen) according to the manufacturers instructions. cDNA was constructed from total RNA using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) following the manufacturers instructions. In order to verify that the presumed intron was removed from the transcript, a portion of the Phred and Phrap were used to call bases and produce an alignment (∼10×) and Consed was used for manual verification of quality Nephtys sp. (Accession number EU293739), Trichoplax adhaerens [NC_008151], Thalassiosira pseudonana (ORF718) [YP_316587.1], Schizosaccharomyces octosporus (ORF786) [NP_700371.1] and Chlorokybus atmophyticus (ORF845) [NC_009630.1] were incorporated within the Zimmerly et al. Nephtys's ORF and T. adhaerens was calculated using the amino acid sequence included in the analysis with the program WU-blastp (http://www.proweb.org/proweb/Tools/WU-blast.html).In order to evaluate the evolutionary history of the introns themselves, the intronic ORFs within Nephtys sp. and related mtDNAs, the protein coding genes of six annelids  and two mollusks  were aligned with CLUSTAL X In order to evaluate the rates of change for