One of the least common types of alternative splicing is the complete retention of an intron in a mature transcript. Intron retention (IR) is believed to be the result of intron, rather than exon, definition associated with failure of the recognition of weak splice sites flanking short introns. Although studies on individual retained introns have been published, few systematic surveys of large amounts of data have been conducted on the mechanisms that lead to IR.TTo understand how sequence features are associated with or control IR, and to produce a generalized model that could reveal previously unknown signals that regulate this type of alternative splicing, we partitioned intron retention events observed in human cDNAs into two groups based on the relative abundance of both isoforms and compared relevant features. We found that a higher frequency of IR in human is associated with individual introns that have weaker splice sites, genes with shorter intron lengths, higher expression levels and lower density of both a set of exon splicing silencers (ESSs) and the intronic splicing enhancer GGG. Both groups of retained introns presented events conserved in mouse, in which the retained introns were also short and presented weaker splice sites.Although our results confirmed that weaker splice sites are associated with IR, they showed that this feature alone cannot explain a non-negligible fraction of events. Our analysis suggests that cis-regulatory elements are likely to play a crucial role in regulating IR and also reveals previously unknown features that seem to influence its occurrence. These results highlight the importance of considering the interplay among these features in the regulation of the relative frequency of IR. Furtheractivity .Drosophila melanogaster's zeste [S. pombe's cdc2 gene [In respect to the mechanistic nature of IR, the most intuitive explanation is that these introns are flanked by weak splice sites that occasionally are not properly recognized. Stamm and coworkers  observed's zeste , S. pombdc2 gene ).S. pombe,C. elegans and Drosophila, for instance), splice site pairs seem to be recognized throughout introns, the intron definition model [Interestingly, all the introns involved in these studies were short (< 274 nt). This may have a mechanistic reason according to current models of splice site recognition. In the exon definition model , splice on model . The preon model . Therefoon model .cis-regulatory elements can indeed counterbalance retention of individual introns [In addition to splice site variation, some studies showed that the expression of the intron retaining and intron spliced isoforms varies along tissues ,14,15 in introns ,16.Although the studies mentioned above provide an experimental basis for IR on specific genes, a generalized model based on a large quantity of data is still missing. This model could reveal how different factors contribute to the frequency of IR. We sought to identify potential sequence features that may be associated with this type of alternative splicing by examining the contrasting features of two sets of retained introns found in human cDNAs. One set corresponded to those cases in which the intron retaining isoform was the minor splice form and the other in which it was the major form. We found that a higher relative frequency of IR is associated with weaker splice sites, genes with overall short intron lengths, higher expression levels and particular densities of regulatory elements.IR events were identified on human cDNA sequences aligned to the genome. An event was considered to be any occurrence of at least one cDNA defining an intron through the existence of its flanking exons plus at least one other spliced cDNA containing at least parts (> 1 nt) of the flanking exons and the intron (see Methods).In order to understand how splice site strength controls IR, we analyzed retained introns partitioned into two categories, according to their relative isoform frequencies (RIF = number of cDNAs with retained intron/number of cDNAs defining the intron). The low-RIF group contained IR events where the intron retaining cDNA was the minor form  and the high-RIF group contained those events where it was the major form . As we wanted to contrast features of the groups of IR events with different relative frequencies of IR, events with an equal number of intron retaining and intron defining evidences  were not considered.The initial set of events (6370 low and 1838 high-RIF) was filtered to produce a higher quality data set. Only those events where both the intron retaining and intron defining forms were confirmed by at least two cDNAs each, presenting no alternative borders (see Methods) and retaining introns longer than 50 nt were accepted. The filtered data set was composed by 1516 low and 296 high-RIF IR events.A few retained introns flanked by non-canonical splice sites were found in the filtered data set (1 in the low and 46 in the high-RIF group). As they were not G(T/C)..AG introns and in several cases different non-canonical splice sites could be obtained by adjustments in the alignment, they were excluded from the filtered data set  and theAnother way to view the dependence of RIF on splice site strength is to compare the frequencies of splice sites with different S&S scores in the two RIF groups Table . As expeDrosophila introns, Talerico and Berget [Based on the study of retention of a 116 nt intron flanked by 30 and 7 nt exons  and obseDrosophila [S. pombe [In organisms like osophila  and S. pS. pombe , intronsS. pombe ,6,19,20 S. pombe . The meanon-retained introns in intron retaining genes. The average length of introns in our data set is 5580 nt , close to previous findings . Genes . Note that the correlation is negative for the low-RIF and positive for the high-RIF group. One explanation is that higher expression levels increase the amount of all transcripts, both of the minor and the major forms, but under these circumstances, very rare forms also appear. Rare splice forms in the low-RIF group will be those with strong splice sites that very rarely retain an intron, resulting in the appearance of events with low retention/non-retention ratios only with higher expression (negative correlation). The reverse would be true for the high-RIF group, in which rare forms will be those with very weak splice sites that only rarely remove the intron, and only with higher expression (positive correlation).We also observed that there is a correlation between RIF and expression levels of genes , intronic splicing enhancers and silencers (ISE and ISS)), play an important role in constitutive and alternative splicing by acting as binding sites for trans-factors that promote (enhancers) or inhibit (silencers) splicing . For exiew, see  and of iiew, see .cis-regulatory elements found in exons or introns are functional, one hypothesis is that they are present in higher densities (motifs/nt) in the sequences they act [As not all motifs corresponding to they act . Recentlthey act  showed tcis-regulatory elements, we scanned retained and non-retained introns, as well as exons of the same genes, for known regulatory motifs .Retained introns were also different from exons, but with slightly higher densities of SELEX-ESEs, intermediary to exons and non-retained introns Table . This trMajor differences between the low and high-RIF groups are those related to the densities of GGG and ESSs. Low-RIF retained introns presented a slightly higher density of GGG than non-retained introns, whereas high-RIF retained introns presented the opposite trend Table . In regai.e. a pseudo-retained intron), and not to retained introns themselves. Analysis of exons of length between 300–600 nt and > 600 nt showed that these sequences do not present the pattern observed for "exon + retained intron + exon" units . Rather, the flanking segments and pseudo-retained introns presented motif densities similar to exons in general and therefore higher than those of retained and non-retained introns, indicating that the observed differences in Table As IR will result in one long exon, being the commonest isoform of the high-RIF group, we evaluated the possibility that such differential motif distribution between flanking exons and retained introns could be actually typical of an architecture of long exons  whereas for high-RIF introns, the inverse would be true. The basis for this argument is that knocking out different GGG triplets in the second intron of α-globin 2 leads to different levels of intron retention [The ESS/GGG densities observed could suggest that etention  and thatetention . This woThe interpretation of the slightly higher SF2/ASF and SC35 densities observed in retained introns is not straightforward. Although it might be only due to a biased nucleotide composition, another possibility is that these ESEs could help defining short "exon + retained intron + exon" units as a single exon (see Discussion), increasing retention. The other possibility is an action in the recognition of splice sites. Although ESEs are exonic by definition, SR proteins binding to introns was shown to both increase and decrease splicing in different studies . If bona fide non-retained introns.One could argue that both the higher density of SELEX-ESEs and lower ESS/GGG density in the high-RIF set of retained introns could be due to selection on sequences that occasionally are exonic, but this possibility is challenged by RESCUE-ESE and class 1 FAS-ESS, whose densities are similar to Evolutionary conservation of given features is often taken as a signal of biological function, under the assumption that negative selection is acting on them. Studying conserved IR events could support the observations made with human data and therefore help understanding the mechanism of intron retention. As the criteria we used to select high-confidence human IR events may have filtered out true events, in this analysis we searched the mouse genome and transcriptome for all the IR events initially detected. With the exception of the number of conserved events, all other data (identities and percentages) were the same when searching only filtered human events.Table Most IR events presented only the major form in mouse  indicates that our data set contains a large fraction of authentic IR events.The main problem in analyzing IR is to guarantee that the events are authentic alternative splicing occurrences and not part of partially processed messages. However, the fact that we found a mechanistically coherent association of splice site strength and differential densities of trans-factors, the formation of secondary structures in the RNA molecule or the dependence of splicing of upstream introns for retention of a specific intron, as observed for the thrombopoietin gene [In agreement with previous experimental observations on individual vertebrate retained introns -9,16 andtin gene .Although cDNA data sets like ours are possibly biased for short retained introns, other observations, not directly based on cDNA data, support the notion that such introns are short , mRNA and EST sequences  were downloaded from the UCSC Genome Browser  and cDNA, mRNA anOnly spliced sequences were used for alternative splicing annotation. Intron retention, exon skipping and constitutive exons were identified in clustered cDNAs by comparison of genomic coordinates with a tool developed by us . An IR eIn order to obtain a high confidence set of IR events, the initial sets of 6370 low and 1838 high-RIF events were filtered to 1515 and 250 events, respectively  and the expression levels were determined through SAGE tag counts . Short SExpression levels: for one mRNA with a poly(A) tail (at least 5 consecutive As in the 3' end of the sequence) from each cDNA cluster, the 3' most NlaIII site was searched and 10 nt downstream were extracted as the bona-fide SAGE tag produced by the given gene [ven gene . The tagComparison of gene expression levels among sets of genes: the global expression level of a group of genes for a given tissue was taken as all normalized expression levels of all libraries available for the tissue. To compare expression levels between a given experimental group  and all other genes, a bootstrap-like process of data randomization was performed. Ten thousand random subsets of the size of the experimental group were generated from the set of expression levels of all genes and had their mean expression level compared to that of the experimental set. If the random expression was different than  that of the experimental set, the counter was increased by 1. Expression levels were considered different when at most 5% of the random subsets compared had an expression level higher  than or equal to that of the experimental set.cis-regulatory elements from the literature were searched in exon, non-retained intron and retained intron sequences. Four scoring matrices for SF2/ASF, SC35, SRp40 and SRp55 (SELEX-ESEs) and their default cut-off scores [Putative and experimentally tested f scores  were usef scores  were scaf scores , ESSs frf scores   to the mouse genome sequence (build 33) obtained from UCSC  [i.e., the two exons defining the mouse intron could have a maximum gap of 20 nt in the cDNA, to ensure the intron was not simply a long alignment gap. When multiple hits existed, the one with the lowest E-value was preferred. Introns found in mouse were further globally aligned to human retained intron sequences with needle [th Blast    and homoh needle  using deConservation of IR events: human cDNA blocks corresponding to "exon + retained intron + exon" segments were aligned to mouse ESTs and mRNAs. Matches corresponding to the coordinates of the retained intron plus parts of the flanking exons (at least -20 nt upstream and +20 nt downstream) were accepted as evidence of intron retention in the mouse . Matches corresponding to exon + exon where the exon/intron borders were similar by ± 10 nt were taken as evidence of the intron being excised in a given mouse cDNA. The cDNA also had to be continuous (gap < 20 nt). Splice sites were identified by aligning mouse cDNAs without the retained intron to the genome with SIM4, using the same procedure applied for human IR events.5'ss – 5' splice site3'ss – 3' splice siteESE – exonic splicing enhancerESS – exonic splicing silencerFAS-ESS – fluorescence-activated screen for exonic splicing silencersIR – intron retentionISE – intronic splicing enhancernt – nucleotideRIF – relative isoform frequencyNJS conceived the study, proposed and performed the analyses, interpreted the data and wrote the manuscript. SJS contributed in the conception of the study, suggested analyses and revised the manuscript. All authors read and approved the final manuscript.Comment on retained introns with non-canonical splice sites, Discussion on the presence of non-canonical splice sites in the intron retention data sets, whether they are authentic or not and how they could be recognized by the major spliceosome.Click here for fileLengths of retained introns and flanking exons, Table S1 summarizes data for lengths of exons, retained and non-retained introns, Table S2 presents data on lengths of retained introns and flanking exons of individual cases from the literature, Table S3 presents the distribution of the frequency of introns in the data sets analyzed categorized by length. Figure S1 shows the distributions of lengths of "exon + intron + exon" units for retained and non-retained introns.Click here for fileAnalysis of gene expression; Tables S1-S3 present the statistical analysis of gene expression per tissue of housekeeping, intron retaining genes and all other genes. Figure S1 shows that gene expression is higher for higher expression breadths, Figure S2 shows the distribution of cluster sizes of intron retaining genes in comparison to all genes.Click here for fileDistribution of the frequency of ESE densities; Figure S1 displays 6 distributions of ESE frequencies  for retained and non-retained introns as well as for exons and pseudo-retained introns.Click here for filecis-regulatory elements in long exons; Table S1 shows the distribution of cis-regulatory elements in exons with lengths in the range of 300–600 nt and Table S2 for exons > 600 nt. Tables S3 and S4 show data for the same long exons split with an additional criterion, the presence of pseudo-splice sites. Table S5 displays data for the impact of nucleotide composition in SELEX and RESCUE-ESE densities.Density of Click here for fileSequence logos of splice sites of retained introns conserved in mouse, Sequence logos of conserved mouse IR splice sites.Click here for fileExamples of intron retention events, contains 4 figures showing examples of intron retention events in real cDNAs from the low and high-RIF groups.Click here for fileList of low-RIF events, list of low-RIF IR events with GenBank accession numbers of sequences that retain/splice the intron, splice sites and RIF.Click here for fileList of high-RIF events, list of high-RIF IR events with GenBank accession numbers of sequences that retain/splice the intron, splice sites and RIF.Click here for file


Probiotics, live microorganisms administered to improve health, interact in as yet poorly understood ways with the complex microbial communities that inhabit the human host. Complex microbial communities, such as biofilms in the oral cavity and lumenal and mucosal communities in the gastrointestinal tract, play prominent roles in human health and disease . MicrobiThe microbiota of animals and humans begins to be established as soon as the newborn is exposed to the environment at birth; its complete establishment in the mammalian intestine requires months. Pioneering bacteria that appear in the mammalian intestine within the first several days of life are followed by a succession of colonizing species in a process that continues at least through weaning ,5. Diet,New molecular tools are facilitating a more detailed view of microvariation in structure and function that may be important both for the microbial community and for metazoan physiology ,7. DiffeLactobacillus populations. Lactobacillus spp. have featured prominently as probiotics. Interleukin-10–deficient mice predisposed to colitis contained predominantly Lactobacillus johnsonii, whereas mice with an intact interleukin-10 gene lacking evidence of colitis had greater Lactobacillus species diversity and featured L. reuteri as a predominant species [Lactobacillus species differences were correlated to functional differences in the abilities of these commensal bacteria to suppress host cytokine production. Intestinal L. reuteri isolates from healthy mice were able to suppress tumor necrosis factor-alpha  production by macrophages, whereas L. johnsonii isolates from diseased animals were not able to affect microphage TNF-α production[Given the complexity of both the communities themselves and the range of factors known to influence them, experiments with model systems have made important contributions to our understanding of host–microbe interactions. In particular, the use of conventionally reared, microbe-free, and gnotobiotic mice have allowed controlled experiments that would be impossible in humans. Many of our current insights into the workings of probiotics come from mouse studies. Mouse models of colitis have described important compositional and functional differences between intestinal  species ,10. Lactroduction.The human microbiome contains roughly 100 times as many genes as does the human genome.Helicobacter hepaticus to mice resulted in reproducible, long-lasting shifts in intestinal microbial composition and reduced overall diversity of the intestinal microbiota [The administration of a single bacterial species in large doses to an animal with an established, complex, indigenous intestinal microbiota with hundreds of species does not necessarily lead to successful colonization. Studies in humans have showed that probiotic strains generally persist in the human intestine for only a short time beyond when they are actually being ingested . Howevercrobiota . The chaIn general, there are both direct and indirect mechanisms by which the introduction of one organism might favorably affect a second organism. The first organism might provide a nutrient or other product (such as an enzyme) that can be used or recognized by the second, resulting in changes in gene expression and metabolism. In addition, the first might provide binding sites or other factors necessary for colonization by the second. Less-direct mechanisms might involve effects by the first organism, mediated by the community  or by the host , resulting in more favorable environmental conditions for the second organism.Bacillus thuringiensis produces a potent insecticidal toxin and has been used to manage insect populations that interfere with the practice of forestry and agriculture. After it is ingested by an insect, B. thuringiensis requires the presence of commensal midgut bacteria to produce insecticidal activity, as demonstrated by the loss of activity following pretreatment of moth larvae with antibiotics [Recent studies are shedding light on the important role of the intestinal microbiota in modulating the impact of exogenously administered bacteria. The bacterium ibiotics . FunctioL. paracasei to Trichinella spiralis–infected mice restored profiles of host energy metabolism, fat mobilization, and amino acid metabolism to those seen in mice without T. spiralis infection [The deliberate manipulation of microbial communities may result in sustainable changes of aggregate microbial and host physiologic functions. The administration of the probiotic nfection . The admnfection . Some ofHomo sapiens with a vast set of genes that effectively increases the human physiological repertoire [The combined functional genomic repertoire of entire microbial communities  can now be accessed with metagenomic approaches, providing new opportunities for scientists to explore mammalian physiology in a host-wide integrated manner. Using such a metagenomic approach, Gill et al. recently found that the human distal gut microbiome is enriched for many genes contributing to glycan, amino acid, and xenobiotic metabolism, and that this microbial genetic assemblage provides pertoire .PLoS Biology by Sonnenburg et al. [Bacteroides thetaiotaomicron, and an organism commonly used as a probiotic, Bifidobacterium longum, in a mouse model. Changes in function are examined at a global level by using a variety of genomic approaches both to monitor transcriptional changes and to characterize habitat-associated carbohydrates. Ba. thetaiotaomicron appears able to target a greater diversity of polysaccharides for degradation when Bi. longum is present. When present, the probiotic also induces an array of host innate immunity genes. Prior work by the same group described functional genomic changes in commensal bacteria that enabled them to use host glycans when dietary carbohydrates were scarce [The manuscript in this issue of g et al.  examinese scarce . IntestiTechnical approaches for understanding the composition and functions of the intestinal microbiota are being expanded to meet the challenges of many tall tasks ahead. The rapid completion of many microbial genomes provides ample DNA sequence information for functional metagenomics. Additional metagenomic studies of intact microbial communities are generating genetic information that is not found in completed or draft single-organism genome projects. The “mapping” of gene sequences and their predicted products to “clusters of orthologous groups” or on known metabolic pathways  allows predictions to be made regarding the functional capabilities of a microbial community . As the Fundamental questions are being explored regarding the functional capabilities of microbial communities that directly affect the physiology of the mammalian intestine and the entire host. Sustainable effects on the host may depend on lasting changes in microbial community composition, its metagenome, and, more specifically, on alterations in the metabolic profile of the community. As we improve our understanding of how probiotics might alter the functions of the resident microbiota, clinicians may improve their abilities to select candidates  for probiotic therapy, with the goal of enhancing or restoring the health of patients.


A knowledge of the positions of introns in eukaryotic genes is important for understanding the evolution of introns. Despite this, there has been relatively little focus on the distribution of intron positions in genes.i.e. between codons 1 and 2. This effect is seen in a wide range of eukaryotes: Vertebrates, arthropods, fungi, and flowering plants. Proteins carrying this start codon intron are found to comprise a special class of relatively short, lysine-rich and conserved proteins with an overrepresentation of ribosomal proteins. In addition, there is a peak of phase 0 introns at position 5 in Drosophila genes with signal peptides, predominantly representing cuticle proteins.In proteins with signal peptides, there is an overabundance of phase 1 introns around the region of the signal peptide cleavage site. This has been described before. But in proteins without signal peptides, a novel phenomenon is observed: There is a sharp peak of phase 0 intron positions immediately following the start codon, There is an overabundance of phase 0 introns immediately after the start codon in eukaryotic genes, which has been described before only for human ribosomal proteins. We give a detailed description of these start codon introns and the proteins that contain them. The "introns-early" school believes that introns were present in the last universal common ancestor of pro- and eukaryotes, and that intron loss is responsible for the lack of introns observed in bacteria. The "introns-late" school, on the other hand, believes that introns have appeared during the evolution of the eukaryotic lineage, and that intron gain is a frequent event in the evolution leading to the gene structures we see today. This debate continues to generate a huge amount of literature; for a recent review, see Rogozin et al. .et al. [Arabidopsis thaliana). In the unicellular organisms Saccharomyces cerevisiae and Plasmodium falciparum, which have relatively intron-poor genomes, there was a marked 5' bias for all introns. Mourier and Jeffares [Plasmodium where Sakurai et al. [On this background, it is surprising that the question of intron position distribution in eukaryotic genes has received relatively little attention. As an exception to this, it has been observed that introns are not uniformly distributed over the entire gene, but tend to be more abundant close to the 5' end. This locational bias is especially seen for genes with only a single intron. Sakurai et al.  found thJeffares  found thi et al.  had seeni et al.  investigi et al. ,3.et al. [donor site, and the 3' end as the acceptor site. The position of the donor and acceptor sites relative to the reading frame is referred to as the phase of the intron: a phase 0 intron is positioned between two codons, while a phase 1 intron disrupts a codon after the first position and a phase 2 intron after the second position. In most coding regions, phase 0 introns are the most common, followed by phase 1 introns and then phase 2 introns as the least common [Also intron gain seems to occur preferentially in the 3' end, Sverdlov et al.  reportedt common ,7. In prt common .et al. [Introns are recognised by the spliceosome, a complex of several small ribonucleoprotein particles (snRNPs) . There iet al.  did a coIt has been found that introns with the non-canonical sequence "at...ac" comprise a special group recognised by its own spliceosome, where the rare U11 and U12 snRNPs have replaced the standard U1 and U2 particles [et al. [There is also some weaker sequence conservation on the exon side of donor and acceptor sites. The consensus sequence for the exon-exon junction is "ag|g" where the "|" denotes the position of the intron . This haet al.  addresseet al. [et al. [et al. [Another question is whether shared intron positions reflects evolutionary conservation or parallel gain of new introns. Qiu et al. , using a [et al.  construc [et al.  used a m [et al. ; a resulet al. [et al. [et al. [It is a matter of debate whether intron position correlates with protein structure. If the "introns-early" theory, also called the "exon theory of genes" is correct, proteins should have evolved by assembly of small autonomous modules or structural domains, and one should expect that exons in proteins will correspond to boundaries of such modules. Stoltzfus et al.  tested t [et al.  found th [et al.  reported [et al.  analysedTordai and Patthy  investiget al. [Recently, Vibranovski et al.  .Introns shorter than this occur in GenBank. The shortest of them are probably annotation errors, but where should we set the cutoff? In order to find a non-arbitrary lower length threshold for introns, the intron length distribution was calculated on the GenBank data – vertebrates, arthropods, fungi, and flowering plants . This is in agreement with earlier observations . Intron In Figure The curves in Figure The distributions of intron positions within the first 100 amino acid positions of eukaryotic genes from the GenBank sets are shown in Figure For proteins with signal peptides , there is an excess of phase 1 introns in a region around 20 for the two animal groups, and between 20 and 40 in plants. This has been described as corresponding to the positions of the signal peptide cleavage sites ,8, cf. ti.e. between the start codon and codon 2. This is what we refer to as the start codon introns. Note that there are no values for phase 0 introns at position 1 – this would be introns occurring immediately before the start codon, and those are not visible in the GenBank coding sequence ("CDS") features the data are derived from (see Methods).In proteins without signal peptides , another phenomenon is seen: a sharp peak of phase 0 introns at position 2, The peak of start codon introns is very high in vertebrates, arthropods and fungi. In plants, it is not higher than the peaks for phase 0 introns between positions 20 and 40, but it is still conspicuous against the relatively intron-poor region between positions 3 and 20.i.e. phase 1 and phase 2 introns in position 1, are relatively rare, especially in plants.Note that introns disrupting the start codon, Drosophila genome shows the same pattern as the arthropod GenBank set.In the genome data sets, both the phase 1 peak for proteins with signal peptides and the start codon peak for proteins without signal peptides are clearly visible . These plots are shown in Supplementary Figure S2 see . In the Drosophila melanogaster. Indeed, the same peak can be seen in the non-homology reduced Drosophila genome  are structural constituents of cuticle . 46 have functional annotation missing or "unknown," and only 4 have an annotation of something other than cuticle. For comparison, only 2.8% of all the D. melanogaster genes with signal peptides are annotated with "structural constituent ... cuticle." In arthropod genes with signal peptides, a peak of phase 0 introns is seen at position 5 Figure . We checp < 1%, Pearson test) downward slopes for all three phases, arthropods only for phase 0 and phase 1. The vertebrate set shows no slope.In arthropods, fungi and plants without signal peptides, a slight downward slope can be seen in the intron frequency. We tested whether this is a real 5' bias by computing the correlation coefficient between position and relative frequency for positions 20 through 100. Fungi and plants have significant ), the background set was limited to phase 0 introns that were the first in the coding sequence.A comparison of intron lengths was inconclusive. For vertebrates, start codon introns are slightly shorter than the background set, but for fungi they are slightly longer. For arthropods and plants, there are no significant differences [see Supplementary Table S1 from p < 10-4, χ2-test, df = 3), but not for arthropods [see Supplementary Table S1 from p < 10-3, χ2-test, df = 15) [see Supplementary Table S1 from When examining the nucleotide distribution, significant differences are found for vertebrates, fungi, and plants . The amino acid frequencies are shown in Table The amino acid distributions were also calculated, and it was found that there are significant differences between proteins with and without start codon introns for all organism groups (p < 10-5). For the alignments of human versus Drosophila, the proteins with start codon introns had 60.9% identities, while the others had 40.2% . Only RBHs where the human gene was predicted not to contain a signal peptide were included in these calculations.In order to assess the conservation of start codon intron proteins, human genome proteins involved in a reciprocal best hit (RBH – see Methods for definition) to mouse or fly were compared. While human proteins with a start codon intron on average have 91.5% identities in the global alignments with mouse, the other proteins have only 84.8% . In the comparison between human and mouse, where the overall conservation of phase 0 intron positions is as high as 95.0%, no significant difference is seen.We also tested whether start codon intron positions are more conserved than other intron positions. All intron positions were mapped onto the global alignments, and in positions where the human protein had an intron it was tested if the mouse or fly protein also had an intron. In the comparison of human versus fly, there are 76 RBHs with a human start codon intron. 45 had a corresponding fly phase 0 intron, and 43 of these (56.6%) were also in a start codon position. For comparison, only 2164 of the 16435 (13.2%) human phase 0 introns in other positions are conserved in fly. This difference is highly significant . The descriptions of all start codon intron proteins from the GenBank data sets can be seen in Supplementary Table S2 [see p < 10-15, χ2-test, df = 1).When looking at the functional annotations, a curious observation was made: in the sets of proteins with start codon introns, ribosomal proteins are overrepresented. This, of course, corresponds well with the fact that ribosomal proteins was the first place where start codon introns were observed. In the vertebrates, there are 16 ribosomal proteins out of the 55 start codon intron proteins, corresponding to 29%, while the background frequency in the non-start codon intron proteins without signal peptides is 2.0%. In the plants, there are 12 out of 91 (13%) against a background frequency of 0.64%, and in arthropods, there are 14 out of 44 (32%) against 2.98%. In fungi, there are only 4 ribosomal proteins out of 45 in the start codon intron set, but the overrepresentation is still significant .Since ribosomal proteins can be expected to be more conserved than other proteins, we investigated whether the higher conservation of proteins with start codon introns could be solely due to the high proportion of ribosomal proteins. The lists of RBHs were constrained to those where the human protein description did not contain the word "ribosomal," and the percent identities were compared again. The difference is still significant, although smaller  and here, the start codon peak was still seen. This strongly suggests that the overabundance of start codon introns is a biological phenomenon. Also, the observation that proteins carrying a start codon intron comprise a special class points in this direction. We have shown that proteins with start codon introns are relatively short, lysine-rich, serine-poor and evolutionarily conserved. In addition, we have shown that the start codon intron positions are often conserved between human and fly. During the peer review process it was suggested that our method for showing the excess of phase 0 introns after the start codon could be questionable. The concern was that that the method of aligning the first 100 aa of all protein sequences, while showing the start codon peak, might fail to detect some other peaks. The reason is that insertions and deletions during evolution would alter the distance between start codon and intron peak, thereby spreading the peak over several positions. In other words, peaks in other positions have been "diluted" over evolution while the start codon peak is retained simply because we use the start codon as "anchor point" for our alignment. This "dilution effect" would be enhanced by the fact that the N-terminal parts of proteins are often less conserved than other parts, with many indels. The reviewer suggested that we specifically analyzed proteins that have their N-terminals conserved over evolution to avoid this suggested effect.Drosophila those global alignments where neither the human nor the fly sequence had gaps within the first 20 positions. This yielded 661 pairs, of which 582 human sequences and 599 fly sequences were predicted to be without signal peptides. We then repeated the intron position statistics for these sequences, and the result is very clear: the phase 0 start codon peaks are even more conspicuous than those in Figure We did this by selecting from the list of RBHs between human and Furthermore, the fact that we can still see the phase 1 signal peptide peak in our start codon alignments despite the length variation of signal peptides suggests that the "dilution effect" does not completely wipe out peaks relatively close to the N-terminus, but only makes them broader. The reviewer suggested that the reason could be that proteins with signal peptides may have their N-terminals conserved over evolution, but this is not likely to be the case, since signal peptides are known to evolve rapidly .The function of the start codon intron may be to allow the 5' UTR to participate in exon shuffling, so that different genes can exchange regulatory information. As described in the Background section, phase 0 introns are often found at the boundaries of evolutionary modules in proteins without signal peptides ,26, and If the start codon intron really is a boundary of an evolutionary module, one should expect that the second intron would often be a phase 0 intron, too, since modules in exon shuffling most often have introns of the same phase in both ends . We trieet al. [Another explanation for the start codon introns could be that the nucleotide context of codons 1 and 2 provide a proto-splice site. The codon before is necessarily "atg," so the two nucleotides before the intron is "tg" instead of the canonical "ag." However, the "a" is not very well conserved, and "t" is the next most common nucleotide in the -2 position relative to the intron ,18. Alsoet al.  in orderDrosophila [Indeed, "g" is enriched in position +4 (relative to the translation start site) in eukaryotes . Howeverosophila , so if tet al. and Lin and Zhang [The 5' bias we observed in the position of introns within the non-vertebrate genes is in contrast to Mourier and Jeffares , who repnd Zhang ,4 did fifunctional class of proteins that are not related by descent (the data sets were strictly homology reduced). To be absolutely sure our homology reduction procedure had not let any pairs of homologous sequences through, we performed an all-versus-all global alignment of the sequences with start codon introns in all the four GenBank organism groups. No pairs showed more than 28.5% identity in the global alignment. We also made phylogenetic trees of the start codon intron sets and found no clusters (the trees look just like stars). Plots of % identity versus alignment score and the trees can be seen in Supplementary Figure S4, section 1 [see The fact that ribosomal proteins are strongly overrepresented in start codon intron proteins is striking, because it is an example of an intron position being conserved in a on 1 see . Drosophila. Initially, we thought that it could be a gene finding artifact, since it was specifically seen in one genome. However, the extremely high proportion of cuticle constituents convinced us that this too is a biological phenomenon. It is also not entirely unknown in the literature: Rondot et al. [Tenebrio molitor, found that both had a single intron within the signal peptide coding region . In addition to their own observation, they remarked that such an intron localization  has been described in cuticle protein genes from many different species, including Drosophila. Again, note that this is not simply a gene family with a conserved intron position, since the peak is also seen in the heavily homology-reduced set. In order to test this further, we did an all-versus-all global alignment of the 20 sequences in the arthropod peak and found no pairs with more than 32.1% identity [see Supplementary Figure S4, section 2, from Another example of this is the position 5 phase 0 peak in t et al. , while det al. [The start codon peak of phase 0 introns is so conspicuous that it is quite remarkable that it has only been described in a single sentence in the literature before. As mentioned above, Yoshihama et al.  saw the Magnoliophyta) were both found in gbpln. Observe that Magnoliophyta comprises both monocots and dicots.Data sets were extracted from GenBank release 149.0 . VertebrInformation about introns in the sequences were taken from "CDS" feature lines with a "join(...)" function in the feature position. "CDS" features on the opposite strand, marked by "complement(join(...))", were also parsed.Before analysis, the GenBank entries went through a filtering process. Genes were skipped for the following reasons (listed in the order the tests were applied): if the gene was a pseudogene ; if the gene was marked "/evidence=not_experimental"; if the sequence was incomplete, indicated by ">" or "<" in the positions; if the argument to "join" contained a cross-reference to another entry ; if the argument to "join" contained "complement" (this was a rare occurrence that we did not bother to parse); if the amino acid sequence contained "X" (unknown amino acid) or "U" (selenocysteine); if the amino acid sequence lacked an initial methionine; if the gene contained introns or exons of zero or negative length; if there was an annotated "gap" feature within the region of the "CDS" feature; or if the length of the amino acid sequence did not match the length inferred from the argument to "join".At this point, the data were used for calculating intron length statistics. After getting the results from that analysis, two more filtering steps were applied: genes with an intron shorter than the found cutoff were skipped, as were genes with introns that did not conform to the splice site consensus "gt...ag".et al. [Non-consensus splice sites are, as described in the Background section, known to exist, but they are rare. Burset et al.  estimateThe number of genes discarded for the various reasons can be seen in Table Drosophila entries is often not found in GenBank itself – the "/product" qualifier contains a code such as "CG4111-PA" – but can be found via cross-referencing to the NCBI Gene database, see below. A protein sequence without a functional annotation can be marked in several different ways: by having no "/product" qualifier, by having the value "unknown," or by having the value "hypothetical protein" or "putative protein."Functional annotation of the proteins in the GenBank data sets were extracted from GenBank by searching for the "/product" qualifier. In general, the vertebrate set is much more annotated than the other organism groups, having a protein name in 74% of the "CDS" features. The corresponding figures for fungi and plants are 40% and 19%. For arthropods, the situation is more complicated, since functional annotation for The GenBank data sets are provided for download at our website .Homo sapiens), mouse (Mus musculus) and fly (Drosophila melanogaster), was downloaded from NCBI  [Genomic data for human (rmation)  in the fIn all cases, the chromosomal GenBank format files were parsed using the FeatureExtract software  to colleDrosophila genome, all genes with ambiguous exon positions were removed, as were genes with "codon_start" annotated to be 2 or 3.For the human and mouse datasets, the following cleaning strategy was followed: Only genes whose protein-product is represented in the curated RefSeq database   was derived directly from the "CDS" comments  in the case of human and mouse, and by cross-referencing the GeneID to the NCBI Gene database  in the cFor the results presented in Supplementary Figure S2 see , we remoThe whole-genome data sets are provided for download at our website .Homology reduction of the GenBank data sets was carried out in order to avoid gene families with conserved intron positions showing up as peaks in the intron position plots.All the amino acid sequences in each data set were aligned to each other using BLAST version 2.2.9 . Filterif(x) = ln(-ln(1 - Pscore≥x)). Alignment scores for unrelated sequences are expected to follow an extreme value distribution [The threshold was found in the same way as for the translation initiation site dataset used in developing NetStart . Brieflyribution  and so sNote in Figure et al. [After finding the threshold, the removal of homologous sequences was carried out using algorithm 2 of Hobohm et al. . The cutTo further ensure that no protein families have escaped our homology reduction, we analyzed the sequences in the start codon peaks and the arthropod position 5 peak in the following way: For each dataset, all protein sequences were pairwise globally aligned against the rest of the set using the "align" program from the FASTA package , and aliThe whole-genome data were not homology reduced.Signal peptide prediction was done with SignalP v. 3.0 . Sequencvice versa. If the best hit for gene A in human was gene B in mouse or fly, and the best hit for gene B simultaneously was gene A, a Reciprocal Best Hit (RBH) between gene A and gene B was recorded. 8089 RBHs were found between human and mouse, and 4223 between human and fly. The pairs of proteins involved in each RBH were then globally aligned using the program "align" from the FASTA package [Conservation of proteins were investigated using the whole-genome sets. All human sequences were aligned to all mouse and fruit fly sequences using BLAST . For eac package  in order2-tests, Yates' continuity correction was used for 2-by-2 contingency tables. Two-sample t-tests were always preceded by an F-test to compare the two variances; if the variances were significantly unequal, Welch test was used.Statistical tests were done in R . When doThe project idea was conceived by HN, who also made all initial work on the GenBank data, the statistical analysis, and the main discoveries. RW provided and cleaned the genomic data, did the all-vs-all alignment analysis and set up the supplementary material. The manuscript was drafted by HN with RW providing details about the genomic analysis. The manuscript was continuously discussed and evaluated by both authors.Additional file 1 which has been uploaded with this manuscript is a PDF document of 3.3 MB (17 pages). It contains Supplementary Figures S1–S4 and Supplementary Tables S1 and S2 which have been referred to in the text. The data sets used in this study are deposited at our website , where yClick here for file


One of the most surprising discoveries in molecular biology was that a gene's coding region is broken up into smaller pieces (the exons) interrupted by noncoding portions called introns. After the DNA is transcribed into RNA, and before the RNA can leave the nucleus, the introns must be cut out and the exons spliced together.Since introns were discovered in 1977, the details of the splicing operations have been a major object of study. For splicing to occur, the ends of an intron must be brought into close proximity, and a number of proteins have been identified that aid this process. However, the function of one group of these proteins, called the hnRNP proteins, which are known to associate with prespliced RNAs, has not been clear. To date, the most accepted role for a subgroup of these proteins, the hnRNP A/B proteins, has been a negative one, since binding of these proteins to certain exons can prevent their inclusion in the mature RNA. In this issue, Rebecca Martinez-Contreras, Benoit Chabot, and colleagues show that when hnRNP A/B or hnRNP F/H proteins bind to intron sequences near splicing signals, they can stimulate splicing.The authors began by making long artificial RNA segments, which are poorly spliced due to the more than 1,000 nucleotides separating the two ends of their introns. By inserting hnRNP A/B–binding sites in the intron near the future splice junctions, they could increase splicing efficiency 4-fold. The binding sites did not have to be on the RNA itself, as long as they stayed close to the ends of the intron, as shown when the authors tethered short pieces of RNA to each end of the intron. These short RNAs contained the binding sites on their tails, which hung loose in approximately the right place next to the intron. This allowed the hnRNP A/B proteins to take up position near the ends of the introns, and splicing efficiency was increased. When binding sites were placed well into the interior of the intron, either on the intron itself or on an RNA tail, splicing was inefficient. Similar results were found when binding sites were inserted for a different binding protein group, hnRNP F/H.The authors propose a model for these results in which the bound hnRNP proteins interact with one another, clasping the two ends of the intron together, forming a loop to help the splicing machinery remove the intron. The authors further support their model by showing that splicing could also be stimulated just by inserting complementary RNA sequences at each end of the intron. These have the ability to bind to one another, forming the intron into a loop.However, they note that in some introns, only one hnRNP A/B site, positioned on the upstream end, is needed to promote splicing, and it does so nearly as well as when sites at both ends are present. The reason may lie in the particular introns that display this behavior—they contain a sequence which may itself bind an hnRNP A/B protein, thus providing the missing binding site and leading to loop formation. Confirmation or refutation of this hypothesis will have to await future experiments. Notwithstanding, the model is generally appealing because the two ends of many human introns are enriched in binding sites for these proteins. Overall, this mechanism suggests that hnRNP proteins can remodel the structure of prespliced RNAs, a property, the authors suggest, that could be important for both splicing and alternative splicing in a wide variety of genes.


Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516) has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2) and a homing endonuclease gene (NaHEG). NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA.Ribosomal DNA of several species of the free-living Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, contains hallmarks of a degenerated NaHEG. Phylogenetic analyses performed on separate data sets representing NaGIR1, NaGIR2, NaHEG, and ITS1-5.8S-ITS2 ribosomal DNA are consistent with an overall vertical inheritance pattern of the intron within the Naegleria genus.To elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria evolution with subsequent vertical inheritance. The intron was lost in the majority of isolates (70%), leaving a widespread but scattered distribution pattern. Why the apparent asexual Naegleria amoebae harbors active intron homing endonucleases, dependent on sexual reproduction for its function, remains a puzzle.The Nae.S516 twin-ribozyme intron was gained early in the  Naegleria is a common genus of soil and freshwater free-living amoeba of the vahlkampfiid family [Naegleria apparently lack a sexual reproduction cycle since meiosis never has been observed or proven experimentally. Subsequently, a number of genetically-defined variants have been isolated in nature and proposed as distinct species [Naegleria amoeba cell contains a distinct nucleus with a large and predominant nucleolus containing as much as 3000–5000 copies of an approximately 14-kb ribosomal DNA (rDNA) plasmid [Naegleria isolates have been reported to contain group I intron insertions at conserved sequence sites, both within the small subunit (SSU) and large subunit (LSU) rRNA genes [E. coli rRNA gene) and at positions 1921, 1926, 1949, and 2563 in LSU rDNA [d family . Naegler species ,2. A typLSU rDNA .Naegleria rDNA introns is Nae.S516. Group I introns at position 516 in SSU rDNA are relatively common among eukaryotic microorganisms with more than 250 cases reported so far [Group I introns are autocatalytic genetic elements carrying a ribozyme domain responsible for the intron self-splicing reaction, and occasionally a homing endonuclease gene (HEG) encoding an endonuclease protein directly involved in intron mobility at the DNA level ,13. A grd so far , and witd so far .Naegleria S516 intron has a twin-ribozyme organization and represents the most complex class of all group I introns known [Naegleria S516 intron have been reported. The splicing ribozyme (NaGIR2) is responsible for the autocatalytic activity that generates intron excision and exon ligation, as well as full-length intron RNA circle formations [Naegleria HEG (NaHEG) encodes a 245 amino acid protein that belongs to the His-Cys box family of homing endonucleases [Naegleria contains and maintains homing introns.A typical ns known . Nae.S51rmations . The abirmations ,20,21. Tucleases ,23. The ucleases . In geneucleases ,27 and iNaegleria and in vitro transcribed intron RNA [The expression of the NaHEG is dependent on a functional NaGIR1 ribozyme, which defines the 5' end of the homing endonuclease mRNA by internal processing and modification of the excised Nae.S516 intron ,28,29. Ptron RNA ,28 are ctron RNA . Thus, ttron RNA .Naegleria twin-ribozyme intron makes it interesting to investigate the evolutionary origin of the different intron domains, as well as the inheritance pattern within the Naegleria genus. Here we report several new intron variants and have performed sequence and phylogenetic analyses providing new insight into fundamental questions such as intron structure, intron-host biology, and the origin and evolution of intron HEG and ribozyme domains. The Naegleria twin-ribozyme intron serves as an attractive model system in the characterization of evolutionary processes behind a recently gained, but vertically inherited, selfish genetic element.The complex and unique structural organization of the Naegleria (Table 1) was performed to gain insight into the genetic relationships among strains and species. A phylogenetic tree based on the NJ method is presented in Figure Naegleria isolates [Naegleria isolates , show aia Table  was perfN. byersi NG597), possess the twin-ribozyme group I intron organization previously reported [Naegleria splicing ribozyme (NaGIR2) is responsible for intron excision and exon ligation during precursor rRNA processing in the Naegleria nucleolus, as well as for the generation of circular intron RNAs [Tetrahymena ribozyme [Naegleria introns. The most prominent differences between the Naegleria and Tetrahymena ribozymes are the lack of a P9.2 segment in Naegleria, the presence of an optional tetra-loop in L5b, and a large sequence insertion (approximately 950 nt) in P6b harboring the homing endonuclease gene (NaHEG) and the capping ribozyme NaGIR1 . All introns, but one  His-Cys endonuclease that recognizes and cleaves the intron lacking allele sequence in rDNA . An aligproteins ,31,34, sproteins . This poproteins  are highTetrahymena intron. We have previously performed phylogenetic analysis of various nuclear S516 group I introns, including five NaGIR2 variants, and recognized the Naegleria introns as a monophyletic clade among the group IC1 introns [Naegleria. Furthermore, we also include a host rDNA analysis based on their corresponding ITS-rDNA sequences. The intron phylogeny was based on 356 sequence positions within NaGIR2, strictly aligned according to the structure diagram in Figure N. italica AB-TF-3  and without bootstrap and Bayesian supports in the MP, ML, and BAY analyses, respectively. Thus, we infer there are no experimental support of horizontal intron transfer and conclude that the NaGIR2 domain, representing the Nae.S516 intron, is vertically inherited within the Naegleria genus.NaGIR2 represents the splicing-ribozyme domain of Nae.S516 and thus corresponds to the sequences present in a prototype group I intron such as the 3 Figure , that coNaegleria twin-ribozyme intron is highly unusual among group I introns in that it contains two distinct structural domains (NaGIR1 and NaHEG) inserted into the same peripheral region (P6) of NaGIR2 . We speculate that the exposed residues in P6a could be involved in RNA-RNA interactions as GNRA receptors. However, these sequences do not fit any known consensus motifs, suggesting that new motifs are yet to be experimentally identified.The primary function of GNRA tetraloops is to participate in long-range RNA-RNA interactions by specific binding to a receptor motif. A variety of receptor motifs, ranging from 4 to 12 nt, have been recognized experimentally -43. FiguNaegleria twin-ribozyme group I introns have been reported previously [Naegleria that includes each of the domains NaGIR1, NaGIR2, and NaHEG, and corroborates a previous study based on 5 intron sequences [Naegleria genus, but after the Cluster 6 branching  The intron was subsequently lost , resulting in complete deletion of the NaHEG as well as its regulatory NaGIR1 capping ribozyme (e.g. N. byersi NG597) [Evolutionary aspects on the structural organization of eviously . Whereaseviously , the NaGeviously . Here weequences . Based oNaegleria S516 introns? There are only two reported examples addressing the biological role of nuclear group I intron HEGs in experimental settings. In sexual matings between intron-containing and intron-lacking strains of either the myxomycetes Physarum polycephalum or Didymium iridis, group I introns were shown to be mobile due to the double-strand-break-repair pathway induced by intron-encoded homing endonucleases [Naegleria. However, the Naegleria intron endonucleases possess hallmarks linked to a function in intron homing. First, sequence comparisons show that the Naegleria enzymes belong to the same His-Cys homing endonuclease family as the known homing endonucleases I-PpoI and I-DirI encoded by the mobile Physarum and Didymium group I introns [Naegleria endonucleases cleave only intron lacking alleles flanking the intron insertion site at the SSU rRNA gene [Naegleria endonuclease and its intron in yeast generate intron homing intermediates consistent with a homing endonuclease function [Naegleria may occasionally perform sexual reproduction in nature since Pernin and co-workers [N. lovaniensis, including chromosomal recombination. Both haploid and diploid strains of the N. gruberi NEG isolate have been described based on both amoeba DNA content and UV-sensitivity [N. carteri NG055 ; N. clarki RU42 (ITS-rDNA and Nae.S516); N. pringsheimi 1D (ITS-rDNA and Nae.S516); N. philippinensis RJTM (ITS-rDNA and Nae.S516); N. carteri NG055 (ITS-rDNA and Nae.S516); Naegleria sp. NG647 (ITS-rDNA and Nae.S516); Naegleria sp. NG358 (ITS-rDNA and Nae.S516); Naegleria sp. NG393 (ITS-rDNA and Nae.S516); Naegleria sp. NG498 (ITS-rDNA and Nae.S516); Naegleria sp. NG169 (ITS-rDNA);Naegleria sp. NG336 (ITS-rDNA); Naegleria sp. NG491 (ITS-rDNA); Naegleria sp. NG492 (ITS-rDNA). The strains without designation are under revision and will be proposed proper species names based on phylogenetic analyses of ITS1-5.8S-ITS2 sequences . A complete list of all 70 Naegleria isolates included in this study is presented in Table 1. DNA samples of Naegleria strains were prepared as described previously [®-T Easy Vector System I (Promega). Individual clones where DNA purified and sequenced with the ABI PRISM BigDyeTerminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer), running on an ABI Prism 377 system (Perkin-Elmer), or using the sequencing service from MWG Biotech [The following  Biotech . Two or Multiple alignment of sequences were performed by using Megalign (Version 5.06) included in the Lasergene package from DNASTAR, Inc , BioeditNaegleria isolates, only 287 nucleotide positions could unambiguously be aligned due to high sequence variation in ITS2. However, when the analysis was restricted to the 14 intron-containing Naegleria isolates we extended the ITS-rDNA region to 415 nucleotide positions. Based on the multiple sequence alignment a NJ tree was constructed with the Kimura-2 evolutionary model of substitution (K2), with pair wise deletion of gaps and bootstrapped with 2000 replications with a cut-off value of 50%. Similarly, intron trees are constructed with NJ-K2 parameters. The robustness of the tree topologies were tested by the NJ-K2 parameter , MP branch and bound search criteria , and ML with the HKY+G model of substitution selected by Modeltest 3.7 [Two different data sets of the internal transcribed rDNA spacer region (ITS-rDNA: ITS1-5.8S-ITS2) were used. In analysis with all the 70 test 3.7 , all froNaegleria isolates. SDJ directed the research and wrote the paper in collaboration with OGW.OGW did the sequencing of ITS-rDNA, and group I introns in collaboration with CE. OGW performed the phylogenetic analyses. JFD contributed with DNA preparations of


The structural and functional features associated with Simple Sequence Proteins (SSPs) are non-globularity, disease states, signaling and post-translational modification. SSPs are also an important source of genetic and possibly phenotypic variation. Analysis of 249 prokaryotic proteomes offers a new opportunity to examine the genomic properties of SSPs.SSPs are a minority but they grow with proteome size. This relationship is exhibited across species varying in genomic GC, mutational bias, life style, and pathogenicity. Their proportion in each proteome is strongly influenced by genomic base compositional bias. In most species simple duplications is favoured, but in a few cases such as Mycobacteria, large families of duplications occur.Xanthomonas oryzae. Asparagine is abundant only in SSPs of low GC species. Aspartic acid is abundant only in the non-SSPs of Halobacterium sp NRC1. The abundance of Serine in SSPs of 62 species extends over a broader range compared to that of non-SSPs. Threonine(T) is abundant only in SSPs of a couple of species. SSPs exhibit preferential association with Cell surface, Cell membrane and Transport functions and a negative association with Metabolism. Mesophiles and Thermophiles display similar ranges in the content of SSPs.Amino acid preference in SSPs exhibits a trend towards low cost of biosynthesis. In SSPs and in non-SSPs, Alanine, Glycine, Leucine, and Valine are abundant in species widely varying in genomic GC whereas Isoleucine and Lysine are rich only in organisms with low genomic GC. Arginine is abundant in SSPs of two species and in the non-SSPs of Although SSPs are a minority, the genomic forces of base compositional bias and duplications influence their growth and pattern in each species. The preferences and abundance of amino acids are governed by low biosynthetic cost, evolutionary age and base composition of codons. Abundance of charged amino acids Arginine and Aspartic acid is severely restricted. SSPs preferentially associate with cell surface and interface functions as opposed to metabolism, wherein proteins of high sequence complexity with globular structures are preferred. Mesophiles and Thermophiles are similar with respect to the content of SSPs. Our analysis serves to expandthe commonly held views on SSPs. Simple Sequence Proteins (SSPs) are composed of various types of amino acid repeats such as amino acid runs , regularAnother major interest in simple sequences stems from the observation that they constitute an important source of genetic (and possibly phenotypic) variation . SequencPreviously, we developed a measure to analyze protein sequences and classify proteomes in a binary mode into two categories: SSPs and non-SSPs. This measure considers the entire protein sequence and SSPs are identified according to the proportion of simple sequences carried in them . Our appeff) in this work refers to the number of proteins of length greater than 45 amino acids. Because the number of such small proteins is very low, it is unlikely to affect the general analysis. The relationship between the number of SSPs and the effective proteome size from 249 proteomes is shown in Figure P < 0.0001). On an average, the number of SSPs in a proteome is approximately proportional to 1/30th of the proteome size although there is considerable variation in the dataset and ranges from 1.62% in Thermoplasma acidophilum to as high as 34.1% in Thermus thermophilus. These observations show that although SSPs are a minority, they tend to grow with proteome size. This relationship is exhibited across species varying in genomic GC content, mutational bias, life style and pathogenicity (See A proteome of a given species can be considered as a collection of protein sequences of that species . From thcity See .P < 0.0001). These results show that, while there is a general trend of SSPs to grow with proteome size across all types of species, genomic GC bias can strongly influence this trend.Although a general growth trend is apparent, a significant variability can be noticed. Two potential factors contributing to this variability are genomic base composition bias and gene duplications. In order to assess the base bias effect, we determined the relationship between the proportion of SSPs in each species and its genomic GC Figure . It is eab initio creation. An approach to examine the role of duplicative processes is by following the growth of the number of paralogous pairs among the SSPs of each species with increasing number of SSPs.According to the model of Qian et al. , genomesStreptomyces coelicolor A3(2) with 80 paralogs.The relationship between the number of paralogous pairs among the SSPs and the total numbers of SSPs of each species is displayed in Figure ad ≥ 0.7 . On the other hand, Glutamic acid(E) displays similarity in abundance in SSPs and non-SSPs with respect to genomic GC content. The abundance of Serine(S) in SSPs of 62 species extends to an upper limit of 60% GC whereas it is restricted to 50% GC in non-SSPs of 19 species. Threonine(T) is abundant only in SSPs of a couple of species.It is apparent that the aliphatic amino acids Alanine(A), Glycine(G), Leucine(L) and Valine(V) display similar abundance patterns in SSPs and non-SSPs of organisms varying widely in genomic GC content. Interestingly, the abundance of Glycine in SSPs persists even in low GC (33.5%) species, whereas its abundance in non-SSPs is restricted to the lowest limit GC content of 45%. The abundance of amino acids Isoleucine(I) and Lysine(K) are restricted to organisms with low genomic GC content in both SSPs and non-SSPs. Asparagine is abundant only in SSPs of species of low GC. Arginine(R) is abundant in SSPs of two species and in the non-SSPs of The restricted abundance of Isoleucine and Lysine in species of low genomic GC content and of Arginine in species of high genomic GC content correlates positively with the AT rich and GC rich base composition of their respective codons. On the other hand, the abundance of Alanine, Glycine, Leucine and Valine in species varying widely in genomic GC content suggests that this phenomenon relates to the evolutionary age of these amino acids instead of base compositional bias of the genomic DNA. The codons of Alanine (GCN) belong to the family of GCT triplets and those of Glycine (GGN) belong to a point change derivative of the GCT family. It has been proposed that the GCT triplets may have expanded during ancient period of evolution of nucleic acids .st(earliest) in the consensus chronological order of amino acids [It is therefore likely that the observed abundance of Alanine, Glycine, and Valine emerges from the abundance of their respective codons as a consequence of the earliest expansions since Glycine and Alanine co-rank 1no acids . Persistth position in chronological order. Interestingly Glutamic acid displays similar patterns of abundance in SSPs and non-SSPs as does Leucine. Miller's imitation experiment of primordial mixture contained Leucine [The abundance of Leucine presents itself as an interesting case. Majority of the codons (4/6) for Leucine are AT rich and Leucine co-ranks with Glutamic acid at the 5 Leucine . Althoug Leucine .Plasmodium falciparum, an AT rich species [The dominance of Asparagine in SSPs of species of low GC mirrors the trend observed in the low complexity sequences of  species  and poin species .P <= 0.0001 to avoid potential erroneous conclusions that may arise from small sample sizes.In order to examine the preferential association of SSPs towards a specific functional class, the SSPs from all the organisms were classified into seven broad functional classes namely, C: Cell Wall, Cell Membrane and Transporters, D: Cell Division, I: Information , L: Translocation and secretion, R: Stress, S: Signaling and Communication and M: Metabolism See . The staThe number of organisms falling into different functional classes with significant positive association of SSPs is shown in Table The number of Simple Sequence Proteins tends to grow with proteome size and their proportion in each proteome is strongly influenced by genomic base compositional bias. In most species, simple duplications is favoured. In a few species such as Mycobacteria, several SSPs are organized into large sized families with role in host-pathogen interactions. Amino acids with low biosynthetic cost are preferred in SSPs. The abundance of amino acids is controlled by multiple factors including biosynthetic cost, base composition of their respective codons, evolutionary age, wide usage in many biological processes and post-translational modifications. SSPs preferentially associate with Cell Wall, Cell Membrane and Transporters. The proportion of SSPs in a given species does not appear to be governed by its growth temperature (unpublished data) and is in agreement with other observations .SSPs either adopt well structured non-globular shapes or may have a propensity to exhibit disordered conformation ,34. The c, which is proportional to the fraction of low complexity sequence (simple sequence) present in the protein. Protein sequences with Fc value ≥ 15 are low complexity proteins and were considered to be SSPs [Complete proteome sequences of 226 bacteria and 23 archaea available in NCBI as on September 2, 2005 were retrieved from the NCBI ftp site . These s be SSPs ,19. The  be SSPs . This deSimple sequences have significant biases in amino acid or nucleotide composition. Collectively, these regions exhibit a very broad range of compositional properties and lengths, and most of them have unknown structures, dynamics and interactions. The sequence simplicity varies from extreme, as in homopolymeric tracts, to very subtle as in some non-globular domains of proteins. Locally abundant residues may be contiguous or loosely clustered, irregularly spaced or periodic. They tend to evolve rapidly, reflecting mutational processes such as replication slippage, unequal crossing-over, and biased nucleotide substitution .c  for identifying a low complexity protein (simple sequence protein). This principle is analogous to that used previously [c ≥ 15 were observed to be non-globular whereas proteins with lower values of Fc were globular. The Sensitivity and Specificity of this procedure was 99.4% and 71.4% respectively. Cases of counter-examples were re-examined with the program SEG using default parameters. We found that SEG produced the same inferences and conclusions as ScanCom [27) or charge clusters (for example RDDRPRDDRPRDDRPRDDRPRDDRPRDDRPRD), other types (for example GGAGGAGGKAGLLFGSGGAGGSGGA).Previously, we had used the structural information available from the non-homologous proteins with high resolution structures in PDB to ascertain the value of Feviously . Protein ScanCom ,19  from small duplications  by computing the number of pairs in each cluster and summing them. The number of pairs in each cluster is given by nC2 = {n(n-1)/2} where n is the number of paralogs in a given cluster. Duplex clusters with 2 members will have one pair whereas multiplex clusters will have large number of pairs. Species with paralogs organized predominantly into duplex clusters (simple duplications) will yield low number of pairs, whereas species with paralogs organized into multiplex clusters (large duplications) yield high number of pairs. A plot of the number of total number of paralogous pairs against the total number of SSPs describes the nature of duplications present in the SSPs of a given species.BLASTCLUST program ,40 for pThe percent amino acid content of all amino acids of SSPs and non-SSPs were computed to examine general preferences in the two datasets. Further, the average percent frequency of amino acids of SSPs or of non-SSPs of each species was computed according to the formula:ni(j) = Number of amino acid of ith type in jth SSP; ℓ(j) = length of jth protein (SSP or non-SSP); N = Total number of proteins (SSP or non-SSP)where, The top three ranking amino acids in each of the species were considered for further analysis.Aeropyrum pernix K1, Agrobacterium tumefaciens str. C58 (Cereon), Halobacterium sp. NRC-1, Listeria innocua Clip11262, Listeria monocytogenes EGD-e, Mannheimia succiniciproducens MBEL55E, Mycobacterium avium subsp. paratuberculosis K-10, Mycoplasma gallisepticum R, Mycoplasma hyopneumoniae 232, Mycoplasma hyopneumoniae 7448, Mycoplasma hyopneumoniae J, Nanoarchaeum equitans Kin4-M, Onion yellows phytoplasma OY-M, Pasteurella multocida subsp. multocida str. Pm70, Streptococcus agalactiae NEM316, Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis could not be classified by ARC. This is due to either incomplete annotation or rarely used gene symbol annotation. These species were dropped for this analysis. Details are displayed in , L: Translocation and secretion, R: Stress, S: Signaling and Communication and M: Metabolism using an automated open source software program ARC . ARC with its associative keyword library, uses a text word match approach to classify proteins. Since most annotation groups use automated approach in genome centers, the success rates (85%) for classification using our strategy is high. The proteins of d in See . A full P <= 0.0001 in order to eliminate potential erroneous inferences due to small sample sizes. The interactive statistical calculation page's website [The Correlation coefficient with statistical test was computed to examine the strengths of relationships. Statistically significant positive association (over representation) of SSPs with functional classes for each species were identified by testing the difference between observed proportion and the expected proportion computed from the entire proteome in the same species. Binomial proportions test was used applying a stringent cut off of  website  was used website  and Corr website ) using aSSPs: Simple Sequence Proteins.SR conceived the idea, helped in critical assessment and writing the manuscript, MBS implemented and carried out the study, MG assisted in functional classification and manuscript revisions. The contributions of MBS and MG may be considered equal.Microbial Organism Information, containing information on the species Taxonomy ID, Organism Name, Super Kingdom, Group Sequence Status, Genome Size, GC Content, Gram Stain, Shape, Arrangment, Endospores, Motility, Salinity, Oxygen Requirement, Habitat, Temperature range, Pathogenic host and Disease caused.Click here for fileParalog clusters in species marked in Figure Click here for fileadditional function predictions of paralogous proteins using other Bioinformatics softwares CDD and SPAAN (see manuscript text for references) detailing the functional characteristics of these proteins in species marked in Figure Click here for fileFunctional class codes, designations and associated keywords used by ARC computer program (see methods section) for functional classification of proteins into the respective functional class.Click here for fileAdobe Acrobat Document, contains the list of all SSPs analyzed in this work.Click here for file


Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing.Here we investigated intron dynamics in the serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.Multiple intron acquisitions were identified in  Spliceosomal introns are key attributes of most eukaryotic genes. Their origin is still unclear, though descent from group II self-splicing introns seems to be likely ,2. All cvice versa, was proposed to foster functional diversification of serpins [The serpins are a superfamily of proteins that cover a highly divergent spectrum of functions ,13. The  serpins ,15.serpin genes are often arranged in tandem arrays and they constitute a substantial fraction of mammalian genomes. During diversification of vertebrates the superfamily has undergone considerable expansion [Serpin genes of basal metazoans, in contrast, are not generally intron-rich, and their exon-intron structures are not conserved along the lineages leading to vertebrates. Sporadic investigations of various species revealed radically different intron patterns in serpin genes, indicating that, during diversification of eumetazoans, massive changes in gene architectures have occurred [serpin genes from various vertebrates  and Branchiostoma floridae (B. floridae), representing a group of extant cephalochordates (phylum: Chordata). Experimental approaches disclosed several genes and cDNAs coding for serpins in B. lanceolatum, further superfamily members were detected by mining the genome of the closely related B. floridae [serpin genes (L1–L3) with distinctly different intron patterns were observed . The L2 genes contain introns at positions ~86b, 151c, 223b, 283c and 339c. The L3 genes exhibit common introns at positions 73b, 125b, ~175c and 339c, however, there are also introns that are unique to individual group members . Another intron in Bflor_Spn10 is located outside the conserved serpin scaffold (gene-specific numbering of position: 29a). There are several additional serpin-like sequences in the B. floridae genome, but it is currently not discernible whether they represent intact genes (not shown); however, it is clear that serpin genes from lancelets and vertebrates differ largely with respect to their exon-intron organizations. In fact, just a single intron location (position 339c) is shared. Apparently, major changes affecting the exon-intron patterns of serpin genes have occurred since the cephalochordate/vertebrate split.We first studied floridae . Altogetn genes L–L3 with n genes L–L3 with serpins in vertebrates, we turned to lampreys, a group of basal, jawless fishes. Lampreys, in sharp contrast with lancelets, depict at least four of the six canonical groups of vertebrate serpins. A survey of cDNA and genomic sequences from Lampetra fluviatilis  and from Petromyzon marinus  revealed representatives of groups V2, V4 and V6 . Members of groups V3 and V5 were not identified.Having established lancelets as appropriate outgroup for evaluating evolution of L. fluviatilis and P. marinus , among other features, definitely reveals this protein as member of the serpin superfamily.Inspection of lamprey serpin sequences disclosed the presence of angiotensinogen and heparin cofactor II (HCII), two prominent members of group V2. All known angiotensinogen proteins depict a conserved decapeptide sequence close to the N-terminus that, after controlled enzymatic cleavage, gives rise to formation of peptides (angiotensin I-IV) involved in blood pressure regulation and other important physiological processes . ClearlyP. marinus (see below). We also recognized a lamprey serpin exhibiting the exon-intron pattern of group V6 . Characteristic features of all HCII sequences are the highly conserved Arg/Lys-rich helix D that is involved in GAG binding and the acidic N-terminal extension that mediates GAG accelerated thrombin inhibition ,26. Thesollagens ,27. A haollagens  and its serpin genes, the dynamics of their exon-intron patterns was analyzed. The list of species investigated included: man, chicken , the clawed frog , and several fishes including Danio rerio (D. rerio), Oryzias latipes (O. latipes), Gasterosteus aculeatus (G. aculeatus), Tetraodon nigroviridis (T. nigroviridis), and Takifugu rubripes (T. rubripes). Several serpin genes, though clearly members of one of the six canonical groups, were found to deflect from the standard organizations. Like their counterparts in lampreys, angiotensinogen genes of tetrapods and D. rerio exhibit the canonical gene architecture of group V2, the T. rubripes orthologue, however, contains two extra introns that split the exonic sequences at positions 77c and 233c, respectively . Though also found in O. latipes and G. aculeatus, this intron here is not considered any further, due to its location outside the serpin scaffold. Examination of lamprey HCII also revealed unique introns. The 83c intron (α1-antitrypsin numbering) is embedded in a well-conserved region; its origin, however, is difficult to evaluate. The others (correctly predicted?) map to the N-terminal extension (gene-specific numbering of positions: 38b and 118c).The  mammals ,30, chicSpn_94a, with a surplus intron at position 94a . The origin of these genes is unclear; the unique surplus intron suggests that they share a common ancestor , all of which are equipped with the standard set of introns , which probably arose as a consequence of genome duplication events in the stem lineage of ray-finned fishes [Dan_HSP47_1, as indicated by the conserved gene order . In G. aculeatus, a second intact HSP47 homologue, Gast_HSP47_2, was identified. Dan_HSP47_2 and Gast_HSP47_2 are orthologous to each other, since they share a set of flanking markers ; Gast_HSP47_2, in contrast, merely possesses the default introns. These findings suggest that the novel introns, which are restricted to HSP47_1 orthologues, were acquired by group V6 during evolution of ray-finned fishes after divergence of the D. rerio lineage, though an intron loss scenario cannot be excluded. Two intron gains or a single, coupled intron gain event are/is sufficient to explain the exon-intron patterns found in group V6; the intron removal scenario, in contrast, requires multiple intron loss events. If such losses had occurred, they must have affected several taxa, including lampreys, tetrapods and fishes. Moreover, this scenario also demands that the same two introns (positions 36b and 102c) were always deleted in parallel, while all other introns were unaffected. The parallel emergence of introns at positions 77c and 233c in angiotensinogen genes , the only member of group V5, is a potent thrombin inhibitor in the presence of heparin. Among other characteristic features, AT orthologues are easily discernible through a highly conserved sequence centering around helix D . Intron 262c is a standard attribute of group V1 that is believed to share a common ancestor with group V5 [AT genes of tetrapods. We were not able to identify this gene in P. marinus; further tracing of the 262c intron was therefore not possible.In contrast with several intron gains, we detected a single case of probable intron loss during evolution of vertebrate group V5 . We therserpins from groups V3 and V4, deviations from the standard structures were not observed (not shown). All genes from group V3 analyzed featured introns at positions ~86a-90a, 167a, 230a, 290b, 323a, 352a, and 380a. The intron pattern characteristic of group V4  was also conserved.In T. rubripes [Spn_94a orthologues cannot be aligned without introducing gaps , referred to as proto-splice sites [serpins of ray-finned fishes  were purchased from the Alfred-Wegener-Institut für Polar- und Meeresforschung, Helgoland, Germany. European river lampreys (L. fluviatilis) were obtained from the Bundesforschungsanstalt für Fischerei, Hamburg, Germany.Adult lancelets (The isolation of poly(A)-RNA and genomic DNA, the synthesis of cDNA, PCR amplification of serpin cDNA fragments using various sets of degenerate primers, and cloning of 5'- and 3'-cDNA ends followed published procedures ,51. DNA serpins from human, chicken [X. tropicalis [ D. rerio [ G. aculeatus [O. latipes [T. rubripes [T. nigroviridis [P. marinus [). Sequences from the B. floridae genome were gathered from the JGI genome browser . EST and cDNA data mining included searches in the NCBI trace archive  and in the UCSC genome browser [Genomic data for  chicken , X. tropopicalis , D. reriD. rerio , G. aculculeatus , O. lati latipes , T. rubrrubripes  and T. noviridis  were extoviridis , or in t marinus , from Pr browser , applyin1-antitrypsin as described [i.e. positions 33 to 394 of human α1-antitrypsin) were considered.All intron positions predicted by gene models were examined visually, corrected and amended manually, if necessary. Whenever cDNA or EST sequences were available, intron positions were checked by means of GENEWISE . Proteinescribed . All int)) and with RepBase Censor  [Sequences of non-canonical introns were searched for repetitive elements with the RepeatMasker package (version 3.2.6; (ensor   using dePhylogenetic analysis was performed using the Neighbor-Joining method  conducteHR designed the study, performed part of data analyses, and wrote the paper. AK accomplished data analyses. KK, YW, CB, MAF, NP and OK generated data and contributed to data evaluation.Mapping of intron positions to aligned serpin sequences. Figure depicting intron positions of serpin genes mapped onto the aligned amino acid sequences.Click here for fileserpin genes analyzed in this study and their accession numbersList of . Table listing accession numbers of genes, cDNAs and ESTs investigated in this study.Click here for fileSpn_94a genesChromosomal gene order reveals orthology of . Figure showing chromosomal synteny of Spn_94a genes.Click here for fileserpin genesSequences of non-canonical introns from vertebrate . Figure depicting the sequences of non-standard introns.Click here for file


Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups.Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost.U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost. In eukaryotes mature RNA is formed by the removal of introns from a primary transcript. Splicing is catalyzed by a multicomponent complex, the spliceosome . Two intU2 introns have characteristic properties at the 5' splice site (AG/GURAGU), 3' splice site (YAG/G) and branch site . There is also a pyrimidine rich region between the branch and 3' splice sites. Much of the specificity in the splicing reaction is accomplished by pairing with snRNAs. Thus, the 5' splice site pairs with U1 RNA and the branch site pairs with U2 RNA.ACU, where the underlined A is the branch point adenosine) are more conserved than their counterparts in U2 introns, whereas the 3' splice site is more variable. In addition, U12 introns lack a pyrimidine rich region. Whereas the vast majority of U2 introns have the dinucleotides GT and AG at their 5' and 3' ends, respectively, some U12 introns have the dinucleotides AT and AC in these positions [The U12 introns have consensus sequences that are different from U2 introns. The 5' splice site (/RTATCCTTT) as well branch site , Wormbase , JGI , from Broad Institute , from TIGR (Entamoeba histolytica 2004 version) and from TraceDB (Phakopsora pachyrhizi and Acanthamoeba castellanii).EST sequences were retrieved using NCBI Entrez. Genomic sequences were downloaded from WUSTL T. spiralis, a total of 25,268 EST sequences were retrieved using NCBI Entrez and used to query a database of T. spiralis genome sequences. The genome sequences contained 15544 contigs with a total of 115,634,429 nt. Only hits with sequence identity at least 98% and HSP length at least 35 nt were considered for further analysis.Introns were identifying from BLAST searches  where ESWhenever an EST matched to more than one genomic contig sequence we selected for further analysis the contig with the most extensive match to the EST sequence. As BLAST is often not able to unambiguously identify the exact location of the splice site, we considered all possible sites and the most probable one was identified by screening with position weight matrices (PWMs) as described below.http://katahdin.cshl.edu:9331/SpliceRack/index.cgi?database=spliceNew. For the 5' splice sites the PWM covers 13 positions where the first 3 positions are the 3' end of the exon, and for the 3' splice site the window has 17 positions when the last 3 positions are in the exon part. The branch site PWM has a length of 12 and corresponds to a location falling into the range of  upstream of the 3' splice site of the intron. PWMs were available for C. elegans, D. melanogaster, A. thaliana, H. sapiens and M. musculus but only the first three of these were used for the scoring of T. spiralis sequences as these PWMs are more appropriate to nematodes as well as to the other species examined here [PWMs for 5', 3' and branch sites of the GT-AG U12, GT-AG U2, AT-AC U12 and GC-AG U2 type of introns from five different species were obtained from the Splicerack database ned here .C. elegans, D. melanogaster and A. thaliana to score all possible intron locations as inferred by BLAST. Each possible position of the intron therefore generated three different sets of 5' and 3' scores. We finally selected the intron position where both 5' and 3' scores were the greatest or, in cases where this was not applicable, where the sum of both scores was the greatest.We used 5' and 3' matrices from http://katahdin.cshl.edu:9331/SpliceRack/ and log-odd matrices were then obtained. In the case of the branch site scoring we used only one PWM, as the PWMs of the five different species were identical. For identifying the most likely branch site every segment of length 12 within the range  relative to the 3' splice site was scored with U12 GT-AG and AT-AC matrices.For identification of U12 introns using the method of Sheth et al , pseudocT. spiralis classified with the method from Sheth et al. we used the branch site which achieved the best score with a GT-AG U12 PWM. All the 14480 branch sites obtained in this way were used to construct a frequency matrix.For prediction of U12 introns using the Burge et al method  we used Sss 5'and Sbps by subtracting the sample mean and dividing by the standard deviation.The scores for each splice or branch site were then computed as described in Burge et al. . The 5' After scoring all introns we tried to separate the putative U12 dependent introns from the U2 dependent ones with respect to their normalized scores. The lower thresholds for U12 type introns were empirically defined with respect to the minimum values of a reference set of minor introns which were used by Burge et al . The tesWe also analyzed all predicted introns with respect to previously known consensus features of U12 introns as referred to by Russell et al . In addiC. elegans that are homologous to the U12 introns in T. spiralis the C. elegans genome sequence  was retrieved from Wormbase ftp://ftp.wormbase.org/pub/wormbase/genomes/c_elegans/sequences/dna/.For identifying introns in C. elegans protein database  with 23962 proteins was also downloaded from Wormbase ftp://ftp.wormbase.org/pub/wormbase/genomes/c_elegans/sequences/protein/.A C. elegans proteins corresponding to the T. spiralis U12 intron genes. Genomic positions of the corresponding gene in C. elegans were then inferred using TBLASTN.BLASTX was first used to identify We have previously examined the phylogenetic distribution of U2 and U12 spliceosomal components . The resC. reinhardtii, which has a distribution of introns lengths with a mode value which is approximately 195 nt, consistent with previous observations [Introns were identified by matching of ESTs to genomic sequences using BLAST . The sizrvations .P. sojae. In order to discriminate between U12 and U2 type introns we used a cutoff based on a reference set of U12 introns as used in Burge et al [P. sojae three different introns, one of the type GT-AG and two of the type AT-AC, fulfilled these criteria.In order to discriminate between U2 and U12 introns we used methods described by Burge et al , Zhu andge et al . Thus, fge et al . The ploT. spiralis. Molecular phylogenetic analysis places Trichinella close to the root in the nematode tree, i.e. more deeply branching than species such as C. elegans of the Rhabditida branch [We examined in more detail the prediction of U12-type introns in the nematode a branch -16 whichT. spiralis we identified a total of 15402 introns. Out of these, 14866 were of type GT-AG, 218 were of type GC-AG and 8 were AT-AC type introns. In addition, 315 introns were identified with non-canonical terminal dinucleotides.In Using the method of Sheth et al , U12 GT-T. spiralis U12 introns we examined the homologous genes in C. elegans where U12 introns are believed to be absent. Of the 16 EST sequences that we identified using the Sheth et al method as being associated with U12 type introns, 3 had no matches to entries in protein databases and could not be associated with a C. elegans gene. Another 6 ESTs matched only partially to the homologous C. elegans gene and for this reason we were not able to examine the fate of the homologous T. spiralis intron. For the remaining ESTs we were able to compare the T. spiralis U12 intron to the corresponding C. elegans intron. Four of these had identical splice sites in the two species as shown in Table T. spiralis introns were changed from U12 to U2 type in C. elegans.In order to study the fate of T. spiralis AT-AC-type U12 introns the intron was completely lost in C. elegans  but as this is the only U12 intron predicted in this species and because we have failed to identify any U12 snRNAs in this species the evidence of U12 splicing is so far very poor.There is also a weak candidate for a U12 AT-AG intron in R. oryzae (S13), P. blakesleeanus (S13), P. sojae (L31), P. infestans (L13) and P. polycephalum (L4).Although U12 introns are very rare they occur in ribosomal protein genes in five of the species examined here, R. oryzae there are at least three non-identical versions of the S13 gene. These genes encode nearly identical proteins  of T. spiralis, E. histolytica, A. castellanii, P. tricornutum, M. brevicollis, and C. reinhardtii. Lower panel: Mean intron lengths for all species with U12 introns considered in this work.Click here for fileSequences of introns. Sequences of introns referred to in Tables R. oryzae ribosomal protein S13 genes and introns.Click here for file