An analysis of human-chimpanzee intron divergence shows strong correlations between intron length and divergence and GC-content. Introns, which constitute the largest fraction of eukaryotic genes and which had been considered to be neutral sequences, are increasingly acknowledged as having important functions. Several studies have investigated levels of evolutionary constraint along introns and across classes of introns of different length and location within genes. However, thus far these studies have yielded contradictory results.i) can be interpreted as the footprint of different intensities and directions of the pressures of natural selection. Our main findings are as follows: there was a strong positive correlation between intron length and divergence; there was a strong negative correlation between intron length and GC content; and divergence rates vary along introns and depending on their ordinal position within genes .We present the first analysis of human-chimpanzee intron divergence, in which differences in the number of substitutions per intronic site (KWe show that the higher divergence of first introns is related to their larger size. Also, the lower divergence of short introns suggests that they may harbor a relatively greater proportion of regulatory elements than long introns. Moreover, our results are consistent with the presence of functionally relevant sequences near the 5' and 3' ends of introns. Finally, our findings suggest that other parts of introns may also be under selective constraints. Introns are neither neutrally evolving sequences nor junk DNA, as they were once considered to be. Increasing amounts of evidence show that they harbor a variety of untranslated RNAs, including microRNAs, small nucleolar RNAs, and guide RNAs for RNA editing . IntronsDrosophila data. Marais and coworkers ; GC short = 0.522, GC long = 0.425 [P < 10-5]). This therefore confirms an intrinsic length effect.Finally, and as an additional way of ensuring that the higher divergence of first introns was not due to their higher average size, we separated them into 'long' and 'short' categories according to their median size. In this way, and only for this analysis, long first introns were those above 2,020 nucleotides and short first introns were those equal to or below this length. When comparing the 2,921 long and 2,920 short first introns classified according to this criterion, we observed that short first introns were significantly more conserved and significantly richer in GC content than were long first introns, following exactly the same trends as described above for nonfirst introns .To test this hypothesis, we divided all introns into three fragments: the first 150 nucleotides from the 5' end, the last 150 nucleotides from the 3' end, and the remaining central part. We also split first introns into three fragments: the first 1,000 nucleotides at the 5' end, the last 150 nucleotides at the 3' end, and the remaining part. Because all the comparisons on these fragments were performed on the unmasked dataset , the raw values of Ki between long and short introns  diverge much less than the central part of the introns , there is apparently almost no difference between first and nonfirst introns , the correlation being observed in both short and long introns . We also confirm that recombination positively correlates with GC content . Finally, there is no overall correlation between intron length and recombination . When performed within each class of size (short and long), the correlations between recombination and length are significant, but their signs are different. That is, recombination rate does not have a linear relationship with length; it is negatively correlated with length for short introns , but positively correlated - albeit weakly - with length for long introns .As expected, divergence and recombination are significantly correlated in the masked dataset .Recombination rates are higher in first and in short introns Table . That isSome of the analyses presented above might have been biased by factors that are known to affect rates of divergence and/or intron length. For example, if genes in the X chromosome had shorter and less divergent introns, then this could artefactually give rise to some of the patterns we detected. To ensure that this is not the case, we repeated our main tests after controlling for these factors . This analysis revealed a few biases, some of which are conservative . For example, introns of chromosome 19, which are highly divergent, tend to be shorter than introns elsewhere in the genome. Also, introns located in telomeres and centromeres are shorter than introns outside these regions but, in contrast, divergence rates go in opposite directions, being higher in telomeres and lower in centromeres . At any rate, our results remain the same after removing genes located in these regions, meaning that introns of different classes are equally affected by these factors. This indicates that the differences in divergence between short and long introns that we reported above are not due to a higher proportion of certain intron classes in given chromosomes or genomic regions.The overall picture that emerges from our findings is that, as revealed by human and chimpanzee divergence, different introns and different parts of introns may have been subjected to different evolutionary forces, among which is natural selection. Our first series of results are related to intron length and nucleotide composition, showing a negative correlation between intron size and GC content. A steep decrease in GC content with intron length had previously been reported in the human genome ; in contAnother series of results involves patterns of divergence. GC content is positively correlated with intron divergence. However, as mentioned above, intronic regulatory sequences are expected to be enriched in GC. Therefore, the higher divergence of GC-rich introns may seem paradoxical, because we would expect GC-rich regulatory motifs to be selectively constrained. However, the positive correlation between intron size and divergence that we detected suggests that the density of conserved sequences is lower in long introns. This may explain why long introns are, simultaneously, GC poorer and more divergent. A class of constrained sequences that could account for this effect are splicing control sites, located close to exon-intron boundaries. However, after removing the outmost 150 nucleotides at both ends of all introns, divergence is still lower in short introns, so their relative higher density of splicing control sites cannot explain the positive correlation between intron size and divergence.Thus, other factors need to be invoked to explain the lower divergence of short introns. First of all, it is possible that other classes of regulatory elements, in particular not GC-based motifs, that we did not take into account are distributed all over the introns, and are not only located in the 150 nucleotides close to intron-exon boundaries. This would be consistent with previous experimental work describing some such elements ,26. If tAs mentioned above, CpG islands are associated with gene expression regulation. They are also constitutively hypomethylated, and lack the mutagenic effect seen in their methylated CpG counterparts . We founA potential factor directly linking intron length and divergence is recombination. In agreement with previous studies ,29, we fDrosophila reported by Haddrill and coworkers [Another hypothesis to explain the relationship between size and divergence in our data is that the class of short introns is enriched in introns from housekeeping genes, because introns are substantially shorter  and GC roworkers  also supMany studies have shown that selectively constrained noncoding DNA and intron-associated control elements are more frequently found in first introns than other introns -11,20, eDrosophila. Apart from the fact that the difference in distribution of intron size between Drosophila and human/chimpanzee makes it difficult to compare the two sets of findings , the discrepancy must be somehow related to the fact that forces acting on nucleotide composition are very different in different lineages. Indeed, Aerts et al. [Thus far we have tried to describe the patterns of intron divergence between humans and chimpanzees, and to propose hypotheses regarding the forces that act on intron evolution, comparing our results to findings from other species. In many cases, these results are contradictory to ours. An example of such contradiction is the positive correlation between GC content and divergence that we report here, which is in conrast to the results reported by Haddrill and coworkers  on Drosos et al.  detecteds et al. . These as et al.  showed tDrosophila species, Presgraves suggested that intron length evolution is affected by chromosome-specific and lineage-specific forces. Using Drosophila yakuba as an outgroup, he showed that in D. melanogaster X-linked introns have slightly increased in size, whereas autosomal ones have slightly decreased in size. In contrast, in D. simulans both autosomes and the X chromosome have decreased in size since their divergence from D. yakuba. Presgraves' conclusion was that this observation could not easily be explained by a single general model of intron length. These examples highlight the difficulties in comparing modes of intron evolution between distant groups of species. If such different trends can be observed in sister species, then it is only to be expected that results between more distant species are even more dissimilar. In the data analyzed here, we found X chromosome introns to be shorter and more divergent than autosomal introns. Comparing pairs of paralogous introns in the human genome, Cardazzo and coworkers [A further example of contradictory data coming from different species is reported by Presgraves . In thatoworkers  also fouThe importance of functional elements in noncoding sequences of the genome is becoming increasingly acknowledged. Conserved noncoding sequences have been shown to be selectively constrained . Among tWe showed that, even after correcting for some potentially confounding factors, long introns have higher divergence between humans and chimpanzees than short introns, whereas GC content and length are negatively correlated. Another pattern is that divergence rates are higher in first introns than in nonfirst introns. The higher divergence of first introns is partly related to their longer length. This may reflect a high proportion of functional elements distributed along their sequence, separated by unconstrained regions. Finally, we also show that the 5' and 3' ends of introns, which are known to contain regulatory elements and splicing control sites, have lower divergence than the remaining parts of introns. The best explanation for all these patterns is that purifying selection has a strong effect on shaping intron sequence evolution. It is also possible that divergence patterns and rates between human and chimpanzee introns have been affected by positive selection. To follow up this work, the next step would be to determine whether this is true and, if so, to identify which are the introns that may have undergone positive selection.We generated a first dataset, composed of sequences obtained from the RefSeq database . The seqTo avoid biases introduced by misalignment, every individual intron was aligned with the local alignment tool BLAST 2 Sequences , which uTo perform some analyses and comparisons (such as the exact determination of intron-exon boundaries), a second, nonmasked dataset was obtained by gathering the unmasked sequences of any intron that had passed the filtering process above.i, using the distmat application from the EMBOSS package [i values, most likely due to false orthology assignments. The dataset was therefore filtered by removing all Ki values three standard deviations above the mean. The filter was applied to the masked dataset and any intron with Ki more than 3.025% was removed from both the masked and the unmasked datasets. This implies that some introns in the unmasked dataset may have higher divergence than this 3.025% limit if their masked version had a divergence below this threshold.For every intron, human-chimpanzee divergence was measured applying the Jukes-Cantor correction to the number of substitutions per intronic site, K package . AlthougAfter this process, a total of 51,674 nonredundant introns was left, for which we know their order in the gene, length, GC content, and level of divergence between humans and chimpanzees. Their size in humans varies from 49 to 955,099 nucleotides and from 49 to 592,440 nucleotides after removing the masked repetitive elements. In chimpanzees, intron size varies from 49 to 974,461 nucleotides, and from 49 to 566,414 nucleotides after removing masked repetitive elements.Ancestral intronic GC content was also estimated for every gene as the average of the current human and chimpanzee GC content. The positions of CpG islands were downloaded from the CpG island UCSC annotation database . The ovei for each segment independently , all intron fragments with Ki above 3.025 were again filtered out to make the new file similar to the one of the global sample. For the same reason, all the fragments of the central part  that were less than 49 nucleotides long  were removed from the analysis.To study the divergence and GC content measurements in fragments of introns that are of particular interest , a set of PERL scripts was written to cut up introns into fragments and measure their divergence and GC content. Because we were interested in these regions and because most known regulatory elements are composed of repetitive sequences, we performed this part of the study on the unmasked dataset. Also, to make sure that we were not losing regulatory elements, we only kept for analysis introns for which the alignment started between nucleotides 1 to 15 from the exon-intron boundary. Introns for which alignment started beyond that boundary were removed from this part of the analysis. After computation of KIntrons were classified into two categories according to size. We followed the criteria used by Haddrill and coworkers , who defTo study factors that are known to affect divergence rates, introns were further classified into five categories according to their genomic location: within the sex chromosomes, human chromosome 19, telomeres (10 Mb from both ends of the chromosome in either species), centromeres (5 Mb from the centromeres in either species), and/or segmental duplications .P values obtained after 1,000 permutations were above 0.01). P values are the proportion of times that the difference in the averages of two categories in a permuted dataset was equal or larger than the observed difference. Correlation tests (Pearson's product-moment correlations) were performed with R, version 1.9.0 [Divergence, length, and GC content were compared among introns belonging to different ordinal categories  or different classes of length  by means of pairwise permutation tests .The following additional data are available with the online version of this paper. Additional data file i and mean lengths for the main known factors affecting divergence rates.A table listing the mean KClick here for filei for short and long introns.A figure representing the mean GC content and mean KClick here for fileDrosophila.Comparative distribution of intron length between human and Click here for fileHuman RefSeq of the introns included in the analysis, with the main factors and variables we study here: Ki, GC, first, and length (lengths are given for the sequences after masking).Click here for file


Martin & Koonin recently proposed that the eukaryote nucleus evolved as a quality control mechanism to prevent ribosome readthrough into introns. In their scenario, the bacterial ancestor of mitochondria was resident in an archaeal cell, and group II introns (carried by the fledgling mitochondrion) inserted into coding regions in the archaeal host genome. They suggest that if transcription and translation were coupled, and because splicing is expected to have been slower than translation, the effect of insertion would have been ribosome readthrough into introns, resulting in production of aberrant proteins. The emergence of the nuclear compartment would thus have served to separate transcription and splicing from translation, thereby alleviating this problem. In this article, I argue that Martin & Koonin's model is not compatible with current knowledge. The model requires that group II introns would spread aggressively through an archaeal genome. It is well known that selfish elements can spread through an outbreeding sexual population despite a substantial fitness cost to the host. The same is not true for asexual lineages however, where both theory and observation argue that such elements will be under pressure to reduce proliferation, and may be lost completely. The recent introduction of group II introns into archaea by horizontal transfer provides a natural test case with which to evaluate Martin & Koonin's model. The distribution and behaviour of these introns fits prior theoretical expectations, not the scenario of aggressive proliferation advocated by Martin & Koonin. I therefore conclude that the mitochondrial seed hypothesis for the origin of eukaryote introns, on which their model is based, better explains the early expansion of introns in eukaryotes. The mitochondrial seed hypothesis has the capacity to separate the origin of eukaryotes from the origin of introns, leaving open the possibility that the cell that engulfed the ancestor of mitochondria was a sexually outcrossing eukaryote cell. In two papers published this year . Like tThe second premise is that group II introns would have proliferated in the host genome. Both papers ,2 argue A further key feature of this model is that the source of group II introns invading any host genome is the mitochondrion; spread of group II introns between individuals (i.e. intergenomic spread) is not invoked. The model permits any host genome within the population to receive group II introns from their mitochondria (endosymbiont gene transfer), so transfer is iterative and ongoing. However, the main source of intron proliferation is spread of introns already integrated into the host genome (intragenomic spread). According to the model, the small effective population size of this new 'prekaryotic chimera' means it is not possible to eliminate group II intron proliferation by purifying selection; drift will dominate, and individuals in which intron proliferation is occurring will be fixed, in spite of their reduced fitness.That genetic elements can proliferate at the expense of the fitness of the host in which they are found is now widely accepted. However, this is only predicted to occur under certain circumstances. A model published by Hickey , illustrf) of a transposable element in a population as:What follows is a brief summary of the relevant aspects of Hickey's model, which I will relate back to the Koonin/Martin model. The first point concerns the nature of intragenomic spread. Hickey defines the average copy number per cell .where f can increase via an increase in either a or b. If f increases due to an increase in a, this is intragenomic spread; the element is increasing in frequency only because those genomes containing copies now contain even higher numbers of the element. In this case, which describes an asexual population, elements do not spread to new genomes. Consequently, those individuals with harmful elements (as per Koonin/Martin) will be at a selective disadvantage relative to those without, and element-carrying individuals are not predicted to spread within the population.b is not irrelevant to the Koonin/Martin model. While the model does not include intergenomic spread between individuals, endosymbiont gene transfer of group II introns enables b to increase in the absence of intergenomic spread. Therefore, it is possible for group II introns to be fixed in the short term, by drift or by high rates of endosymbiont gene transfer.The parameter This is unsurprising in that we know that group II introns exist in both bacterial and archaeal lineages, and are able to spread via horizontal gene transfer. However, what is not observed is massive proliferation within these asexual lineages, even in the presence of horizontal gene transfer.Methanosarcina acetovorans and Methanosarcina mazei [Methanosarcina spp. and that these species are sufficiently 'garden-variety', one would expect a similar rate of proliferation as in the Koonin/Martin model. However, none of the group II introns are inserted in archaeal open-reading frames, hence do not result in ribosome readthrough. Instead, these genes have a tendency to insert into the reverse transcriptase genes encoded by other group II introns, generating nested introns. I have no idea as to the effective population size of these two archaea, but that these elements have neither gone berserk , nor inserted into archaeal protein-coding genes does not serve to strengthen the model presented by Koonin.A case in point is the recent discovery of group II introns in two species of archaea, na mazei ,8; in boMethanosarcina spp. have not inserted into coding regions is probably consistent with this. Likewise, assuming some cost associated with element presence, individuals that completely lose the elements will be at an advantage [Rather, this is what one may expect for selfish elements in asexual lineages, even with horizontal gene transfer. Upon insertion, such elements will end up in linkage disequilibrium with the other genes in the genome (there is no meiotic recombination and no outbreeding). Consequently, in the long term, even if drift fixes the presence of an element that imparts a cost to the host, elements that evolve to be less harmful will be at a selective advantage; the fitness of the host and the element are identical. Thus, rather than spreading wildly, the result will be more cautious mechanisms of maintenance or spread. That the group II introns from dvantage ,9.Giardia lamblia, which is not known to be sexual [If an element does proliferate wildly in an asexual population, the cost to all individuals may become so high as to lead to population extinction. Indeed, it has recently been argued that element overload is one probable cause of extinction of obligately asexual lineages that have evolved from sexual lineages . Survivae sexual , and whie sexual ,14.The bottom line is that even if endosymbiont transfer can ensure an increase in the proportion of individuals carrying one or more genomic copies of an element, there will still be a selective advantage for attenuation, even if complete loss does not occur.Assuming introns-late, these elements have must have proliferated at some stage during early eukaryote evolution. Again, Hickey's paper explains under which circumstances this can occur, even when the element has a deleterious effect on the fitness of the host. In an attempt to avoid reproducing the entire paper here, the key point is that this will happen in a diploid, sexual, outbreeding population.Individuals carrying the element, despite having a lower fitness relative to element-free individuals, will nevertheless dominate the population. Considering just a single locus, when the frequency of the element in the population is close to zero, the element will double in frequency within the population because most zygotes receive a single copy (they begin as heterozygotes) but pass on twice as many copies per gamete (transposition makes them homozygous for the element). If they are only half as fit as element-free individuals , the number of copies of the element that are passed on is the same for a normal nontransposable gene. Consequently, provided the cost to the host is < 0.5, and with high efficiency of transposition, the element will spread. Hickey's model illustrates that, as the element increases in the population, selection against individuals can rise significantly above 0.5, yet this will not impede further spread.What this is telling us then is that meiotic sex and outbreeding are prerequisites for introns to have proliferated massively under introns-late. For Koonin's theory to work over a long enough time scale for several complex systems of quality control to emerge, I would argue that he should at least have invoked the emergence of facultative meiotic sex. The problem with this is that sex, with its two-fold reproductive cost, must be invoked under a scenario where there is a population of primed selfish elements 'waiting' to spread. While the level of sexuality can be increased in a facultatively sexual population, this is not so for an asexual population . As the Invoking a garden-variety  archaeon as Koonin does serves to weaken the introns-late hypothesis because it eliminates the possibility of the mitochondrion entering an early eukaryote stem lineage that had already evolved meiosis  – all 'archezoa' either still have, or have lost, this organelle . TherefoOne has to accept the necessity of stem-lineage eukaryote ancestors irrespective of the absence of extant primitively amitochondriate eukaryotes. By stem lineage I do not mean the extant eukaryotes that were formerly designated archezoa. I mean lineages of eukaryotes that have gone extinct and which diverged from the lineage leading to the Last Eukaryotic Common Ancestor  subsequent to the mitochondrion clearly involve eukaryotes , and modMethanosarcina have not suffered from massive intron expansion seems to confirm this suspicion.In summary, I am not convinced that the host in the endosymbiosis leading to modern eukaryotes was an archaeon. This should be detectable phylogenetically, picking out a specific group of archaea as the closest relatives of eukaryotes in exactly the same way as this is possible for the mitochondrion. Second, no archaea have been identified which carry endosymbiont bacteria, so accepting Koonin's assumption would require that all extant archaea have subsequently lost this capacity. Nor am I convinced that the transfer of group II introns into an archaeal host from its bacterial endosymbiont would have led to massive expansion of group II elements. This does not fit with our current knowledge of selfish element spread under an asexual reproductive mode. That modern methanogenic archaea of the genus Importantly, even if eukaryotes had evolved from archaea, the model still runs counter to a considerable body of knowledge on selfish element proliferation. By invoking a sexual, eukaryotic host (as a later step in evolution from archaea), the spread of introns in eukaryotes can better be accounted for under our current understanding of selfish element spread in sexual populations. Without the conditions created by intron spread the selection pressure to drive the emergence of the eukaryotic nucleus and features for quality control must come from some other source.This comment article began as a referee report I wrote when reviewing Koonin . Dr. Koo


Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera).The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 gene was sequenced from the sponge Tetilla sp. (Spirophorida). This fragment suggests the presence of a 1143 bp intron. Similar to all the cnidarian mitochondrial introns, the putative intron has group I intron characteristics. The intron is present in the cox1 gene and encodes a putative homing endonuclease. In order to establish the distribution of this intron in sponges, the cox1 gene was sequenced from several representatives of the demosponge diversity. The intron was found only in the sponge order Spirophorida. A phylogenetic analysis of the COI protein sequence and of the intron open reading frame suggests that the intron may have been transmitted horizontally from a fungus donor.A 2,349 bp fragment of the mitochondrial Little is known about sponge-associated fungi, although in the last few years the latter have been frequently isolated from sponges. We suggest that the horizontal gene transfer of a mitochondrial intron was facilitated by a symbiotic relationship between fungus and sponge. Ecological relationships are known to have implications at the genomic level. Here, an ecological relationship between sponge and fungus is suggested based on the genomic analysis. Podospora anserina (accession number: NC_001329) contains 33 introns located in nine different genes, including 15 introns in the cox1 gene, which encode the subunit 1 of cytochrome c oxidase (COI). However, none of these introns are obligatory and some fungi do not include introns in their mitochondrial genome . In Metazoa, mitochondrial introns have only been described in Cnidaria of the subclass Zoantharia  alSequences were aligned using ProbCons  with thrcox1 genes. MI initiated the study and was responsible for the collection and identification of the sponges studied. DH coordinated the study, performed the molecular analysis and wrote the paper. All authors contributed to the writing and the revision of the manuscript.CR and IG extracted the sponge DNAs and sequenced the cox1 geneLocation of introns in the . DNA sequence alignment (in FASTA format) used to create Figure Click here for fileSequencing primers. Name, sequence and direction of the primers used to sequence the cox1 gene.Click here for fileCOI protein alignment. Protein sequence alignment (in FASTA format) used to reconstruct the phylogenetic tree present in Figure Click here for fileLAGLIDADG protein alignment. Protein sequence alignment (in FASTA format) used to reconstruct the phylogenetic tree present in Figure Click here for file


The pckA regulatory region has target sequences for many other temperate phage repressors, and thus we suggest that down-regulation of the host pckA pathway increases lysogen fitness by lowering the growth rate of lysogens in energy-poor environments, perhaps as an adaptive response to the host predation system or as an aspect of lysogeny that must be offset by down-regulating pckA.Temperate bacteriophage parasitize their host by integrating into the host genome where they provide additional genetic information that confers higher fitness on the host bacterium by protecting it against invasion by other bacteriophage, by increasing serum resistance, and by coding for toxins and adhesion factors that help the parasitized bacterium invade or evade its host. Here we ask if a temperate phage can also regulate host genes. We find several different host functions that are down-regulated in lysogens. The  Lysogenic bacteriophage such as lambda integrate into their host genome, but do they regulate specific host genes? This study shows that they do, thereby increasing the fitness of the lysogen. A central question in the biology of host–parasite interactions is how a balance between the costs and benefits to both is achieved. If the burden to the host is too high, the parasite will go extinct, and for this reason it is often postulated that parasites confer some benefit upon their hosts, thereby arriving at an equilibrium in the competition between parasite-free and infected host populations.Escherichia coli; the β toxin produced by Corynebacterium diptheriae, the causative agent of diphtheria; endotoxin production by Clostridium botulinum; staphylococcal endotoxins; and cholera toxin produced by Vibrio cholerae, to name a few. Then too, prophage often code for functions that allow the lysogen to successfully colonize the animal host 1/2, a measure of the brightness of the fluorescent signal; Columns I, J, and K: Statistic based on the pooled data from 48 individual measurements using 12 different samples taken from exponentially growing cells. The E. coli MG1655 primer set from Sigma-Genosys  was designed to amplify 4,539 open reading frames and is therefore very heterogeneous in target length. The λ set of 73 primer pairs contained some duplicate targets, for example, LMD__rexA__L and LMD__rexA in which the L set was approximately 300 nucleotides long and the other set contained approximately 70 nucleotides. The LMD primer set is available from the corresponding author .Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genes and gene products discussed in this paper are b0557 (GeneID 948980), b2002 (GeneID 946514), cI (GeneID 1238683), E (GeneID 2703482), ea22 (GeneID 2703469), ea47 (GeneID 2703536), hmpA (GeneID 947018),int (GeneID 2703470),kil (GeneID 2703471), nin221 (GeneID 2703476), orf-64 (GeneID 2703478),pabC (GeneID 946647), pckA (GeneID 945667), rexA (GeneID 2703537), rexB (GeneID 2703517), rlpB (GeneID 946257), xis (GeneID 2703504), yjgQ (GeneID 1037318), and λ genes (NC 001416). The GenBank accession number for the E. coli MG1655 primer set is U00096.The GenBank (


The specific and covalent addition of ubiquitin to proteins, known as ubiquitination, is a eukaryotic-specific modification central to many cellular processes, such as cell cycle progression, transcriptional regulation, and hormone signaling. Polyubiquitination is a signal for the 26S proteasome to destroy earmarked proteins, but depending on the polyubiquitin chain topology, it can also result in new protein properties. Both ubiquitin-orchestrated protein degradation and modification have also been shown to be essential for the host's immune response to pathogens. Many animal and plant pathogenic bacteria utilize type III and/or type IV secretion systems to inject effector proteins into host cells, where they subvert host signaling cascades as part of their infection strategy. Recent progress in the determination of effector function has taught us that playing with the host's ubiquitination system seems a general tactic among bacteria. Here, we discuss how bacteria exploit this system to control the timing of their effectors' action by programming them for degradation, to block specific intermediates in mammalian or plant innate immunity, or to target host proteins for degradation by mimicking specific ubiquitin/proteasome system components. In addition to analyzing the effectors that have been described in the literature, we screened publicly available bacterial genomes for mimicry of ubiquitin proteasome system subunits and detected several new putative effectors. Our understanding of the intimate interplay between pathogens and their host's ubiquitin proteasome system is just beginning. This exciting research field will aid in better understanding this interplay, and may also provide new insights into eukaryotic ubiquitination processes. Ubiquitination is a fundamental post-translational protein modification for all eukaryotic organisms. It controls several critical aspects of cell metabolism, such as cell cycle progression, transcriptional regulation, signal transduction or recognition, and resistance to pathogens –3. UbiquMany Gram-negative pathogenic bacteria of both animals and plants have evolved type III and/or type IV secretion systems (T3/4SSs) as an essential virulence determinant. These secretion systems are large protein complexes spanning the bacterial envelope that are dedicated to the transfer of protein or DNA substrates into target cells to subvert host defense and other signaling cascades for the benefit of the invading pathogen. We refer the reader to some excellent reviews on these topics –17. In tIn this review we focus on the growing number of T3/4SS effectors from both intracellular and extracellular plant and animal bacterial pathogens that specifically exploit their host ubiquitin proteasome system (UPS) . We wantSalmonella enterica serovar Typhimurium (S. typhimurium) T3SS effectors. This facultative intracellular bacterium is an important enteric pathogen of humans, causing gastrointestinal inflammation. Two T3SSs, encoded by the Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2), are essential for its pathogenicity and are used during different stages of infection for entry into intestinal cells and subsequent replication of the intracellular bacteria, respectively -DNA), resulting in genetic colonization of the host [Arabidopsis homologues of the yeast SKP1 protein, ASK1 and ASK2, were isolated as interactors of VirF [Arabidopsis protein VIP1 was identified as an interactor of VirE2 [A. tumefaciens infection as a molecular adaptor between VirE2 and karyopherin-α that results in nuclear uptake of the T-complex [Arabidopsis proteins have been identified as putative targets of VirF . Among these are proteins that have been shown to be involved in host defense–related processes. Although the interaction of some of these proteins has been confirmed in vitro, the relevance of these interactions during infection and their VirF-mediated degradation remains to be confirmed. As described above, a function in disarming host proteins involved in defense against bacterial attack and suppression of the immune response seems to be a general mode of action for effectors of both mammalian and plant pathogens.the host –95. VirFthe host ,97. VirFthe host . F-box pthe host . The Ara of VirF . The F-b of VirF . VirE2 i of VirF ,102, whe of VirF ,104 and  of VirF . VirE2 c of VirF , but the of VirF ,107, whiof VirE2 . This pr-complex . Recentl-complex . A host-Ralstonia solanacearum uses a T3SS to promote “bacterial wilt” on a variety of plant hosts [Arabidopsis SKP1-like proteins (ASKs). Like A. tumefaciens VirF, GALAs interact with ASK1 and ASK2, but also interact with other ASKs, in a manner that is reminiscent of plant FBPs [R. solanacearum on Arabidopsis or tomato [GALA genes is significantly less virulent on tomato and Arabidopsis [Medicago truncatula, another host plant, a single mutant for the GALA7 gene appears dramatically affected in its virulence. The virulence capacity of this single mutant is restored by complementation with a full length GALA7 construct, but not by a GALA7 gene construct deleted of its F-box domain. These results support a model, similar to VirF, in which specific GALAs (GALA7 on Medicago truncatula) and combinations of GALAs could form bacterium/plant composite SCF-type E3 Ub ligases in specific host cells, possibly to ubiquitinate and subsequently degrade mediator(s) of plant defenses.The plant pathogen nt hosts –112. Amont hosts –112 is ant hosts . Indeed,nt hosts . We furtant FBPs ,115. Patbidopsis  suggestsA. tumefaciens and recently R. solanacearum were the first prokaryotes shown to harbor proteins with an F-box that is essential for virulence [Mesorhizobium loti was also demonstrated to be transported into plant cells in a heterologous translocation assay by the A. tumefaciens T4SS [Legionella pneumophila strains Paris and Lens enabled the annotators to identify three genes that likely encode FBPs, and one gene that encodes a protein with two U-box domains [http://www.ebi.ac.uk/interpro, data release 13.0). We searched for the eukaryotic-specific putative E3 Ub ligase U-box (IPR003613) and F-box (IPR001810) motifs. In addition to the L. pneumophila U-box protein Lpp2887 that was recently annotated [Candidatus Protochlamydia amoebophila UWE25, an obligate endosymbiont of free-living amoebae [A. tumefaciens VirF protein [R. solanacearum GALA proteins [Mesorhizobium loti msi061 protein [L. pneumophila, however, our analysis found not only the three already annotated FBPs [Coxiella burnetii, the agent of Q-fever [L. pneumophila, also contains three genes encoding putative FBPs  target specific signaling intermediates involved in mammal or plant innate immunity, as performed by Yersinia sp. YopJ/P, or (iii) mimic specific host-like UPS components, illustrated by the bacterial FBPs.A number of animal and plant pathogenic bacteria have evolved type III and type IV effectors that, once translocated into the host cell, have the capacity to interfere directly with ubiquitin signaling, a mechanism fundamental to the eukaryotic host cell. These bacteria have developed different strategies to exploit the host cell ubiquitin/proteasome system to their advantage to (i) control the timing of action of their virulence effectors, as exemplified by the The variety of examples presented in this review illustrates the effectiveness of pathogens in interfering with host Ub signaling pathways. It also illustrates that each bacterium has developed a different mode of interference with the UPS by its effectors, depending on its infection strategy. Some bacteria suppress their host's immune response , wheras others control the host cell actin cytoskeleton for bacterial internalization. This exciting area of research is advancing at high speed; future research will certainly result in the discovery of more effectors that interfere with the UPS and the identification of specific host targets and the host functions affected. Such discoveries will supplement research on other (eukaryotic and prokaryotic) pathogens and on mechanisms other than T3/4SS that bacteria have evolved to interfere with the host's UPS. It will undoubtedly result in a better understanding of this intimate host–pathogen interaction, as well as provide new insights in eukaryotic ubiquitination processes. In addition, it may also form the basis for the development of a specific new class of antimicrobials.


To do so, we have developed a new open-source software library called CGL (for “Comparative Genomics Library”). Our results demonstrate that change in intron–exon structure is gradual, clock-like, and largely independent of coding-sequence evolution. This means that genome annotations can be used in new ways to inform, corroborate, and test conclusions drawn from comparative genomics analyses that are based upon protein and nucleotide sequence similarities.We have used the annotations of six animal genomes  Just as protein sequences change over time, so do gene structures. Over comparatively short evolutionary timescales, introns lengthen and shorten; and over longer timescales the number and positions of introns in homologous genes can change. These facts suggest that the intron–exon structures of genes may provide a source of evolutionary information. The utility of gene structures as materials for phylogenetic analyses, however, depends upon their independence from the forces driving protein evolution. If, for example, intron–exon structures are strongly influenced by selection at the amino acid level, then using them for phylogenetic investigations is largely pointless, as the same information could have been more easily gained from protein analyses. Using 11 animal genomes, Yandell et al. show that evolution of intron lengths and positions is largely—though not completely—independent of protein sequence evolution. This means that gene structures provide a source of information about the evolutionary past independent of protein sequence similarities—a finding the authors employ to investigate the accuracy of the protein clock and to explore the utility of gene structures as a means to resolve deep phylogenetic relationships within the animals. Sequence alignment and comparison have revealed much about evolution at the nucleotide and amino acid level, but much less is known about the structural evolution of genes—how their intron–exon structures, intron lengths, alternative splicing, and untranslated regions change over time. Genome annotations comprise an invaluable resource for answering such questions because they describe the essential parts of a gene and their relationships to one another —informatAlthough the origins and mobility of introns are still subjects of debate, previous studies ,3 have eThe utility of gene structures as materials for phylogenetic analyses, however, depends upon their independence from the forces driving protein sequence evolution. If, for example, intron–exon structures are strongly influenced by selection at the protein level, then using them for phylogenetic investigations is largely pointless, as the same information could have been more easily gained from protein analyses. Also needed is a better understanding of the rates at which different aspects of gene structures evolve. Clearly, more slowly evolving aspects of gene-structure—intron positions –11, for Drosophila simulans and D. melanogaster, to the more than 500 million years that have elapsed since the Cambrian explosion.In order to address these issues, we have characterized the number, position, and length of introns and exons in 11 individual genomes representing four phyla. These data provide a panoramic perspective from which to investigate the evolution of gene structures on a variety of timescales—from the less than 5 million years since the divergence of We show that evolution of intron lengths and positions is largely—though not completely—independent of protein sequence evolution. Thus, gene structures provide a source of information about the evolutionary past independent of protein sequence similarities. We use this fact to investigate the accuracy of the protein clock and to explore the utility of gene structures as a means to resolve deep phylogenetic relationships within the animals.http://www.yandell-lab.org/cgl.In order to facilitate the use of genome annotations as substrates for computational analyses, we developed an open-source software library (CGL) for comparative genomics using genome annotations. The software and a tutorial on its use are available at of Homo sapiens  / (Li + Lj); and Li and Lj refer to length of orthologous introns i, and j, respectively. If the two introns are the same length, Lc equals 1. If one member of the pair is twice the length of the other, Lc equals 0.5. Thus Lc provides a simple means to associate a similarity value with each pair of orthologous introns. For purposes of display 1 − Lc is plotted so that two introns having exactly the same length, flanked by exons with a Ka/Ks = 0 will lie at the graph's origin.2 = 0.0015). No significant Spearman correlation coefficient was observed for these data.Orange line, best-fitting linear regression Click here for additional data file.


Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria.Synechococcus sp. C9, and the other two are in the LSU gene of Synechococcus lividus strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria.Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG), in large subunit (LSU) rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of bacterial gene (multiple group I introns have been reported in at least one phage gene and one prophage gene). The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown.We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same  A common feature of these introns is the presence of LAGLIDADG homing endonuclease genes in peripheral stem-loop regions of the group I ribozyme. To our knowledge, this is the first discovery of multiple group I introns in a single chromosomal gene of a bacterium . We anaSynechococcus lividus strain C1 and Synechococcus sp. C9, both originally isolated from a hot spring habitat in Yellowstone National Park, Wyoming, USA , app, app31]]thyA gene (encoding thymidylate synthase) of Bacillus mojavensis and in the nrdE gene of a prophage of Bacillus anthracis and other Bacillus species . One caPCC 6803 , was recPCC 6803 . The totnt paper  many HNHSynechococcus lividus strain C1 and Synechococcus sp. C9, are distant relatives based on phylogenetic analyses of small , it , it 41]]Fuligo septica harbors 12 group I introns . In . In 46]] introns ), which Synechococcus sp. C9 contains three group I introns, at positions L1917, L1931, and L2593, whereas the S. lividus strain C1 LSU rRNA gene contains similar introns at L1931 and L2593. This finding is surprising because the vast majority of bacteria contain no rDNA introns and the few others that have any introns possess only one. The intron-encoded HEGs belong to the LAGLIDADG family, and contain one copy each of the conserved amino acid motif that defines this family . Phylogenetic analyses show that the cyanobacterial introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. Finally, from previous studies it is estimated that approximately 3% of nuclear group I introns contain HEGs. In our survey of group I introns and HEGs in the literature we estimate that at least half of organellar group I introns contain HEGs, and that about one third of bacterial group I introns contain HEGs.We found multiple HEG-containing group I introns in cyanobacterial LSU rRNA genes. Specifically, the LSU rRNA gene of Synechococcus lividus strain C1 and Synechococcus sp. C9 were a gift from David Ward, MontanaState University, Bozeman. These cyanobacterial strains wereoriginally isolated from microbial mat communities in Octopus Spring, Yellowstone National Park, Wyoming, U.S.A. Cells were scraped fromthe slants and DNA was isolated with the Puregene kit  following the manufacturer's protocol.Axenic slant cultures of ad hoc and not necessarily consistent with inferred phylogenetic relationships.The bacterial nomenclature used in this study is not addressed here other than to point out that the cyanobacterial names in this paper are of botanical origin and have not been validly published under the rules of the Bacteriological Code, unlike the other bacterial names in this report. Therefore they should be considered Approximately 2.8 kb of the 23S rRNA gene was amplified from genomic DNA by polymerase chain reaction (PCR) using primers 36F and 2763R [see Additional file DQ421379–DQ421380.Temperature and cycling conditions were as follows: one 95°C denaturation cycle for 3 min, followed by 35 cycles of 95°C denaturation for 15 sec, primer annealing at 49°C for 15 sec, and elongation at 72°C for 90 sec. Four μL of the amplified products were visualized on 1.5% agarose minigels and the remainder was purified using 30,000 NMWL low-binding, regenerated cellulose membrane filter units (Millipore). Agarose plugs were sometimes taken of weak PCR products and reamplified at 51°C using the same conditions. Both strands of purified PCR products were directly sequenced in 10 μL reactions using the sequencing primers listed in Additional file The central paired elements  in group I introns were identified by comparing the intron sequences to available secondary structures of related introns (identified by BLAST searches). Secondary structures of peripheral regions were predicted using Mfold .The number of available small and large ribosomal RNA gene sequences of known origin was determined by searching the NCBI (GenBank) databases, restricting the search to prokaryote organisms and excluding sequences determined from bulk environmental DNA. The search was further restricted to complete or nearly complete gene sequences, at least 900 nucleotides in the case of small subunit (16S) rRNA sequences and at least 2000 nucleotides in the case of large subunit (23S) rRNA sequences.Synechococcus intron DNA and HE protein sequences were added to previously published sequence alignments [The five ignments . Only inignments  and 3. Pignments , and wilHEG, homing endonuclease gene; HE, homing endonuclease; LSU, large subunit, rRNA, ribosomal RNA; rDNA, ribosomal DNA; ORF, open reading frame; SSU, small subunit.Synechococcus strains, carried out the phylogenetic analyses, compiled the list of group I introns with HEGs in bacteria, and drafted the manuscript. DB participated in the analysis and interpretation of the data and in manuscript preparation. JDP conceived of the broader study using LSU rDNA to examine the phylogeny of cyanobacteria, and assisted with data interpretation and manuscript preparation. ST participated in the broader phylogenetic study, in the experimental design that led to the discovery of the introns reported here, in GenBank searches, and in the drafting of the manuscript. LAL assisted with data analysis and interpretation, and in manuscript preparation. KMP planned and coordinated the study, designed primers, sequenced the LSU rRNA data reported here (including the introns and their encoded HEGs), and assisted with drafting the manuscript. All authors have read and approved the final manuscript.PH reconstructed the putative secondary structures of rRNA group I introns in Synechococcus.Oligonucleotides used in this study. Primers used for amplication and sequencing 23S rRNA (including introns and homing endonuclease genes) in Click here for fileGroup I intron dataset. Nexus formatted intron data set.Click here for fileHoming endonuclease data set. Nexus formatted homing endonuclease data set.Click here for file


The somatic DNA molecules of spirotrichous ciliates are present as linear chromosomes containing mostly single-gene coding sequences with short 5' and 3' flanking regions. Only a few conserved motifs have been found in the flanking DNA. Motifs that may play roles in promoting and/or regulating transcription have not been consistently detected. Moreover, comparing subtelomeric regions of 1,356 end-sequenced somatic chromosomes failed to identify more putatively conserved motifs.We sequenced and compared DNA and RNA versions of the DNA polymerase α (pol α) gene from nine diverged spirotrichous ciliates. We identified a G-C rich motif aaTACCGC(G/C/T) upstream from transcription start sites in all nine pol α orthologs. Furthermore, we consistently found likely polyadenylation signals, similar to the eukaryotic consensus AAUAAA, within 35 nt upstream of the polyadenylation sites. Numbers of introns differed among orthologs, suggesting independent gain or loss of some introns during the evolution of this gene. Finally, we discuss the occurrence of short direct repeats flanking some introns in the DNA pol α genes. These introns flanked by direct repeats resemble a class of DNA sequences called internal eliminated sequences (IES) that are deleted from ciliate chromosomes during development.Our results suggest that conserved motifs are present at both 5' and 3' untranscribed regions of the DNA pol α genes in nine spirotrichous ciliates. We also show that several independent gains and losses of introns in the DNA pol α genes have occurred in the spirotrichous ciliate lineage. Finally, our statistical results suggest that proven introns might also function in an IES removal pathway. This could strengthen a recent hypothesis that introns evolve into IESs, explaining the scarcity of introns in spirotrichs. Alternatively, the analysis suggests that ciliates might occasionally use intron splicing to correct, at the RNA level, failures in IES excision during developmental DNA elimination.This article was reviewed by Dr. Alexei Fedorov (referred by Dr. Manyuan Long), Dr. Martin A. Huynen and Dr. John M. Logsdon. Spirotrichous ciliates are intriguing because they carry out elaborate genomic DNA rearrangements when they develop their somatic nucleus, or macronucleus, from their hereditary nucleus, or micronucleus. In the hereditary nucleus, macronuclear destined sequences (MDSs) in the germline DNA are separated by noncoding, A-T rich, internal eliminated sequences (IESs), each bounded by a pair of direct repeats. Tens of thousands of IESs must be removed during macronuclear development, while the MDSs are "sewn together" leaving one copy of the pair of direct repeats at the "stitch" Fig. . After t4A4)nC4 telomeric repeats [The somatic macronucleus of spirotrichous ciliates contains millions of short macronuclear chromosomes ranging in size from ~400 to ~15,000 bp  and flan repeats ,4. Macro repeats . A typicS. histriomuscorum  also yielded similar conclusions [cis-elements in macronuclear chromosomes have been experimentally confirmed: the heat shock responsive elements in Sterkiella nova (formerly Oxytricha nova)[Euplotes species [Stylonychia lemnae [cis-motifs deduced from multiple sequence comparisons include the putative chromosomal breakage signal 5'-HATTGAAaHH-3' (abbreviated using the IUPAC symbol H = not G) in Euplotes crassus [Relatively little is known about the transcriptional structure and regulation of genes in spirotrichous ciliates, including their promoter sequences, transcriptional start sites, and signals that direct transcriptional regulation and post-transcriptional processing. A study of 66 putative protein-coding macronuclear molecules in spirotrichs revealed that the 5' and 3' flanking sequences are less than 200 bp and A-T rich (> 70%) . A more clusions , suggestclusions  has not clusions ,11,12. Tcha nova) and seve species  and the a lemnae ,16. Othe crassus  and a co crassus . However crassus , with th crassus .Spirotrichous ciliates generally contain few introns . Known iIn this study, we sequenced the genes and determined the mRNA ends of nine diverged orthologs encoding the large subunit of DNA polymerase alpha (pol α) in spirotrichous ciliates. We found a conserved motif aaTACCGCB (B = not A) located upstream from the transcription start site in each species. In addition, we also identified putative polyadenylation signals -10 to -15 bp upstream of the polyadenylation sites. We experimentally confirmed that introns in these genes were generally short, with the exception of two longer introns in euplotids. By comparing the locations of these introns within the coding region, our results suggest that independent gains/losses of introns have occurred within the spirotrichous ciliate lineage. Finally, we investigated the recurring and curious observation in our data of direct repeats overlapping or present near the exon-intron boundaries. Some of the larger of these repeats are similar in length to those that function in IES excision. We provide a statistical and bioinformatic analysis that shows that some of these direct repeats are more common than expected when compared to biologically constrained randomized genes. This result could be interpreted as strengthening a recent hypothesis (elaborated in ) that inEuplotes spp. and Eschaneustyla sp. are newly reported in this study. We determined the mRNA ends from seven of the nine orthologs and re-sequenced the corresponding macronuclear regions in order to avoid possible discrepancies between RNA and DNA data due to strain differences or alleles within a species. RNA samples from U. grandis and Eschaneustyla sp. were not available; thus putative features were inferred from sequence comparison to other species.Features of the DNA polymerase α (pol α) gene of 9 ciliate orthologs are listed in Figure Holosticha, these sites are within the first 100 bp of subtelomeric DNA at the 5' end. In Holosticha, two other genes are present on the same macronuclear chromosome, upstream of the DNA pol α gene [U. grandis and in Eschaneustyla sp.  were found on adenosine residues, preceded by a thymine base and followed by an A-T rich region Fig. . With thl α gene . We searl α gene ,11. Howesp. Fig.  and 3b. muscorum  also faiE. octocarinatus to 1,524 aa in S. lemnae . Several previous studies have shown that the eukaryotic poly(A) addition signal AAUAAA [cis-element, a GU-rich or U-rich element downstream of poly(A) sites, has been shown to help guide the cleavage of pre-mRNA at the 3' end before poly(A) tail addition [The mRNA polyadenylation (poly(A)) sites of all seven DNA pol α genes were found at an adenine residue preceded by a thymine base, except in l AAUAAA  is not ul AAUAAA ,11,12,18l AAUAAA -32, mousl AAUAAA , and maml AAUAAA  expressel AAUAAA . At leasaddition , and thiEuplotes . Through DNA sequence analysis, we infer the presence of the first intron in Eschaneustyla sp., but not the second intron. There is also no indication that either of these two introns is present in the early diverged stichotrich U. grandis.The translation initiation sites of spirotrich DNA pol α genes have been the subject of some controversy . Ribosomiew, see ), or sup. lemnae , S. hist S. nova ,38. Howe S. nova  and diveies Fig. , intron E. aediculatus and E. octocarinatus. The position of these introns in the protein alignment is 10 nt (or 3.33 aa) downstream from the position of the first 5' intron found in the other six stichotrichs (data not shown). Moreover, the intron is 159 bp in E. aediculatus and 1,271 bp long in E. octocarinatus .All of the introns in our dataset contained canonical eukaryotic splice signals 5'-GT...AG-3' but they did not consistently contain the putative extended spirotrich ciliate splice signals 5'-GTAAG...TAG-3' referred to in ,21. HalfOur null models make the following assumptions: fixed amino acid sequences of the DNA pol α proteins in each species; fixed codon usage bias in the genes of each species with independent evolution of codons; and fixed base composition of each intron independently with independent evolution of bases and possible splicing constraints.Holosticha and the first introns in Uroleptus and Urostyla are significantly rare in the eukaryotic splicing constraint null model with a false discovery rate of 10%. This implies that if the repeats flanking these four introns were to be investigated further, the expected risk is less than or equal to 10% that one of them is just a random occurrence as represented in this null model. In contrast, the eukaryotic splicing constraint null model could not be rejected for the generally shorter repeats flanking the other 10 introns with this FDR. Although the first three introns in Table We then measured in the null models for each of the 14 introns how often we observed exact repeats of any word as long as or longer than the naturally observed repeats at the exact same positions bridging the exon/intron boundaries. The relevant results are shown in Table As mentioned, we also calculated the frequencies of repeats in different null models of intron splicing constraints, one with no splicing constraints, and the other with extended splicing constraints. Even with extended "Ciliate" splicing constraints, the expected false discovery rate rejecting the null model for the same four introns is bounded by 25% (FDR ≤ 0.25). In fact, because of the evidence for this extended signal in our data and elsewhere ,21, the Uroleptus intron was more likely to be flanked by repeats than the first Holosticha intron. The reason for this is that the Holosticha repeat contains a C, which is relatively rare in AT-rich ciliate genes, where Uroleptus presents a T. The fact that this substitution is mirrored in both repeat copies is consistent with their function in putative IES removal. Curiously, the TGTA repeats in the second Holosticha intron were significant in the eukaryotic model, despite a length of only four nucleotides. The reason for this is that the intron in question contains only one G, other than that of the intron splice acceptor. In fact, as discussed above and shown in Table In all three null models we examined, the first Urostyla 3' intron repeats were still significant by these criteria (data not shown). However, all other repeats we studied, besides this one, fall tightly over the intron-exon boundaries, making the 15 bp window not applicable.We extended our results by looking for occurrences of the same repeat pair in the actual sequence data within 15 bp windows surrounding the intron/exon boundaries. We learned that these repeats occur only if they overlap the intron/exon boundaries in our randomized sequences, and almost always at exactly the same locations as in the actual sequences. As such, the simulated repeats were always frame-preserving when the natural repeats were frame-preserving. In searching for any repeat pair as long as or longer than those observed in the natural data, we found such repeat pairs of any sequence to to be enormously common. Only the 8-bp It is interesting to ask under what conditions repeat sequences in protein-coding regions can function dually either in IES removal or intron splicing, while preserving reading frame in both pathways. Considering only the generic eukaryotic splice signals GT and AG (since AT-AC introns have not been identified in ciliates), all possible variations are shown in Table 4A4 telomeric sequences , transcription factor binding sites, and poly(A) signal(s) have been difficult to find [E. crassus with a gene for neomycin resistance and showed that this recombinant gene had the same TSS as the wide type α-TBP gene [cis-elements to guide and regulate gene expression.While the regulation of gene expression in spirotrichous ciliates is poorly understood, and  to find ,11,12, m to find ,11. BendTBP gene . FurtherS. lemnae [In this study, we compared gene sequences for DNA polymerase α from nine spirotrichous ciliates. We also sequenced RNA versions of seven of these orthologs and determined the mRNA 5' and 3' ends. We identified a motif of aaTACCGCB present 5 – 34 nt upstream from the TSS. This motif may serve one or more of the following functions: (i) promoter; (ii) copy number regulation, (iii) transcriptional regulator, (iv) chromosomal breakage signal. However, because this motif is absent from most other sequenced macronuclear chromosomes , it is unlikely to be a chromosomal breakage signal. Furthermore, the previously identified chromosomal breakage signals HATTGAAaHH in euplotids ,50 and T. lemnae ,16 have Uroleptus)  signal located at the 3' ends of macronuclear chromosomes have been reported ,11,12. P. lemnae  resembleE. octocarinatus is approximately five times longer than the longest intron that has been previously reported in spirotrichous ciliates. In addition to the intron paucity in spirotrichs observed by Prescott [Paramecium [E. octocarinatus derived from a transposon, a pathway that has been suggested by Rautmann et al. [At 1,271 nt, the 5' intron in the DNA pol α gene in ramecium  are generamecium ,54, the n et al. . Howevern et al. ,41 nor an et al. . Thus, tWe also noticed a quirky tendency of some introns in this study to be flanked by direct repeats, reminiscent of the pairs of pointer repeats that flank IESs and may facilitate their removal. In some cases the observed repeats flanking introns overlap intron donor and acceptor splice signals. Five repeats we found of length five or greater were biologically significant, in that they would preserve protein-coding information if they were to function in IES removal, and they also satisfy other criteria such as rarity in the sequence and linear contiguity in a single macronuclear-destined segment Table . FurtherIt is tempting to speculate that some ciliate introns may evolve into IESs by providing a nucleus for the evolution of extended repeats, which then lead to the elimination of the enclosed sequence during macronuclear development. This could help explain the observed intron paucity and some intron loss in spirotrichs during evolutionary time, as well as the shared locations of some introns and IESs ,57. TablOur results suggest that conserved motifs, perhaps participating in gene regulation, are present in both 5' and 3' untranscribed regions of the DNA pol α genes. Our data also suggest that several independent gains and losses of introns in DNA pol α genes have occurred in the spirotrichous ciliate lineage. Finally, we found statistical and bioinformatic support for the hypothesis that direct repeats flanking introns might have an additional function in IES removal. This may favor the scenario in which introns evolve into IESs, which would explain the observed ciliate intron scarcity. Alternatively, some IESs might actually be spliced as introns, as a back-up in case they fail to be removed during macronuclear development. Further experimental pursuit of both hypotheses would be fruitful, noting that the two scenarios are not mutually exclusive and that both may contribute to the evolvability and robustness of information in ciliate genomes.Euplotes aediculatus, Eschaneustyla sp., Holosticha sp. [Uroleptus sp. [Paraurostyla weissei [Stylonychia lemnae strain Y and Euplotes octocarinatus were generous gifts from Dr. Hans Lipps. Sterkiella histriomuscorum strain JRB310 was a generous gift from Dr. Glenn Herrick. DNA of U. grandis was a generous gift from Dr. David Prescott. Ciliate species were grown in conditions published elsewhere [icha sp. , Uroleptptus sp. , and Par weissei  were isolsewhere . Macronulsewhere .U. grandis type I: AY008387; U. grandistype II: AY008386[Holosticha sp.: AY293851[Uroleptus sp.: AY293852; P. weissei: AY293806[E. aediculatus: DQ060373; E. octocarinatus: DQ060372; Eschaneustyla sp.: DQ060374; S. lemnae: AF194338[S. histriomuscorum: U59426[Plasmodium falciparum (PFD0590c); Toxoplasm gondii (AF093136).Parts of the macronuclear DNA polymerase α (pol α) genes were first PCR amplified by using degenerate primer sets published elsewhere ,37. The  AY008386; Holosti AY293806; E. aedi AF194338; S. histCiliate cells were fed with green algae one day prior to harvesting for RNA isolation. Cells were then concentrated by filtering through 10 μM sieve (Sefar American). RNA was extracted by using the Trizol LS reagent (Invitrogen) following the manufacturer's protocol. The quantity and purity of RNA were assessed by measuring UV absorbance at 260 nm/280 nm. 5'- and 3'- rapid amplification of cDNA ends (RACE) were carried out by using the FirstChoice RLM-RACE kit (Ambion) following manufacturers' protocols.The alignments of DNA poly α genes were generated by using Accelrys (formerly GCG) software package. Sequence management under Windows OS was assisted by using the software Bioedit . The 9 bPrograms were written in Perl to generate permutations of the six sequences with observed intron-flanking repeats of length four or greater according to different constraints and to thereby test, by a randomization procedure, the statistical significance of the occurrence of repeats surrounding introns. Sequence manipulation was done in BioPerl . PermutaCodons were permuted within and among exons, preserving the amino acid sequence of the protein (using the UAR:Gln genetic code) and the codon usage of the gene. Bases were permuted within each intron either freely or fixing bases inside splice junctions, so as to preserve their lengths, base compositions, and potential splicing constraints.For each repeat pair, permuted exons and introns were combined to generate simulated genes in which the occurrence of repeats was monitored.For other species and introns with shorter repeats, the equivalent probabilities of repeats in our null models were computed exactly by hand from the probabilities of sampling words without replacement from multinomial distributions based on intron compositions and the codon frequencies of exons.Intron be the phase on removal of an intron and φIES be the phase after IES excision from repeats that flank such an intron in such a way that this IES excision potentially disrupts the splicing of that intron (a disrupting pair). We say that the repeats are frame-preserving if φIntron = φIES. Let the 5' and 3' coordinates of the intron be , the 5' coordinates of a disrupting pair of repeats be A and C, and the 5' coordinates of a differently located disrupting pair of repeats be a and c. We then have φIntron = (j - i + 1)mod3, and φIES = (C - A + 1)mod3 or φIES = (c - a + 1)mod3. If the first pair of repeats are frame-preserving then (j - i + 1)mod3 = (C - A + 1)mod3, similarly for the second pair of repeats. The quality of frame-preservation is transitive; if both pairs of repeats are frame-preserving, then we know that (c - a + 1)mod3 = (C - A + 1)mod3. Since both (c - a + 1) and (C - A + 1) are congruent modulo 3, we know by definition that (c - a - C + A)mod3 = 0. We used this last relation to test whether permuted repeats were frame preserving given that natural repeats are frame-preserving, without knowing the length or coordinates of the introns.Let φThe author(s) declare that they have no competing interests.Euplotes spp.. LFL designed experiments and helped to draft the manuscript. All authors read and approved the final manuscript.WJC designed experiments, characterized DNA polymerase α sequences, analyzed genealogies of introns, and drafted the manuscript. VMA characterized mRNA and introns in stichotrichs. EA programmed and performed the statistical analysis. DHA inferred the presence of introns bioinformatically, designed the statistical analysis of repeats, derived the results in Table Alexei Fedorov, Department of Medicine, Medical University of Ohio (nominated by Manyuan Long)This paper represents very interesting results and hypothesis, so I have no reservation in recommending it for publication. However, many places in this MS are not reader-friendly. I suggest a revision in the style of presentation (Minor revision prior to publication). In a nutshell, only a very small group of scientists have a good expertise in ciliates. Thus, a good brief introduction to the problem is desirable for a broader audience....We have now reordered and rewritten the background section for clarity and added a new figure .In a very straightforward manner, the results indicate some clear sequence motifs that are likely involved in transcription of these genes. This is a valuable contribution to the field since such signals are not well understood in these organisms. These ciliates are also somewhat depauperate of spliceosomal introns, making the authors' discovery of 2 or 3 introns per gene of some interest on its own. The presence of spliceosomal introns was verfied by cDNAs in most cases and reveals five separate sites that harbor introns. The two Euplotes species both have two introns (at shared positions) that are not shared in position with the introns in the other species. The three remaining introns are variably present among the other seven species: two are widely present (6/7 and 5/7), and one less widely (3/7). At face value, these data indicate that introns are being gained and/or lost in this lineage.In considering a possible mechanistic relationship between spliceosomal introns and IESs, the authors evaluate some potentially shared signals between these processes. These statistical analyses suggest that introns flanked with direct repeats  could be lost via IES excision (at the DNA level). The results are consistent with this interesting and novel hypothesis for intron loss, but it does not seem to explain many of their own observations. It is only the last (most 3') intron that requires an inference of loss in this dataset: this intron is present in Urostyla grandis, Eschaneustyla sp., and Uroleptus sp., and missing in all of the other taxa shown. Notably, this intron is missing in Holostricha sp., and in the P. weissei/Sty. lemnae/Ste. histrio. clade which, given the phylogeny, would indicate its separate loss in these two lineages. The other four introns can be parsimoniously explained by single gains with no losses.Considering the putative intron losses from the Holostricha sp., and P. weissei/Sty. lemnae/Ste. histrio. clades, there is available data from some of these species on the micronuclear arrangement of this gene (PNAS 102: 15149). In these species, is there an IES in the same location as the "lost" intron? How about the other intron positions? In general, it might be useful to provide a "map" of the IESs relative to the intron positions.In the PNAS paper we did provide a map of IES locations (where pointers are) relative to intron positions. In some cases IESs coincide precisely with putative intron loss positions, e.g. an IES in P. weissei is present at the same position of the 3' intron in Uroleptus; numerous IESs surround the 5' intron regions in U. grandis.Author response: Also, the authors do not consider the converse idea: that some intron gain could be explained by the coincidence between splicing signals and IESs. That is, some unremoved IESs could function as spliceosomal introns.Although we had included this alternative interpretation in the conclusion, as it fits with our earlier work on the subject , it was not expressed clearly enough in the version you reviewed. We agree with the reviewer about the importance of this alternative hypothesis and have now stated it more clearly throughout the paper.Author response: Although the authors argue that one of these introns (the first one) is shared between ciliates and animals (H.sapiens and C.elegans), the data presented are unconvincing. The alignment segment shown Fig  does notINTRON POSITIONS/PHASE SHOWN ABOVE SEQUENCE)The apparent gap in the C. elegans sequence casts doubt on the alignment (and homology assessment) of the intron-containing sites in animals. If the introns are not shared between animals, then there is very little reason to consider this position as "ancient" or even old. Thus, the apparently coincident position in the ciliate and human gene  would more easily be explained as separate gains into that site. In order to make a stronger case for the antiquity of this (or any other) intron, a much more phylogenetically broad survey of the available gene sequences and their introns will need to be performed and presented. If so, a more extensive alignment of the amino-acid sequences would be needed to assess the homology inferences at each of the intron-containing sites.As for the other introns that are nearby known positions in orthologs from other species  there is relatively little known about these species with their peculiar genome organization. That their IES machinery is somehow related to their splicing machinery opens new possibilities for research. Having said that, the statistical support for the "non"-randomness of the direct repeats that the authors observe flanking the introns is rather weak. Furthermore not all the introns are flanked by repeats. The authors first identify the repeats and then test their significance. Nevertheless, the hypothesis is tantalizing and I do hope the authors will be able to find more support for it in future studies.We thank Dr. Huynen for his prompt response and his suggestion. As stated above, we would conduct a more extensive research with other genes.Author response:


The signals that determine the specificity and efficiency of splicing are multiple and complex, and are not fully understood. Among other factors, the relative contributions of different mechanisms appear to depend on intron size inasmuch as long introns might hinder the activity of the spliceosome through interference with the proper positioning of the intron-exon junctions. Indeed, it has been shown that the information content of splice sites positively correlates with intron length in the nematode, Drosophila, and fungi. We explored the connections between the length of vertebrate introns, the strength of splice sites, exonic splicing signals, and evolution of flanking exons.A compensatory relationship is shown to exist between different types of signals, namely, the splice sites and the exonic splicing enhancers (ESEs). In the range of relatively short introns , the enhancement of the splicing signals for longer introns was manifest in the increased concentration of ESEs. In contrast, for longer introns, this effect was not detectable, and instead, an increase in the strength of the donor and acceptor splice sites was observed. Conceivably, accumulation of A-rich ESE motifs beyond a certain limit is incompatible with functional constraints operating at the level of protein sequence evolution, which leads to compensation in the form of evolution of the splice sites themselves toward greater strength. In addition, however, a correlation between sequence conservation in the exon ends and intron length, particularly, in synonymous positions, was observed throughout the entire length range of introns. Thus, splicing signals other than the currently defined ESEs, i.e., potential new classes of ESEs, might exist in exon sequences, particularly, those that flank long introns.Several weak but statistically significant correlations were observed between vertebrate intron length, splice site strength, and potential exonic splicing signals. Taken together, these findings attest to a compensatory relationship between splice sites and exonic splicing signals, depending on intron length. GU(A/G)AGU in vertebrates, the first two nucleotides of the intron are underlined) at the donor splice site is complementary to the 5' end of U1 small nuclear (sn)RNA, and the interaction between the donor site and U1 is thought to be the major requirement for splicing [CAG|G consensus sequence (the last two nucleotides of the intron are underlined) preceded by a polypyrimidine tract is typical of the acceptor splice site which is recognized by the splicing factor U2AF [Most protein-coding genes in multicellular eukaryotes are interrupted by multiple introns that are removed by the spliceosome in a complex succession of concerted hydrolysis and ligation reactions -4. Obviosplicing -8. The Ctor U2AF . The brator U2AF ,10.However, the classical splice signals are insufficient for correct splicing, at least in vertebrates where intron lengths vary within an extremely broad range, and many introns are long. Moreover, it has been estimated that, while the canonical, intronic signals are sufficient for accurate splicing by "intron definition" in nematodes and flies, these signals contain only approximately one-half of the information required for correct splicing of even short introns in mammals and plants . AdditioDrosophila [A variety of observations indicate that the splicing process is not indifferent to intron length. Long introns might hinder the activity of the spliceosome through interference with the proper positioning of the intron-exon junctions. Thus, it could be expected that additional mechanisms and/or signals might be required for splicing of long introns. Indeed, it has been shown that the information content of splice sites positively correlates with intron length in the nematode , Drosophosophila , and funosophila . We wereWe first tested the hypothesis that long introns are associated with strong flanking splice sites in mammalian genomes. For each genome, we derived a position-specific weight matrix for both donor and acceptor splice sites. Using these matrices and background base frequencies, a log-odds score for each splice site was computed. The total splice site score for each intron was calculated as the sum of the scores of its flanking donor and acceptor sites.R = 0.124, alternative R = 0.145) but highly statistically significant (P ≈ 0)  but statistically significant  correlations between intron length and splice site strength  and alternative  introns  between the observed ESE density (Eobs) and the expected ESE density (Eexpect), given the base composition of that exon end. Note that Eenrich is in terms of density, and is therefore normalized by exon end length. The resulting Eenrich values had weaker but still significant correlations with intron length, for short constitutive  and alternative  introns. A slightly different test, in which the relative enrichment (Eenrich/Eexpect) was calculated, yielded weaker correlation values for short constitutive  and alternative  introns. The results of these tests suggest a specific increase in hexamer ESE density with increasing intron length among relatively short introns, as opposed to a more general trend in base composition.Whereas for splice sites we observed correlations between signal strength and intron length for long introns Figure , we founs Figure . As withs Figure , it seemHaving detected correlations between intron length and splicing signals for introns in a single genome, we sought to determine if differences in the lengths of orthologous introns in different species were associated with changes in splicing signals. As small changes in intron length are likely to have little effect on splicing, we examined only those pairs of orthologous introns for which the length difference was substantial . When one intron of an orthologous pair was substantially longer than the other, and the various splice signals differed in strength, we compared the strengths signals associated with these introns.p-values for chi-square tests performed on a variety of contingency tables such as that given in Table Table Although the exact locations of ESEs within exons are largely unknown, increased density of ESE motifs has been detected in the 25–30 nucleotides from exon ends, the regions of exons that have the lowest SNP density in humans . We reprP ≈ 0). ESE sites were substituted at a mean rate of 5.4% as compared to 5.9% for non-ESE sites. Excess conservation of ESE sites was found to occur over all intron lengths  would be more conserved than those flanking short introns. Indeed, we found that both synonymous and nonsynonymous sites were more conserved in exon ends adjacent to long introns Figure . The effThe results presented here corroborate and extend previous observations on the connection between intron length and the strength of splicing signals. Generally, we observed that, as expected, longer introns that might present greater problems for intron definition and, consequently, for efficient splicing than shorter introns were associated with stronger splicing signals. However, there was a notable compensatory relationship between different types of signals, namely, the splice sites and the ESEs. In the range of relatively short introns , the enhancement of the splicing signals in longer introns seemed to occur within the exons and was manifest in the increased concentration of ESEs. In contrast, for longer introns, this effect was not detectable, and what was seen instead, was an increase in the strength of the donor and acceptor splice sites. Since the ESEs are located in protein-coding exons, it appears likely that accumulation of A-rich hexamers beyond a certain limit is incompatible with functional constraints operating at the level of protein sequence evolution. Hence the compensation in the form of evolution of the splice sites themselves toward greater strength.The threshold separating "short" and "long" introns used here was different from the boundary at ~200 nucleotides that separates introns spliced via intron definition from those spliced via the exon definition ,34. We aAll of the above relationships consistently held for both constitutive and alternative exons. Curiously, however, the connection between splice site strength and intron length was somewhat stronger for the alternative exons whereas the opposite was true of the dependence between ESE density and intron length, which was most pronounced for constitutive exons. This suggests yet another level of compensation between different types of splicing signals.The correlations between intron length and ESE density that were readily observable for ESE hexamers, were not seen when we analyzed a different class of ESEs that have been defined as octamer motifs ,24 Figu. HoweverIn addition to the above dependences, we observed a correlation between intron length and sequence conservation in the exon ends, especially, in synonymous positions, throughout the entire length range of introns Figure . This suThe greater strength of different types of splicing signals around longer introns could evolve either via a neutral evolution route or as an adaptation. The neutral scenario would apply to a case when an intron becomes shorter as a result of deletion that is followed by splice site amelioration. In contrast, when the length of an intron increases due to an insertion, the splicing signals would evolve under positive selection, adapting to the new situation by restoring splicing efficiency.All the trends in the relationships between the strength of splicing signals, evolutionary conservation of synonymous positions in exon ends and ESE sites, and intron length that are reported here and elsewhere -8 are maOn balance, despite the weakness of the observed correlations, the coherence of different types of signals uncovered in this study, some of which are limited to single-genome analysis (splice site strength and ESE density) whereas others involve sequence conservation in different genomes, strongly suggests that these observations are functionally relevant for the splicing mechanism rather than spurious. In particular, these findings provide an incentive for an experimental search for new types of ESEs.We used the NCBI build 34 assembly (July 2003) of the human genome and the NCBI build 33 assembly (May 2004) of the mouse genome. For single species analyses, intron annotations were obtained from the Alternative Splicing Database (ASD) . Human Rpi,j is the frequency of nucleotide j at splice site position i, qj is the background transcript frequency of nucleotide j, and s(i) denotes the nucleotide at position i within splice site s. The background frequencies of nucleotides within human and mouse RefSeq pre-mRNA transcripts were found to be 27.5% A, 20.8% C, 21.6% G, and 30.1% T.Position-specific weight matrices were constructed for both donor and acceptor splice sites . A donorPutative hexamer ESE sequences for human and mouse, as determined by Fairbrother et al. , were obFor comparative analyses, the RefSeq  mRNA datOrthologous intron pairs were obtained through the use of a 9 vertebrate  multiple whole-genome alignment, obtained from . This alFor the purpose of measuring changes in splice site scores of orthologous introns, position-specific weight matrices were constructed using splice sites from both genomes under consideration. When calculating contingency tables, such as that shown in Table Statistical analyses were performed using the R package . All corCND participated in the data collection, performed most of the data analysis and wrote the first draft of the manuscript; IBR participated in the data collection and study design, and performed some of the statistical analyses; EVK initiated the study, participated in its design and the interpretation of the results, and wrote the final version of the manuscript. All authors read and approved the final version of the manuscript.The complete contingency table used in this analysis and Figures S1-9.Click here for file