The crystal structure of a T = 7 papilloma virion has not yet been determined; however, the crystal structure of a T = 1 particle containing 12 pentamers is known. The T = 1 structure reveals that helix-helix interactions, through three helices–h2, h3, and h4–near the C-terminus of L1, mediate the inter-pentameric bonding that is responsible for T = 1 assembly. Based on the T = 1 crystal structure, we have generated a set of internal deletions to test the role of the three C-terminal helices in T = 7 assembly. We have demonstrated that the h2, h3, and h4 near the C-terminal end of L1 are important for the L1 structure and particle assembly. In particular, we found that h2 and h3 are essential for L1 folding and pentamer formation, whereas h4 is indispensable for the assembly of not only T1, but also of the T7 virus-like particle.The outer shell of the papillomavirus particle is comprised of 72 pentamers of the major capsid L1 protein arranged on a T = 7 icosahedral lattice. The recombinant L1 can form T = 7 virus-like particles  Papillomaviruses are non-enveloped DNA tumor viruses. Infection by papillomaviruses is associated with tumorigenesis in experimental animals and in humans (reviewed in ). The viin vivo and in vitro [in vitro assembly of L1 occurs spontaneously under high salt or low pH conditions [The recombinant L1 capsid alone can assemble into the virus-like particle structure in vitro ,10-12,15The atomic structure of a T = 7 papilloma virion has not yet been determined. However, the crystal structure of a smaller particle structure, a T = 1 particle containing 12 pentamers, has been solved [By fitting the L1 atomic structure of from the T = 1 particle into the cryo-EM reconstruction of BPV , an atomThe crystal structure of the T1 particle of HPV16 L1 capsid protein shows 60 L1 molecules assembled through pentamerization interactions and inter-pentameric bonding . The L1 Because the L1 expression level and solubility could not be easily detected by directly analyzing the cell lysates by SDS-PAGE, we performed partial purification of the GST-fusion proteins from the cell lysates using affinity chromatography. The GST-L1 protein from the supernatant fraction of the lysates was passed through a glutathione resin column. After washing the column with 10× bed volumes of buffer L , the resin was incubated with thrombin to cleave L1 from the GST fusion.The proteins bound to the resins before and after thrombin treatment, as well as those in the eluate after thrombin treatment, were analyzed by SDS-PAGE Fig. . D1 and After thrombin digestion of the GST-L1 fusions on the glutathione resin, soluble D1- and D2-L1 were eluted from the column . For D3-L1, however, no intact protein was present in the elution after thrombin cleavage; D3-L1 may be degraded by thrombin due to an unfolded or misfolded conformation.The L1 proteins eluted from the glutathione column were analyzed using size-exclusion chromatography on Superdex-200 Fig. . A singlSince both D1 and D2 deletion constructs of HPV16 produced soluble L1 proteins, we attempted the equivalent of a D2 deletion on HPV18 L1 Fig. . As expeWe used two methods, electron microscopy (EM) and size-exclusion chromatography, to examine whether the purified proteins from the deletion constructs of HPV16 and 18 assembled into particles. The L1 proteins (at a concentration of ~0.1 mg/mL) were first incubated in a buffer known to promote icosahedral particle assembly [The T = 1 particle structure shows that h2, h3, and h4 near the C-terminal end of L1 are the solely responsible for making inter-pentameric contacts required for the particle assembly , and theE. coli. The mutant proteins with partial or entire h4 deletion, D1-L1 and D2-L2, were similar to the full-length wild-type L1 in protein expression and behavior by size-exclusion chromatography. Like wild type, D1-L1 and D2-L2 were expressed at quite high levels, and a good fraction of each expressed proteins was soluble and could be purified as pentamers  were expressed, little intact GST-L1 fusion protein was detected Fig. . InsteadInterestingly, a small helix at the C-terminus, h5 Fig. , was alsThe deletion of h4 in another papillomavirus, HPV18, resulted in a mutant that behaved like the HPV16 D2-L1 mutant. This result confirmed that the L1 structure is conserved among different HPV types, which is not surprising considering the amino acid sequences of L1 proteins are highly conserved. We predict that the same D2 deletion of any other papillomavirus L1 should have the same properties.When the D1-L1 and D2-L2 proteins of HPV16 and 18 were incubated under assembly conditions, pentamer structures, but no T = 7 particles, were visualized by EM examination Fig. . If the In conclusion, we have demonstrated that the structural elements h2, h3, and h4 near the C-terminal end of L1 are important for the assembly of papillomaviruses into particles. The h2 and h3 regions are essential for L1 folding and pentamer formation, whereas the h4 region is indispensable for the assembly of not only T1, but also of the T7 virus-like particle.E. coli expression vector. The standard molecular cloning methods, including PCR amplification, enzyme digestion, DNA end ligation, and transformation, was used to obtain the deletion mutants of L1 as shown in Fig. The full-length L1 clones of HPV16 and HPV18 in pGEX-2T were used as parent clones for generating the deletion mutants of D1, D2, D3, and D4 as shown in Fig. The protein expression and purification of all L1 deletion mutants was carried out essentially as described preciously . Brieflyin vitro assembly conditions [Wild-type L1 can assemble into particles under known nditions . The assThe author(s) declare that they have no competing interests.BB and JD participated in making some of the constructs and performed the experiments. BB and XC designed the experiments.


PLoS Computational Biology recently published an article about spliceosomal intron evolution by Nguyen, Yoshihama, and Kenmochi ..PLoS Commates of  should ce, an intron is lost with probability ep(1 → 0) and an intron is gained with probability ep(0 → 1) at every site. Assuming a continuous-time Markov process,λ, μ > 0 are branch-specific gain and loss rates, and t > 0 is branch length. Introns are observed at the terminal taxa. An all-absent intron site is never observed, and, thus, the number of potential intron sites must be estimated for correct likelihood optimization. The likelihood can be computed by a dynamic programming algorithm [uconditional likelihood L(x) for every node u and state x ∈ {0,1}: uL(x) is the probability of the observed states in descendants of u, conditioned on the state x at u. One can further define the uupper conditional likelihood U(x) for the observed states outside the subtree of u, which can be computed efficiently by dynamic programming even if the underlying process is irreversible [x at every node u can be computed asTo estimate ancestral intron losses and gains, Nguyen and coauthors use an exponential-time procedure, which is practical only for a few species. In reality, the estimation can be done in linear time , as desclgorithm . The algversible . Feslensversible  reviews x → y on an edge uv is computed asThe posterior probability for state change uvq(0 → 1) and uvq(1 → 0) over all intron sites, respectively. Nguyen and coauthors consider instead all 2N state labeling of N internal nodes to compute the expected numbers of gains and losses. A Java package implements the more efficient procedure, and is publicly available at http://www.iro.umontreal.ca/~csuros/introns/.The expected numbers of gains or losses are obtained by summing the probabilities ep(x → y) for every branch, which can be combined to get exponentially many choices that give the same likelihood function. The continuous-time process of ep(0 → 1) + ep(1 → 0) < 1. Such constraint leads to unique parametrization (except for the root position), and is more natural than the one proposed by Nguyen and coauthors, which is based on the variance of intron gains and losses.Nguyen et al.  reiterat68 possible intron presence–absence patterns for the average gene family, but only 49 intron sites. The argument is not sound: the number of patterns has little to do with inference . It is the number of parameters that matters. Nguyen and coauthors discuss an important study by Qiu, Schisler, and Stoltzfus . Qiu and


Drosophila melanogaster and D. simulans reveals a strongly negative correlation between intron length and divergence and intron divergence and GC content. This suggests that most intronic DNA is evolving under considerable constraint.An analysis of inter-specific divergence in 225 intron fragments in  Introns comprise a large fraction of eukaryotic genomes, yet little is known about their functional significance. Regulatory elements have been mapped to some introns, though these are believed to account for only a small fraction of genome wide intronic DNA. No consistent patterns have emerged from studies that have investigated general levels of evolutionary constraint in introns.Drosophila melanogaster and Drosophila simulans, sampled from a broad distribution of intron lengths. We document a strongly negative correlation between intron length and divergence. Interestingly, we also find that divergence in introns is negatively correlated with GC content. This relationship does not account for the correlation between intron length and divergence, however, and may simply reflect local variation in mutational rates or biases.We examine the relationship between intron length and levels of evolutionary constraint by analyzing inter-specific divergence at 225 intron fragments in Drosophila genome may experience little or no selective constraint, most intronic DNA in the genome is likely to be evolving under considerable constraint. Our results suggest that functional elements may be ubiquitous within longer introns and that these introns may have a more general role in regulating gene expression than previously appreciated. Our finding that GC content and divergence are negatively correlated in introns has important implications for the interpretation of the correlation between divergence and levels of codon bias observed in Drosophila.Short introns make up only a small fraction of total intronic DNA in the genome. Our finding that long introns evolve more slowly than average implies that, while the majority of introns in the  Non-coding DNA makes up a large proportion of the genomes of most eukaryotes, yet little is known about its functional significance and the forces affecting its evolution. The identification of functional regions of the genome has tended to concentrate on coding DNA, yet the recent shift in focus towards non-coding DNA has revealed that introns and intergenic sequences may be subject to considerable levels of selective constraint, implying that they contain functional elements -6. No coet al. [Caenorhabditis elegans and Caenorhabditis briggsae; this was at least in part due to their function in splicing, because constraint appeared to be higher at the edges of introns. Likewise, Bergman and Kreitman [Drosophila melanogaster and Drosophila virilis. In contrast to these studies, Halligan et al. [Drosophila were evolving approximately 17% faster than fourfold synonymous sites. They concluded that these sites were effectively evolving free from selective constraint. The discrepancies among previous studies suggest that no clear conclusions can yet be drawn regarding the levels of selective constraint in non-coding intronic DNA.Several recent studies have attempted to estimate the proportion of sites within introns that is subject to selective constraint. For example, Jareborg et al.  estimateet al.  estimateKreitman  estimaten et al.  found thD. melanogaster; a large number of short introns clustered around a minimal intron length and a broader distribution of longer introns (median intron size of 86 base-pairs (bp), mean intron size of 1411 bp; [Intron size is one possible factor that may explain these conflicting results. Comeron and Kreitman  and othe1411 bp;  showed tet al. [P = 0.03) negative correlation between intron divergence and size for first introns (but not other introns) in the dataset of Halligan et al. [et al. [D. melanogaster and D. simulans) by combining published data for 225 intron fragments sampled from a much broader distribution of intron lengths and positions within genes.If regulatory elements occur frequently in introns, and these are of some minimal size, it follows that size may be an important factor in intron evolution. In agreement with this prediction, Marais et al.  noted a n et al. . Marais  [et al.  suggeste [et al.  and also [et al. . BecauseDrosophila genome. The relationship between intron length and nucleotide divergence for all complete introns and intron fragments surveyed is shown in Figure Rs = -0.388, P < 10-4). We also divided the data into two size classes based on the median intron size of 86 bp in Drosophila [P < 10-4). The correlation between intron length and divergence is somewhat weaker, but still significant within the longer intron class .We investigated levels of divergence at a total of 225 introns  scattered across the osophila ; small . Mean divergences were not significantly different between first and non-first introns when compared within short and long size classes  but not small introns . This finding is consistent with the conclusions of Halligan et al. [Drosophila genome may be evolving under little or no selective constraint, most intronic DNA in the genome is likely to be evolving under considerable constraint.While this is strong evidence for evolutionary constraint on longer introns, short introns do not appear to evolve much more slowly than synonymous sites in n et al.  that intn et al. . Thus, iDrosophila [Rs = -0.345, P < 10-4)   Figure , and a s) Figure . The parDrosophila [Similar to the pattern we observe in introns, a negative association between synonymous site substitution rates and GC content at the third position of codons has previously been noted in osophila  and in mosophila . This paosophila ,23; howeosophila ,24, or tosophila .u and GC mutates to AT at rate ku the equilibrium frequency of GC for neutral sites (neglecting polymorphic sites) is approximated by p = 1/(1 + k), and the equilibrium rate of substitutions is K = 2uk/(1+k) [K = 2u(1 - p), so that the equilibrium rate of substitution is negatively and linearly related to GC content. This formula predicts that the intercept (divergence at zero GC content) is equal to the absolute value of the slope, and so this hypothesis is testable. The regression coefficient of divergence on GC content in the complete dataset is -0.180 (-0.254/-0.106), and the corresponding intercept is 0.157 (0.115/0.163), which at first sight is consistent with the hypothesis that variation in the level of the mutational bias parameter, k, is sufficient to account for the relation between divergence and GC content.The possible role of mutational bias can be examined using the following method. It follows from the standard model of drift and reversible mutation that, if AT mutates to GC at rate uk/(1+k) ,27. Thiset al. [The relationship between divergence and length, however, makes the above test problematic, in view of the wide variation in intron length. If only the 127 short introns (length ≤ 86 bp) are used, which are much more uniform in length, the regression of divergence on GC content is almost unchanged at -0.116 (-0.207/-0.023), and the intercept is 0.150 (0.142/0.162). Note, however, that there is a significant partial correlation of 0.166 (0.041/0.345) between GC content and length for short introns, but not for long introns, so there is still a residual relation between length and GC content in short introns. While we cannot rule out the possibility that biased gene conversion and/or selection in favour of GC versus AT explains the relationship between GC content and divergence, our analysis suggests that variation in mutational bias may be sufficient. If this process also explains the relationship between synonymous site divergence and GC content, tests for selection on codon bias based on negative correlations between codon bias and divergence  lose thet al. .The correlation analyses strongly suggest that longer introns show lower levels of divergence, and that this is not simply caused by mutational rate differences related to GC content, although other sources of mutation rate differences cannot of course be ruled out. So why might longer introns be subject to higher levels of constraint? Introns are known to contain regulatory elements  and marginally significantly lower than for introns of intermediate size . The non-binomial standard deviation in divergence is estimated to be 0.0056 for the very long introns, compared with 0.023 for the 38 intermediate-sized ones for which fragments at least 20 bp shorter than the introns were used for estimating divergence (this ensures that both classes represent samples rather than complete sequences). This is the opposite pattern to what is expected with strong clustering of regulatory sequences. Levels of constraint, and thus the density of putatively funtional regulatory elements, therefore appear to be relatively uniform across longer introns.Are putative regulatory elements in longer introns discrete entities (such as clusters of binding sites for transcription factors), or is this regulatory function more diffuse? If intronic regulatory elements occur in clusters, surrounded by unconstrained regions, we might expect to find higher levels of divergence in the short, several hundred base-pair regions of very long introns (such as those surveyed here), compared to intermediate-sized introns, provided that they have similar total amounts of regulatory sequences. The rationale for this is that, if constrained regulatory elements are clustered into one region, short fragments of very long introns would be unlikely to coincide by chance with a functional element, whereas similarly sized regions from introns of intermediate length would be more likely to coincide with such elements. Such clustering is possible, given that transcription factor binding sites and regulatory elements can range in size from a few base-pairs up to several hundred base-pairs . If D. melanogaster Adh gene reduce splicing efficiency and decrease production of the Adh protein. These authors show that compensatory mutations that restore the secondary structure result in a mutant indistinguishable from the wild type in splicing efficiency and protein production. A hairpin structure in the second intron of this gene also shows striking structural conservation across ten species in three sub-genera of Drosophila [A uniform density of regulatory functions is unexpected if these often involve clusters of, for example, transcription factor binding sites. However, it might be expected, for example, if the regulatory functions of introns often involve the formation of complex secondary structures. Evidence suggesting that intron sequence and length affects the secondary structure of precursor messenger RNA (pre-mRNA) is accumulating. If this secondary structure plays a regulatory role, it is likely to be conserved. Several studies have found evidence for epistatic selection on introns to maintain pre-mRNA secondary structure -39, and osophila . Our finDrosophila are relatively short, but these short introns make up only a small fraction of total intronic DNA in the genome. We demonstrate that levels of selective constraint are higher with increasing intron length. Thus, while the majority of introns in the Drosophila genome may be evolving under little or no selective constraint, the majority of intronic DNA in the genome is likely to be evolving under considerable constraint. We also find that the density of functionally important elements within longer introns does not appear to depend on their length. This suggests that functional elements may be ubiquitous within longer introns and that these introns may have a more general role in regulating gene expression than previously appreciated, possibly via the formation of pre-mRNA secondary structures. This pattern contrasts with that found in mammals, where constraint does not appear to be a function of intron length [Drosophila may reflect mutational biases rather than selection for codon usage.Most introns in n length  . Several previous studies have documented a positive relationship between exon length and synonymous site divergences [As a comparison for levels of divergence at intron sites, we used synonymous site divergences from 102 genes compiled by Betancourt and Presgraves . Single-ergences -59. Thisergences   and GC content . When comparing within size classes (≤86 bp versus >86 bp), however, levels of divergence are not significantly different between autosomal and X-linked introns, and GC content is significantly different for the short intron class, but not the long intron class. The negative correlation between intron length and divergence holds for autosomal and X-linked introns separately  as does the negative relationship between GC content and divergence . The differences in levels of divergence and GC content between autosomal and X-linked introns, therefore, cannot explain the observed relationships between intron length versus divergence and GC content versus divergence.As our data come from three different sources, we investigated possible biases relating to how and why the data were collected. In particular, the studies of Haddrill et al.  and Glin [et al.  surveyed [et al.  containset al. [et al. [et al. [The following additional data are available with the online version of this paper. et al. , Haddril [et al.  and Hall [et al.  data, reAn Excel file listing all introns analyzedClick here for fileet al. [Alignments of the Glinka et al.  dataClick here for fileet al. [Alignments of the Haddrill et al.  dataClick here for fileet al. [Alignments of the Halligan et al.  dataClick here for filePrograms written to carry out partial moment correlations, least-squares regressions and bootstrapping procedures and the data used for these analysesClick here for file


The origin of spliceosomal introns is the central subject of the introns-early versus introns-late debate. The distribution of intron phases is non-uniform, with an excess of phase-0 introns. Introns-early explains this by speculating that a fraction of present-day introns were present between minigenes in the progenote and therefore must lie in phase-0. In contrast, introns-late predicts that the nonuniformity of intron phase distribution reflects the nonrandomness of intron insertions.In this paper, we tested the two theories using analyses of intron phase distribution. We inferred the evolution of intron phase distribution from a dataset of 684 gene orthologs from seven eukaryotes using a maximum likelihood method. We also tested whether the observed intron phase distributions from 10 eukaryotes can be explained by intron insertions on a genome-wide scale. In contrast to the prediction of introns-early, the inferred evolution of intron phase distribution showed that the proportion of phase-0 introns increased over evolution. Consistent with introns-late, the observed intron phase distributions matched those predicted by an intron insertion model quite well.Our results strongly support the introns-late hypothesis of the origin of spliceosomal introns. The origin of spliceosomal introns – "extra" DNA sequences that disrupt the coding regions in nuclear genes of eukaryotes – is still a mystery. Since the evolution of introns is closely related to the evolution of eukaryotic genomes, understanding the origin of introns is vital for understanding the evolution of eukaryotes. There are currently two opposing theories of intron origin. The introns-early theory proposes that introns already existed at the progenote  to facilitate the construction of the first genes -4. The iIntrons can be located in one of three phases: phase-0, -1, and -2 introns are defined as introns located before the first, after the first, and after the second nucleotide of a codon, respectively. The phase of an intron is conserved during evolution, because a variation in intron phase is possible only through simultaneous mutations that alter the 5' and 3' ends of the intron in a complementary manner . The disThe introns-early theory explains the non-uniform distribution by speculating that 35% of modern introns are ancient, i.e., existed at the progenote to facilitate the assembly of the first genes ,11. SincIn this paper, we tested the introns-early and introns-late theories using two independent approaches: (i) by inferring the evolution of intron phase distribution and (ii) by retesting whether intron phase distribution reflects the nonrandomness of intron insertions. The results show that there is a general trend over evolution toward increasing the preponderance of phase-0 introns and that the observed phase distribution of introns can be indeed explained by an intron insertion model. Consequently, our results seem to support the explanation provided by the introns-late theory for the nonuniformity of intron phase distribution.Arabidopsis thaliana and the other from the ecdysozoa ancestor to Caenorhabditis elegans, the differences between phase distributions of gained introns and ancestral introns are statistically significant . In contrast, differences between the phase distributions of lost introns and ancestral introns are not statistically significant for any branch that has data for lost introns. Our result for the evolution of intron phase distribution thus suggests that the nonuniformity of intron phase distribution is more likely to be due to the nonrandomness of intron insertions.Figure Schizosaccharomyces pombe to 8.1 in Homo sapiens. The GC content of the coding regions in the genomes ranges from 24% in Plasmodium falciparum to 56% in Neurospora crassa, and the distribution of phase-0 introns ranges from 38.2% in N. crassa to 57.6% in A. thaliana. In all species the intron phase distributions show an obvious pattern of phase-0 > phase-1 > phase-2; the only exception is A. thaliana, in which the distribution of phase-2 introns is slightly larger than that of phase-1 introns. These results are consistent with previously published results , but did not match for GC-poor species with GC content <50% . For all GC-poor species, the largest errors in prediction occurred in phase-0 and phase-1 introns; the proportions of phase-0 introns were underestimated whereas those of phase-1 introns were overestimated. Note that although most Xenopus tropicalis introns are shared with H. sapiens introns (unpublished data), the GC content is 5% lower and the prediction error is much larger in X. tropicalis. Based on this observation, we speculated that the larger prediction errors in GC-poor species may be due to higher mutation rates.Figure A. thaliana, Oryza sativa, Chlamydomonas reinhardtii, and H. sapiens, and inferred the evolution of GC content and intron density  GC content and its high density of introns. The analysis indicated that 98% of A. thaliana introns already existed in its last common ancestor with O. sativa, and the inferred GC content for this ancestor was 54%. The result suggests that the large reduction in GC content (from 54% to 47%) in A. thaliana is likely to be the main cause for its large prediction error.  It is possible that when introns are inserted, the exon junctions surrounding introns are subjected to a much lower mutation rate than the average mutation rate in the genes of fast-evolving species due to the need for efficient splicing. Consequently, the intron phase distributions predicted using current sequences in fast-evolving species would not match the observed data.To test our speculation that the prediction errors were due to high mutation rates, we compiled a smaller dataset containing 79 orthologs of ribosomal protein (RP) genes from four species: y Figure . The thrl Figure . The outP > 0.05) for H. sapiens, N. crassa, Fusarium graminearum, Cryptococcus neoformans, A. thaliana, and X. tropicalis. There are several lines of evidence for the validity of our mutation correction model. First, for A. thaliana, the GC content at the best mutation rate was 57.6% .  Third, the inferred best GC contents of the two other animals: D. melanogaster and C. elegans were also very close to those of H. sapiens and X. tropicalis. Finally, our result suggests that the human genome is evolving toward decreasing its GC content, consistent with the result of Meunier and Duret [To accommodate this source of error in fast-evolving species, we proposed a simple model for mutation correction and used it to re-predict the intron phase distributions for all species in the genome-wide dataset Figure . The bess Figure . It is pnd Duret .et al. [The introns-early theory explains the excess of phase-0 introns by predicting that a fraction of present-day introns are ancient and these introns were in phase-0. If this explanation is correct, the excess of phase-0 introns should generally decrease during eukaryotic evolution as new introns are inserted into random positions. A direct test of the explanation of the introns-early theory for the excess of phase-0 introns is therefore to infer the evolution of intron phase distribution from observed data. This test was first performed by Roy et al. . Using aet al. ). In conet al.  was applet al. . Anotheret al.  are anciet al. , the reset al.   account quite well for the observed distributions  than in N. crassa (38:34:28). This indicates that the change in intron insertion frequencies has stronger effect on the intron phase distribution than the change in DNA sequences.The intron phase distributions are lineage-specific and may be affected by two factors: changes in DNA sequences and changes in intron insertion frequencies. The latter may reflect changes in the efficiency with which the splicing machinery splices out introns. When the intron insertion frequencies learned from The debate surrounding introns-early versus introns-late remains vigorous ,24. We pD. melanogaster, A. gambiae, H. sapiens, C. elegans, Saccharomyces cerevisiae, S. pombe, A. thaliana, and P. falciparum. We used this database for our analysis, but excluded S. cerevisiae due to its sparse intron distribution. Following our previous analysis [Koonin's group  compiledanalysis , we assuanalysis  assumes analysis  but the analysis ,30.H. sapiens, D. melanogaster, C. elegans, S. pombe, A. thaliana, and P. falciparum) from NCBI [N. crassa, F. graminearum, and C. neoformans) from BROAD Institute's Fungal Genome Initiative website [X. tropicalis from the JGI Eukaryotic Genomics website [X. tropicalis, gene structures were built using annotation. For X. tropicalis, there was no annotation for gene structures, so we first used the cDNA sequences as input to the BLAST program [ad hoc program was written in the C programming language to automate the construction of gene structures.We downloaded data about the genomes of six eukaryotes . Let Oi (i = 1..N) be the count of pattern i among all observed splice sites; Ci (i = 1..N) be the count of pattern i in all coding regions; and Dij  be the count of pattern i appearing at phase j in all coding regions. The preference of intron insertion in pattern i is proportional to Ei = Oi/Ci and the frequency of intron insertion in pattern i, Fi, is calculated by:In the all-pattern intron insertion model, we used patterns of 5-bp length, with 3 bp upstream and 2 bp downstream of the splice sites. The number of patterns, j introns, Pj, is calculated by:Then the expected number of phase-j introns, Wj, is calculated by:Finally, the expected percentage of phase-H. sapiens, A. thaliana, O. sativa, and C. reinhardtii. The RP genes of H. sapiens and A. thaliana were taken from the manually curated Ribosomal Protein Gene database [O. sativa and C. reinhardtii were first collected from the TIGR Rice Genome Annotation website [A. thaliana as queries. Their gene structures were then manually constructed by using both annotation and the gene structures of H. sapiens and A. thaliana as references. When a gene of a species existed in multiple copies, the copy with the most introns was used.We compiled 79 orthologs of RP genes from four eukaryotes: database . The RP  website  and the  website , respectad hoc program was written in C to extract an intron presence/absence matrix and the conserved DNA regions of these alignments. The conserved regions were then concatenated together and the DNAML program of the PHYLIP package [H. sapiens as the outgroup) were used as input to our maximum likelihood method [Multiple sequence alignments for each of these gene orthologs were built using CLUSTAL W , and an  package  was usedd method  to inferCi and Dij taken from the mutated sequences instead of the original sequences. The simulated mutation correction was repeated 20 times and the average of the 20 χ2 values between the predicted and observed intron phase distributions was used as the prediction error for the mutation rate. We then searched for the best mutation rate  in the range  using the Brent search algorithm [We applied a simple model for mutation correction in which only mutations see  that chalgorithm . Intron RP: ribosomal proteinHDN, MY, and NK conceived and designed the research. HDN performed the experiments. HDN, MY, and NK analyzed the data. HDN contributed analysis tools. HDN and NK wrote the paper. All authors read and approved the final manuscripts.A. thalianaVariations of GC content and prediction error as a function of mutation rate in . The file shows the variations of GC content and prediction error as a function of mutation rate in A. thaliana.Click here for fileList of all allowable mutations. The file lists all allowable mutations used for mutation correction.Click here for file


Arabidopsis thaliana has identified introns residing in both coding and non-coding regions of the genome.The majority of introns in gene transcripts are found within the coding sequences (CDSs). A small but significant fraction of introns are also found to reside within the untranslated regions (5'UTRs and 3'UTRs) of expressed sequences. Alignment of the whole genome and expressed sequence tags (ESTs) of the model plant A. thaliana EF1α-A3 gene affects the gene expression and the size of the 5'UTR intron influences the level of gene expression.A bioinformatic analysis revealed some interesting observations: (1) the density of introns in 5'UTRs is similar to that in CDSs but much higher than that in 3'UTRs; (2) the 5'UTR introns are preferentially located close to the initiating ATG codon; (3) introns in the 5'UTRs are, on average, longer than introns in the CDSs and 3'UTRs; and (4) 5'UTR introns have a different nucleotide composition to that of CDS and 3'UTR introns. Furthermore, we show that the 5'UTR intron of the Introns within the 5'UTR show specific features that distinguish them from introns that reside within the coding sequence and the 3'UTR. In the EF1α-A3 gene, the presence of a long intron in the 5'UTR is sufficient to enhance gene expression in plants in a size dependent manner. Introns, first discovered in 1977 ,3. ThereA growing number of plant expression studies on chimeric RNA have demonstrated that such intron sequences can enhance the level of protein expression, a phenomenon termed Intron-Mediated Enhancement (IME) -10. InclArabidopsis thaliana has a compact genome and generally small introns [The plant  introns , consist introns ,20. On t introns . Therefo introns , and it  introns . The ele introns -26.A. thaliana 5'UTR U2 introns supported by EST and cDNA data. Our results show that, firstly, the density of introns in the 5'UTRs is similar to that in the CDSs but much higher than that in the 3'UTRs; secondly, introns within the 5'UTR are not randomly distributed along the UTR but are more likely to be located closer to the ATG; thirdly, the introns that reside within the 5'UTR are, on average, significantly larger than the average intron found in both the CDS and 3'UTR; and finally, the sequences around the splicing junctions show distinct nucleotide bias that distinguish them from CDS and 3'UTR introns. Our findings indicate that 5'UTR introns may be subject to different selective forces from the introns in CDSs and 3'UTRs, possibly due to a specific regulatory role in gene expression. These observations are exposed in the well-annotated and relatively compact Arabidopsis genome.Studies on plant introns have revealed a strong nucleotide bias toward T proximal to the AG intron acceptor site, and throughout the intron there is an A/T bias relative to the adjacent exon . While tA. thaliana gene EF1α-A3 – which has an intron-containing 5'UTR – was undertaken in order to investigate what influence 5'UTR introns have on gene expression, and how this is affected by intron length. We confirm that the presence of the 5'UTR intron in EF1α-A3 increases gene expression [To complement the bioinformatic analysis, an experimental analysis of the pression -15 levelA. thaliana introns was undertaken, using the TAIR (The Arabidopsis Information Resource) database [The presence, frequency, length distributions, and structure of introns and exons have been extensively studied -35. Whildatabase . This st-3, 2.7 × 10-3 and 2.9 × 10-4 in 5'UTRs, CDSs and 3'UTRs, respectively. Since these intron densities are normalized by the total length of sequence, incomplete UTR annotation should not greatly bias these results. Thus the intron density in 5'UTRs is ~60% of the intron density in CDSs and ~5.5 times the intron density in 3'UTRs. In mammals, a marked under-representation of introns in the 3'UTRs may be explained by the requirements for nonsense-mediated decay (NMD) [A. thaliana may indicate that plants utilize a similar NMD pathway.There are 32,955 annotated protein-coding genes supported by EST sequences in the TAIR database. Of these, 18,285 include both the 5'UTR and the 3'UTR, 527 contain the 5'UTR but not the 3'UTR, 1,979 contain the 3'UTR but not the 5'UTR, and 12,171 contain neither. Considerably more genes were found to lack 5'UTR annotation than 3'UTR annotation, probably due to the directional construction of cDNA libraries from the polyA tail. A number of the annotated 5'UTR sequences are expected to be only partial sequences; however, this is not expected to greatly affect the conclusions of this paper (see below). Many protein-coding genes (72.0%) contain introns. While the non-coding regions are less likely to include introns, these are more commonly found in the 5'UTR: 19.9% of annotated 5'UTRs contain introns; by comparison only 5.6% of 3'UTRs are annotated to include introns. The high number of intron-containing 5'UTRs cannot be explained by the 5'UTR lengths, as the average 5'UTR that has been delimited by EST and/or cDNA sequences is considerably shorter than the average 3'UTR region  = 56 nucleotides, UQ (upper quartile) = 175 nucleotides; 3'UTR: median = 208 nucleotides, LQ = 154 nucleotides, UQ = 283 nucleotides). Indeed, Table ay (NMD) ,39. The p-value < 10-15). Notably, there is a significant under-representation of short introns between 50 nucleotides and 150 nucleotides and a notable increase of introns above 200 nucleotides. These results are not expected to be greatly biased by incomplete UTR annotation. The significant differences between the size of introns that reside within the 5'UTRs compared to introns that reside within CDSs and 3'UTRs may be a feature that influences their role in enhancing translation levels. It is also plausible that the large 5'UTR introns function as spacers within the genome, providing an AT-rich stretch of sequence between coding sequence and promoter.Figure trans-factors and the ribosome. We compared the observed 5'UTR intron position distributions with the distributions that would be expected if introns were distributed uniformly (i.e. constant insertion probability after any given nucleotide) throughout 5'UTRs – calculated using Monte-Carlo simulations (see Methods). Figure It has been suggested that the splicing of 5'UTR introns would lead to deposition of EJCs, which interact with translation initiation, resulting in translational enhancement -26. If tAn investigation of the nucleotide preference around the splice junctions allows a more detailed comparison between the UTR introns and the CDS introns. Sequence logos  were creA. thaliana was fused to the firefly luciferase reporter gene in the Ti binary vector pGreenII 0800-LUC [Agrobacterium-mediated transient infiltration into Nicotiana benthamiana leaves, followed by dual-luciferase assay [Renilla luciferase (REN), which provides an internal control and is under the transcriptional regulation of the 35S promoter. Thus, the activity of the LUC can be normalized by the activity of the REN, in order to minimize experimental variability such as differences in expression caused by different plants, leaf age and infiltration volume. Figure 2 = 0.95), the regression gradient and its standard error were taken as the measurement of reporter gene activity. The relative expression of pGEF1I is 0.0340 ± 0.0016 and pGEF1Idel is 0.0139 ± 0.0008, indicating a significant  enhancement of reporter gene activity under the presence of the 602-nucleotide 5'UTR intron. This data is consistent with IME requiring transcription, and acting post-transcriptionally on RNA to influence either stability or translatability.In order to investigate the effects of 5'UTR introns on gene expression, the annotated intron-containing 5'UTR of EF1α-A3 (AT1G07940) from 0800-LUC , resultise assay . The assArabidopsis to confirm the level of IME obtained in transient assay data for the pGEF1I and pGEF1Idel fusions. The plasmids used for our transient assay were modified to contain the plant kanamycin transformation selection gene and transformed into A. thaliana 'Columbia' by the floral dip method [Renilla luciferase gene was inactive, despite being under the transcriptional control of the 35S promoter. Therefore the relative expression of LUC was not determined. It is not unusual for some reporter genes to become inactivated during the transformation process [Renilla luciferase gene to this form of gene-silencing. This transgene silencing is probably because of a post-transformation event, such as methylation-mediated transcriptional silencing [t-test, p-value = 0.0037). This is consistent with the transient expression data. These T1 lines were not analyzed for T-DNA copy number, so some of the variability between transgenic lines containing the same construct could be due to the number of T-DNA insertions in each of the transgenic lines.We also generated transgenic p method . Figure  process , though ilencing . NonetheAgrobacterium cell as the dual-luciferase reporter cassette. The P19 enzyme is a viral silencing suppressor from the tomato bushy top virus (TBTV), which prevents activation of PTGS [Post-transcriptional gene silencing (PTGS) or RNAi is a mechanism that is used to degrade RNA transcripts after they have been transcribed . PTGS is of PTGS . Althougt-tests: 1ABC p-value = 2.8 × 10-5; 2ABC p-value = 6.7 × 10-5; 3ABC p-value = 4.0 × 10-4). On the other hand, if constructs are grouped by their 5' end, there is no obvious correlation between intron length and LUC activity.One of the most prominent observations from the bioinformatics analysis was the dramatic difference in the length distribution of 5'UTR introns compared with both CDS and 3'UTR introns Figure . To testThese observations suggest that IME in EF1α-A3 is mediated by, not one, but three or more elements distributed over the 5' 350 nucleotides of the 5' UTR intron, with relatively little effect from the 3' 250 nucleotides. Indeed, multiple AT-rich stimulatory elements have been previously described in plants ,30-33. aA. thaliana 5'UTR introns in the TAIR database and shown that 5'UTR introns are noteworthy in terms of their nucleotide composition around the splicing donor and acceptor site, the distribution of intron sizes and the position distribution within the UTR and proximity to the ATG start codon. In addition, we have shown that, not only can the presence of an intron in the 5'UTR significantly enhance gene expression in at least one gene, but the length of intron also influences the level of gene expression. These results should be beneficial in determining the mechanism of IME in plants, as well as determining the origin and role of 5'UTR introns. As these introns are not embedded within coding sequence, the flanking nucleotides can be modified without interfering with the open reading frame (cf. CDS introns). We believe that this makes the introns that reside within non-coding sequences a powerful resource to assist in unravelling the role of introns within the genome.A growing number of plant expression studies have revealed that the presence of an intron within the 5'UTR induces enhanced RNA and protein accumulation. However, the intrinsic determinants of 5'UTR IME in plants, especially the role of any sequence motifs within the intron, remain poorly defined. In this paper, we have presented extensive statistical analyses of all the annotated A. thaliana introns were analysed using the TAIR (The Arabidopsis Information Resource) database [In this study, the statistics of database ,55. The database  for sevedatabase , and 2)A. thaliat-tests, Kolmogorov-Smirnov tests etc.) were calculated using R. Also, Figures Data were processed using C-shell scripts, C++ programs, and the statistics package R version 2.0.1 . Basic sThe Monte-Carlo simulation program – written in C++ – was used to calculate the expected position distribution of 5'UTR introns if introns were distributed uniformly (i.e. constant insertion probability after any given nucleotide) throughout 5'UTRs. To do this, all of the original introns were extracted from all of the original 5'UTRs, and then they were randomly re-inserted into the processed 5'UTR sequences. This was repeated 10,000 times, and the average intron position distributions were calculated. Sequence logos were drawn using WebLogo version 2.8 . The inpArabidopsis EF1α-A3 (AT1G07940) gene was isolated from genomic DNA of 'Columbia' using primers RPH-130 (TCTAGAATGGTACCTAATTACTTCAC) and RPH-131 (CTCTTTACCCATGGTTAGAGACTG). The PRH-130 primer altered the sequence at the 5' end of this promoter, introducing a KpnI site as well as an NcoI site at the ATG of this gene. The PCR product was cloned into pGEM-T Easy (Promega) and sequenced to ensure accurate amplification. Similarly, four other promoters were isolated: AT1G10670 (0.651 kb) CAS-001 (GGTACCCACAAATGGAATGGTTGAAG) and CAS-002 (CTTCCTCGCCATGGCAAAACGAAAACTGG); AT1G13980 (2.1 kb) CAS-013 (GGTACCTAGAGGTGTGTATGATAATG) and CAS-014 (CCATGGAATCTGCTCAAATCTTCAGCCAG); AT1G17470 (0.92 kb) CAS-017 (GGTACCTGTAGCGTTTCTACTCTCGT) and CAS-018 (CCATGGTGCTTCACTTGTTTTTGC); AT1G72050 (2.7 kb), CAS-021 (GGTACCATTCGGTCACTGAAGACAC) and CAS-022 (CCATGGTGCGTGATCGAGGCTTACTTGC). In all cases, a KpnI site was introduced at the 5' end of the promoter and an NcoI site at the ATG.The 1.87 kb promoter of the Pwo polymerase (Roche), using the pGEM-T Easy clone of the intron-containing promoter as template. PCR products were then treated with polynucleotide kinase (NEB) and re-circularized with T4 DNA ligase (NEB). Because of the blunt termini created by the Pwo polymerase, the ligated product excludes the intervening sequence between the divergent primers and, in our case, precisely removed the intron sequence as it would be in the processed mRNA. In the same way, intronless versions for 4 other Arabidopsis promoter clones were generated: AT1G10670, CAS-003 (CGTTGAGAGAGAATGGGGGTAG) and CAS-004 (TTTTCGTTTTGCCATGGCGAGG); AT1G13980, CAS-015 (TGTTTCTCCAGCGATTCAGAG) and CAS-016 (TAATGATTGAGTTTGGCCTCTATC); AT1G17470, CAS-019 (AGTGGAATTGGTGAAGGGCG) and CAS-020 (ATAGTAGCAAAAACAAGTGAAGC); AT1G72050, CAS-023 (CTGATGATGAGATTGGATGTG) and CAS-024 (ATGGAACTTGAAGAGGAAAGAG) for intron 1, CAS-025 (CTCGAGCGAATGACTCTGCA) and CAS-026 (GAAATAAATAGCCTTTTGTTT) for intron 2. As AT1G72050 has two 5'UTR introns, two rounds of intron-out-PCR were needed to generate the intronless version of this promoter.Intron-containing promoter-UTR clones were modified to become intronless by a version of inverted PCR. In the case of AtEF1α-A3 (AT1G07940), two primers were utilized. A forward primer, RPH-133 (CTCAGAGATATCGCAAGAGAG), corresponded to the region of the sequence at the 3' end of exon I, the sequence that precedes the 5'UTR intron; this primer is homologous to the complementary strand and primes towards the upstream promoter region. A reverse primer, RPH-134 (ATTTGTTTGACAGTCTCTAAC), corresponded to the 5' region of exon II, the sequence that directly follows the 5'UTR intron. These two primers were used in a PCR amplification with Renilla luciferase (Promega) reporter gene (REN) under the transcriptional regulation of 35S promoter and CaMV terminator [Agrobacterium GV3101 (MP90) according to [NcoI site (CCATGG), the fusion between promoter-5'UTR and reporter gene contains no intervening sequence. The intron-containing and intronless constructs containing the AtEF1α-A3 promoter were converted to stable plant transformation vectors by inserting a nos-kan selection cassette [Promoter fragments were subcloned into the binary vector pGreenII 0800-LUC . This verminator , and a prding to . As the cassette  downstreDeletions within the EF1α-A3 intron were achieved by performing the same PCR method used to remove the whole 5'UTR intron described above. A combination of diverging primers to the 5'  and 3'  of the deletion point were used in generating nine intron deletions. Primer A , Primer B , Primer C , Primer 1 , Primer 2 , Primer 3 .Agrobacterium GV3101 (MP90) [Agrobacterium were cultured in Lennox agar (Invitrogen) with 50 mg/ml kanamycin (Sigma) at 30°C for 3 days. Cells were re-suspended in infiltration media  until OD600 = 0.2 and allowed to incubate at room temperature for 2 hours prior to infiltration. Re-suspended Agrobacterium were infiltrated into the leaves of 3–4 weeks old Nicotiana benthamiana  and the plants were allowed to grow for a further 3 days. Infiltrated patches were ground in 500 μL Passive Lysis Buffer (PLB) (Promega), diluted (5:500 in PLB), and 5 μ'L used for dual-luciferase assay using the Dual-Luciferase Reporter (DLR™) Assay System (Promega). Relative light units (RLU) were measured over 15 seconds following 5 seconds of delay with a Turner 20/20 luminometer. Relative luciferase activity was calculated by performing a regression analysis from 6 independent measurements using the statistics package R version 2.0.1 [Reporter gene plasmids were electroporated into 1 (MP90) . Agrobacon 2.0.1 .A. thaliana 'Columbia' were created via the floral dip method [The transgenic p method  with theArabidopsis promoters, intron deletions and generation of stable transgenic plants. AEF: bioinformatics. CMB: supervision of BYWC. All authors read and approved the final manuscript.BYWC: bioinformatics, data analysis and manuscript preparation. RPH: experimental design, EF1α-A3 intron deletions, transient assays data analysis and manuscript preparation. CS: isolation of


Accumulating evidence indicates that the nascent RNA can invade and pair with one strand of DNA, forming an R-loop structure that threatens the stability of the genome. In addition, the cost and benefit of introns are still in debate.At least three factors are likely required for the R-loop formation: 1) sequence complementarity between the nascent RNA and the target DNA, 2) spatial juxtaposition between the nascent RNA and the template DNA, and 3) accessibility of the template DNA and the nascent RNA. The removal of introns from pre-mRNA reduces the complementarity between RNA and the template DNA and avoids the spatial juxtaposition between the nascent RNA and the template DNA. In addition, the secondary structures of group I and group II introns may act as spatial obstacles for the formation of R-loops between nearby exons and the genomic DNA.Organisms may benefit from introns by avoiding deleterious R-loops. The potential contribution of this benefit in driving intron evolution is discussed. I propose that additional RNA polymerases may inhibit R-loop formation between preceding nascent RNA and the template DNA. This idea leads to a testable prediction: intermittently transcribed genes and genes with frequently prolonged transcription should have higher intron density.This article was reviewed by Dr. Eugene V. Koonin, Dr. Alexei Fedorov (nominated by Dr. Laura F Landweber), and Dr. Scott W. Roy (nominated by Dr. Arcady Mushegian). Introns are intervening sequences that are spliced out of RNA transcripts. Four major classes of introns are recognized: group I introns, group II introns, tRNA/archaeal introns, and spliceosomal introns. Introns are found in all major groups of organisms on earth from bacteriophages to mammals , and reaThe large number of introns in eukaryotic genomes hints that they may confer some selective advantages to overweigh their costs . Variousin vitro transcription of some sequences, 42%–63% of the template DNA molecules form R-loops with nascent RNAs [The R-loop is a structure in which RNA invades and pairs with one strand of DNA to form an RNA-DNA hybrid Fig.  18-20].-20.18-20ent RNAs ,21,22. Aent RNAs ,23. Forment RNAs ,25. In tAs R-loop formation is a transcription-related phenomenon, is highly expressed genes more liable to form R-loops with their transcripts? In transcription bubble, nascent RNA is paired with the DNA template. But such short DNA:RNA hybrids are unlikely the cause of transcriptional R-loops. Some evidence has shown that nascent RNA molecules are separated from the template DNA by RNA polymerase after it has emerged from the exit channel of the RNA polymerase ,27. ThusThe transcription processes in starving cells are likely to be prolonged because of substrate- or energy-limitation. According to the above hypothesis, the genes being transcribed in starving cells are liable to be damaged by transcriptional R-loops. In facts, there are many observations dating back to 1988 showing starved cells experience much (tens or even hundreds of times) higher mutation rates than fast-growing cells -34. ConsThe rate of ectopic recombination between DNA molecules declines as the homology length decreases . SimilarSimilar ideas were previously published by other researchers. The fragmentation of a gene into exons may protect the coding sequence from recombination with its own processed pseudogenes ,14. FedoRecent work has revealed that intron splicing usually occurs coincident with transcription, beginning just after transcription of the sequence to be spliced , readers can find comparisons of this hypothesis with previously ones, and a quantitative estimation for the benefit of R-loop avoidance.The major groups of introns, Group I/II introns and spliceosomal introns, may have the effect of protecting exons from deleterious R-loops. Although speculative and somewhat naive, I propose that the benefit may be selected as a function of introns in evolution. It is also possible that avoiding R-loops by the presence of introns is just a subsequent and secondary property, which came in well after introns and splicing machinery became established. Till now, I am not sure how strong the effect of avoiding R-loops is, and how much the benefit has driven the evolution of introns. Regardless of the quantitative uncertainty, this is the first time to propose that introns may have the effect of protecting exons.The author(s) declare that they have no competing interests.Eugene V. Koonin, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USAWe do not know why all eukaryotes (so far) have introns; what seems, more or less, certain, is that there is a complex web of neutral and selective factors underlying this quintessential feature of eukaryotes. So any reasonable proposal on the raison d'etre of introns is of interest. The hypothesis discussed in this paper, namely, that introns prevent the formation of deleterious R-loops by limiting, via cotranscriptional splicing, the amount of nascent RNA that is available for hybridization with the genomic DNA at any given time, is one such idea, and welcome in that capacity. However, I cannot help thinking that the idea is rather weak. Indeed, introns seem like an awfully expensive way to avoid R-loop formation. Why not simply sequester the growing RNA chain via the polyadenylation complex and the nucelocytoplasmic export machinery? In fact, eukaryotes do just that. Furthermore, there are many virtually intronless eukaryotes  in which introns cannot protect genomes from R-loops but which nevertheless survive just fine. Again, to the extent R-loops are, indeed, a menace, they are avoided by sequestering the nascent transcripts in a variety of complexes. One could argue, with rather good reasons, that these sequestering mechanisms themselves descend from the ancestral splicing machinery, so the role of introns in the avoidance of R-loop formation might have been greater at the early stages of eukaryotic evolution. I believe this is what the author implies toward the end of the paper. Nevertheless, at this stage, I cannot avoid the conclusion that the proposed mechanism, if real, only can be a minor contributor to the evolution of eukaryotic gene structure. I find it commendable that, in the concluding remarks, the author is very candid about the uncertainty with respect to the actual importance of R-loop avoidance.Author response: I agree with the comments. The actual importance of R-loop avoidance by introns is uncertain now. Further studies are required for a conclusion.Alexei Fedorov, Director of Bioinformatics Lab, the University of Toledo, Toledo, OH 43614-5809, USA (nominated by Dr. Laura F Landweber)This paper describes one of the most intriguing and incomprehensible questions in molecular biology – origin and evolution of introns. The author shows deep understanding of multiple problems associated with existence of exon/intron gene structures. After 25-years of intron early-or-late debate it is absolutely clear that nobody can prove or disprove a particular intron evolution hypothesis among a number of proposed ones. Thus, I do not expect a paper to resolve this very intricate problem and welcome any new fresh look on this subject.I read this MS with interest and think that it deserves publication. However, I am disappointed about the absence of any quantitative estimations of the effect of hybridization of transcripts with their DNA matrixes. Even in the conclusion the author writes: "I am not sure how strong the effect of avoiding R-loops is, and how much the benefit has driven the evolution of introns". This is the weakest side of the MS. The author should try to provide as much quantitative estimation as possible. For example, on page 4, in the last paragraph of the Background section, the author writes: "...there are many observations since 1988 that starved cells experience high frequency of mutations." Is it 5–10% or 100–200% increase? This and all similar places must have numerical estimations which would significantly increase the value of the paper and the hypothesis. For another example on the same issue – see page 7 , the statement: "At least the translated regions of most mature mRNAs are unlikely to have stable secondary structures". This statement also lacks any quantification. However, if the author takes modern RNA folding software package  and studies local 2D structures in exons vs. introns; it appears that many exons have energetically stable secondary structures comparable to those inside introns. After examination of thousands of exonic and intronic sequences, I can claim that there is only a subpopulation of exons (about 25–30% of the entire human pool) that do not exhibit strong secondary folding . The rest of human exons are comparable to introns on this property (our yet unpublished results).Author response: Quantitative estimations are expected by any hypothesis advocator. In present case, previous experimental studies provided very little quantitative information. Also limited by my academic capacity, I am not able to do quantitative estimation. Fortunately, Dr. Roy approached a quantitative estimation in his review of this paper. His estimation is a very helpful supplement of my manuscript. I revised this manuscript with more numerical descriptions of previous experimental results.On the stable secondary structures of RNAs, there is another uncertainty. All that we know was proposed by Gowrishankar and Harinarayanan in their paper , but not demonstrated. As the stability of double helix comes partially from base stacking, the short stem-loop secondary structures of tRNAs seem less stable than continuous RNA:DNA double strand. I doubt the importance of R-loop avoidance by short stem-loop structures (like those in tRNA molecules). So I revised the statement.Finally, I agree with the author that introns could help in prevention of hybridization of transcripts with their original matrixes. In fact, we published a similar hypothesis but for RNA world .Author response: I was unaware of that paper. Now I realize that it has similar ideas, and so I cite it in the body of this hypothesis. Meanwhile, I add several other related references.Scott W. Roy, Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand (nominated by Dr. Arcady Mushegian).I have no idea whether Dr. Niu's hypothesis is true, but it is certainly intriguing and deserves to be widely read.To me, a (or perhaps the) central mystery of intron evolution concerns the unique apparent proliferation (as well as transformation) of type II introns in early eukaryotes, with no similar event in any prokaryotic lineage nor perhaps in subsequent eukaryotic evolution. Dr. Niu's hypothesis offers a possible solution to this quandary: intron proliferation would have ameliorated the mutation rate increase associated with separation of transcription and translation brought on by the nucleus.This hypothesis is important in that it is formally different from many previous hypotheses in that it (i) invokes positive selection to explain intron spread, and (ii) proposes that this positive selection solves a problem that would be unique to (early) eukaryotes.The hypothesis is different from many previous attempts to explain intron proliferation within early eukaryotes due either to (i) increased mutation rates ; (ii) decreased population size (as put forward by Lynch and Richardson as well as by Martin and Koonin); or (iii) decreased selection against introns ).At the same time, the hypothesis is different from many other previous ideas that see an advantage for introns, in that it proposes an advantage that would have been (i) immediate, rather than long-term; and (ii) would have been unique to early (or pre) eukaryotic ancestors. Many previous ideas for an advantage for introns  generally rely on subsequent additional mutations  which are expected to occur at low rates and therefore are unlikely to have led to the initial fixation of the intron itself. Other ideas have proposed types of positive selection are not specific to early eukaryotes . Other hypotheses such as Lynch and colleagues' ideas about intron spread being facilitated by NMD invoke eukaryotic-specific processes (NMD), however these processes themselves are likely largely required by introns' presence .By contrast, Niu's idea suggests a reason for general positive selection for intron spread that is specific to early eukaryotes. Given the ubiquity of introns in eukaryotes, the dearth of hypotheses based on positive selection is striking, and therefore any such hypothesis is important and at the very least thought-provoking.Now, to the hypothesis itself. Among the host of possible objections to the hypothesis that I can imagine, I believe that fairly satisfying answers are possible.The first is overkill: faced with the seemingly simple challenge of segregating nascent transcripts from DNA, why would evolution have devised as elaborate and seemingly problematic a mechanism as the spliceosomal system, rather than a simpler and presumably more efficient TREX-like transcript-coating mechanism? However, type II introns were likely available in the early eukaryotic nucleus (likely imported with the mitochondrion); type II intron transpositions that were overall favored would fix, intron numbers (and thus genome-wide transposition rates) would increase, and introns would saturate the genes. The shift towards trans-splicing would then only come secondarily. Given the positive-feedback dynamics of intron proliferation, it could be quite rapid, conceivably requiring less time than emergence of an RNA-coating protein (complex) which would need to distinguish mRNAs from non-coding functional RNAs in the cell. Introns then could emerge as the first line of defense, with TREX-like coating mechanisms only later taking over the role of transcript protection in some lineages.Nes > 1). In this case, the selective advantage to intron presence is related to the decreased mutation rate in the adjacent coding sequence. In the absence of recombination, the selective disadvantage to an allele that changes the mutation rate is roughly equal to the change in rate of mutation to disfavored alleles. So, if the general point mutation rate per generation is u and the difference in rate between intron-containing and intron-lacking alleles is xu per site, the selective advantage for having an intron which protects l adjacent sites, of which a fraction c is constrained by selection, will simply by clxu, and this selection will be sufficient to efficiently distinguish between intron-containing and intron-lacking alleles if Neclxu > 1.The second concern is whether the selective advantage proposed, of reducing the mutation rate in coding sequence upstream of the intron site, is likely to be sufficiently strong to overcome drift. In general, selection will be efficient if the selective advantage is greater than roughly the inverse of the effective population size (Neu) have been made for a range of eukaryotes, and vary from around 10-2 to 10-4 (most recently compiled by Lynch in MBE last year), thus we have the requirement clx > 102-104. For the lower value, this seems quite reasonable – if intron presence reduces the mutation rate by around twofold (i.e. x = 1) for l = 200 nucleotides of which around c = 0.5 are constrained, this would mean clx = 100, and all of these values could be quite conservative. Even values in the range of 104 seem quite not impossible: the condition would be fulfilled if a single intron protected cl = ~10,000 sites, or if x >> 1 (which may be more likely). Importantly, the hypothesis predicts that species with higher estimated Neu values should have more introns, directly opposite to the findings of Lynch .Estimates of the product of the effective population size and the mutation rate (As such, I think that the hypothesis is viable overall and deserves to be widely read. I suspect that the manuscript's most important contribution will be in pointing the way for a new set of hypotheses based on newly positively selected traits of intron presence in early eukaryotes.Author response: I appreciate the comments from Dr. Roy. Frankly, my knowledge on intron and evolution is not enough to think the subject so deeply. This report is a very helpful enhancement of the section "Implications for intron evolution". I have not integrated this report in my manuscript as often done in revising manuscripts submitted to journals with anonymous review. The traditional reviewing model is unfair to anonymous reviewers even if the authors using some grateful words like, "as suggested by the anonymous reviewers, we...". I thank Biology Direct for providing such an efficient way for both authors and reviewers to contribute to the same subject, while both are indicated.


Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be “conserved,” i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution. Genes in eukaryotes are usually intervened by extra bits of DNA sequence, called introns, that have to be removed after the genes are transcribed into RNA. Why do introns exist in eukaryotic genes? What is the reason for the increased intron density in higher eukaryotes? There is much that is not known about introns. This research tries to clarify the evolutionary process by which introns arose by comparing the gene structures of two types of ribosomal proteins; one in cytoplasm and the other in mitochondria of the cell. Since cytoplasm and mitochondria are of archaeal and bacterial origin, respectively, cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) are believed to diverge at the same time with the divergence of archaea and bacteria. Thus, a comparative analysis of CRP and MRP genes may reveal whether introns already existed at the last common ancestor of archaea and bacteria (introns-early) or whether they emerged late (introns-late). The results make it clear, at least, that all of the introns in MRP genes were gained during the course of eukaryotic evolution and therefore lend more support to the introns-late theory. There are two opposing theories regarding when spliceosomal introns originated. The introns-early theory proposes that primordial spliceosomal introns already existed at the beginning of life –3. The eIntron insertions have been proposed to occur primarily in “proto-splice sites” with the consensus sequence MAG|R, where M is A or C, R is A or G, and the vertical line represents the intron insertion site . HoweverAbout 25% to 30% of intron positions are shared between species that diverged in the distant past, such as plants and humans ,12. SomeThe ribosome is a vital component of the translational machinery and therefore of all cellular life. Consequently, ribosomal proteins (RPs) have been highly conserved throughout evolution ,18. Thus were compared with the gene structures of MRPs from five eukaryotes  ((Rickettsia prowazekii and Escherichia coli) were also included in the analyses  . In ordeanalyses . We founanalyses . We clasThe maximum parsimony method was used to infer the most parsimonious scenarios of these nine shared positions. We recently proposed a maximum likelihood approach for inferring the evolution of introns ; howeverK = 1 (see K), all nine of these shared intron positions were predicted to be the result of parallel gains. Even when intron loss was presumed to occur 100 times more easily than intron gain (K = 100), seven out of nine positions were still classified as being the result of parallel gains, whereas only one position  was classified as being the result of position conservation.  However, considering that A. thaliana has a very high rate of intron gain [The costs of the most parsimonious scenario of the two possible cases, intron position conservation and parallel intron gain, were inferred for each shared intron position . For theron gain ,26, the . The estimated average distribution of proto-splice sites at all intron positions in the whole genomes of seven of the nine species, excluding O. laptides and Ci. intestinalis, was 19% (unpublished data). The frequencies of proto-splice sites in the coding regions of 25 CRP genes  and 25 MRP genes  were 3.3% and 2.9%, respectively. The strong tendency to have proto-splice sites at intron positions shared by CRP and MRP genes supports the proto-splice site hypothesis and further supports the above conclusion that these nine shared intron positions are the result of parallel gains. The higher-than-average number of proto-splice sites at intron positions in CRP and MRP genes than in the complete genomes can be explained by the higher degree of conservation of CRP and MRP genes than of other genes.In order to analyze the nucleotide sequences surrounding the nine shared positions, multiple alignments of these sequences were generated , and theWe compared intron positions in genes of MRP origin with those in genes of CRP origin to gain insight into the evolution of spliceosomal introns. Since MRP and CRP genes are of bacterial and archaeal origin, respectively, this comparison was expected to shed new light on this topic. A similar analysis using genes of organelle origin was reported previously, but this generated extensive discussion and, due to the limited numbers of intron positions and species used, no definitive conclusion was reached –29.In this study, no clear case of intron position conservation between CRP and MRP genes was found. Most  MRP introns appear to have been inserted after endosymbiosis . We beliOur results show that parallel gains between CRP and MRP genes account for 2.3% of the total intron positions. A recent simulation estimated that parallel intron gains account for 5%–10% of shared intron positions  , consistIt is known that spliceosomal introns existed in quite high density in the last common ancestor of the three eukaryotic kingdoms: animals, fungi, and plants ,12,26. HH. sapiens, Ci. intestinalis, Dr. melanogaster, Ca. elegans, S. pombe, and P. falciparum, together with the RP genes of E. coli were taken from the manually curated Ribosomal Protein Gene database . The CRP gene sequences of O. laptides were collected from Medaka UTGB (http://medaka.utgenome.org) by performing a BLAST search using human CRP genes as queries. The CRP gene sequences of A. thaliana and Di. discoideum were first collected from the Arabidopsis Information Resource  and dictyBase (http://www.dictybase.org), respectively, by using annotation, and were then confirmed by aligning their sequences with those of other species. The RP genes of R. prowazekii were taken from the National Center for Biotechnology Information  using annotation. Human MRP genes were taken from the HUGO Gene Nomenclature Committee (HGNC) Gene Family Nomenclature's Mitochondria Ribosomal Proteins homepage (http://www.gene.ucl.ac.uk/nomenclature/genefamily/MRPs.html). The MRP genes for other species were selected in a way similar to that used for the CRP genes of A. thaliana. If there was no annotation, sequences were collected by performing a BLAST search using sequences from humans and other species. Genome homepages for the different species we investigated are as follows: Ci. intestinalis ; Dr. melanogaster; Ca. elegans ; S. pombe (http://www.sanger.ac.uk/Projects/S_pombe); and P. falciparum. When a gene existed in multiple copies, the copy with the most introns was used in the analysis. The sequences of the 25 pairs of CRP/MRP genes are available at http://ribosome.miyazaki-med.ac.jp/crpmrp/view.cgi.The CRP genes of six eukaryotes: ClustalW (1.82)  was usedgainsα × losses,β where α and β are the probabilities of gain and loss on each branch, respectively, and gains and losses are the number of gain and loss events, respectively. A scenario with higher probability of occurrence will be preferred, so the following cost function is used:Our maximum parsimony method, which is based on the assumption that intron gain and loss events occur rarely in evolution, accepted the scenario that had the least cost as measured by a function of gains and losses. Assume that the probabilities of intron gain and loss are constant across branches and are much smaller than the probabilities of unchanging intron state; then the probability of occurrence of an intron evolution scenario will grow proportionally with α or β but depends largely on the ratio K = β/α, which means intron loss occurs K times more easily than gain. Therefore, we chose to fix the value of β to 0.1. With this simplification, the cost function becomes:Notice that the relative difference between the costs of two scenarios does not depend much on the particular values of K (K = 1 and K = 100) were used. Unlike the method of Rogozin et al. [Two different values of n et al.  which coDataset S1(8 KB TXT)Click here for additional data file.Figure S1MRP homologs are indicated with light grey shading. Conserved amino acids are highlighted by a dark grey background. Phase 0, 1, and 2 intron positions are highlighted by red, blue, and green backgrounds, respectively.A. thaliana; Ce, Ca. elegans; Ci, Ci. intestinalis; Dd, Di. discoideum; Dm, Dr. melanogaster; Ec, E. coli; Hs, H. sapiens; Ol, O. laptides; Pf, P. falciparum; Rp, R. prowazekii; Sp, S. pombe .At, (243 KB PDF)Click here for additional data file.Figure S2Splice sites that are completely consistent with the MAG|R sequence are shown in red. Conserved nucleotides between CRP and MRP genes are highlighted by a grey background. + indicates the presence of intron.A. thaliana; Ce, Ca. elegans; Ci, Ci. intestinalis; Dd, Di. discoideum; Dm, Dr. melanogaster; Hs, H. sapiens; Ol, O. laptides; Pf, P. falciparum; Sp, S. pombe.At, (47 KB PDF)Click here for additional data file.Table S1O. laptides and Ci. intestinalis, the scaffold number was shown. For Di. discoideum, the dictyBaseID was shown.*For †For genes with multiple copies, only one copy was used. When the number of introns in multiple copies of the same gene was different, the one with the most introns was used.‡Gene structures were determined by a combination of these sequences.§Gene sequences were partial.A. thaliana; Ce, Ca. elegans; Ci, Ci. intestinalis; Dd, Di. discoideum; Dm, Dr. melanogaster; Hs, H. sapiens; Ol, O. laptides; Pf, P. falciparum; Sp, S. pombe. At, (59 KB PDF)Click here for additional data file.


Symbiotaphrina buchneri.Group I introns have spread into over 90 different sites in nuclear ribosomal DNA (rDNA) with greater than 1700 introns reported in these genes. These ribozymes generally spread through endonuclease-mediated intron homing. Another putative pathway is reverse splicing whereby a free group I intron inserts into a homologous or heterologous RNA through complementary base-pairing between the intron and exon RNA. Reverse-transcription of the RNA followed by general recombination results in intron spread. Here we used phylogenetics to test for reverse splicing spread in a taxonomically broadly sampled data set of fungal group I introns including 9 putatively ancient group I introns in the rDNA of the yeast-like symbiont S. buchneri) and intron lateral transfer. There are several examples in which introns, many of which are still present in S. buchneri, may have spread through reverse splicing into heterologous rDNA sites. If the S. buchneri introns are ancient as we postulate, then group I intron loss was widespread in fungal rDNA evolution.Our analyses reveal a complex evolutionary history of the fungal introns with many cases of vertical inheritance (putatively for the 9 introns in On the basis of these results, we suggest that the extensive distribution of fungal group I introns is at least partially explained by the reverse splicing movement of existing introns into ectopic rDNA sites. Escherichia coli gene) to be closely related . How  related .Long-distance intron movement can in principle be achieved by reverse splicing that facilitates intron mobility through an RNA intermediate -16. In rPhysconia perisidiosa). In addition, we included 10 rDNA group I introns from the yeast-like fungal symbiont of anobiid beetles, Symbiotaphrina spp. . In . In 30]] present ,33. This present ,35. The  present . Success present ,37.S. buchneri  contains e.g., ,23,25] w,25 wS. bSymbiotaphrina spp. were acquired for this study: S. buchneri (JCM9740) and S. kochii . DNA was isolated from these cultures using the Puregene Kit (Gentra Systems) following the manufacturer's protocol for filamentous fungi. The nuclear small (SSU) and large subunit (LSU) rDNA were amplified for each strain by PCR. In addition we amplified the RPB2 (second-largest subunit of RNA polymerase II) nuclear gene from S. buchneri JCM9740 and S. kochii CBS589.63. PCR products were purified from agarose gels using GELase (Epicenter) and directly sequenced on both strands using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems), and Big Dye . The PCR primers used in this study came from various sources  and avad ) but sad [e.g., ,23,25,55th generation. To increase the probability of chain convergence, the first 2,000 trees were discarded as burnin and the remaining 1,000 were used to calculate the posterior probabilities.A total of 136 aligned positions were selected for the initial phylogenetic analyses . For these data, we used two different approaches to infer the phylogeny. First, we used the single parameter Jukes-Cantor (JC) evolutionary model  with neiBased on the analysis of the 189-sequence data set, we generated a second reduced intron data set of 116 sequences that maintained the diversity of intron sites in the large data set. A JC-NJ tree was inferred from these data  and Bayesian posterior probabilities were calculated for the tree as described above. In addition, we did an unweighted maximum parsimony (MP) bootstrap analysis of the data. For this method, a heuristic search was used with each of the 2000 bootstrap pseudosamples and starting trees were obtained using random additions 10 rounds) with tree bisection-reconnection branch swapping. The 51-sequence data set  was analyzed with the JC-NJ, MP, and Bayesian methods as described above. In addition, we did a maximum likelihood bootstrap analysis of these data. In this approach, the gamma value (with 4 rate categories) and the transition/transversion ratio were estimated using PAUP*. Bootstrap analyses (100 replicates) were then done using DNAML (PHYLIP V3.6b ) with 1  rounds wVR and FL did the sequencing of group I introns and did the phylogenetic analysis of the fungal host tree and contributed to the manuscript. DMS sequenced fungal introns and contributed to the manuscript. DB conceived of and supervised this study and wrote the manuscript. All authors read and approved the final manuscript.


Giardia and Trichomonas, have very low intron densities, so low that canonical spliceosomal introns have only recently been discovered through genome sequencing. These organisms were formerly thought to be ancient eukaryotes that diverged before introns originated, or at least became common. Now however, they are thought to be members of a supergroup known as excavates, whose members generally appear to have low densities of canonical introns. Here we have used environmental expressed sequence tag (EST) sequencing to identify 17 genes from the uncultivable oxymonad Streblomastix strix, to survey intron densities in this most poorly studied excavate group.Certain eukaryotic genomes, such as those of the amitochondriate parasites Streblomastix genes contain an unexpectedly high intron density of about 1.1 introns per gene. Moreover, over 50% of these are at positions shared between a broad spectrum of eukaryotes, suggesting theyare very ancient introns, potentially present in the last common ancestor of eukaryotes.We find that Streblomastix data show that the genome of the ancestor of excavates likely contained many introns and the subsequent evolution of introns has proceeded very differently in different excavate lineages: in Streblomastix there has been much stasis while in Trichomonas and Giardia most introns have been lost.The  Giardia) and parabasalia (e.g. Trichomonas).One of the prominent features that distinguishes eukaryotic genomes from those of prokaryotes is the presence of spliceosomal introns. Introns are intervening sequences that are removed from expressed RNAs, in the case of spliceosomal introns through a series of transesterfications mediated by a large riboprotein complex called the spliceosome . SpliceoGiardia and Trichomonas, as well as the identification of proteins involving splicing in Trichomonas .CHS analysed the EST data, performed PCR and sequencing, and examined conservation of intron positions in other organisms. PJK collected the termites and purified RNA for library construction. Both authors participated in the writing and editing of the manuscript. All authors read and approved the final manuscript.