Related Articles

Background

Many multicellular eukaryotes have two types of spliceosomes for the removal of introns from messenger RNA precursors. The major (U2) spliceosome processes the vast majority of introns, referred to as U2-type introns, while the minor (U12) spliceosome removes a small fraction (less than 0.5%) of introns, referred to as U12-type introns. U12-type introns have distinct sequence elements and usually occur together in genes with U2-type introns. A phylogenetic distribution of U12-type introns shows that the minor splicing pathway appeared very early in eukaryotic evolution and has been lost repeatedly.

Results

We have investigated the evolution of U12-type introns among eighteen metazoan genomes by analyzing orthologous U12-type intron clusters. Examination of gain, loss, and type switching shows that intron type is remarkably conserved among vertebrates. Among 180 intron clusters, only eight show intron loss in any vertebrate species and only five show conversion between the U12 and the U2-type. Although there are only nineteen U12-type introns in Drosophila melanogaster, we found one case of U2 to U12-type conversion, apparently mediated by the activation of cryptic U12 splice sites early in the dipteran lineage. Overall, loss of U12-type introns is more common than conversion to U2-type and the U12 to U2 conversion occurs more frequently among introns of the GT-AG subtype than among introns of the AT-AC subtype. We also found support for natural U12-type introns with non-canonical terminal dinucleotides (CT-AC, GG-AG, and GA-AG) that have not been previously reported.

Conclusions

Although complete loss of the U12-type spliceosome has occurred repeatedly, U12 introns are extremely stable in some taxa, including eutheria. Loss of U12 introns or the genes containing them is more common than conversion to the U2-type. The degeneracy of U12-type terminal dinucleotides among natural U12-type introns is higher than previously thought.

Background

Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes.

Results

I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the α-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron-rich genome. Thus, it appears that ancestors of spliceosomal introns, indeed, have existed since the earliest stages of life's evolution, in a formal agreement with the introns-early scenario. However, there is no evidence that these ancient introns ever became widespread before the emergence of eukaryotes, hence, the central tenet of introns-early, the role of introns in early evolution of proteins, has no support. However, the demonstration that numerous introns invaded eukaryotic genes at the outset of eukaryotic evolution and that subsequent intron gain has been limited in many eukaryotic lineages implicates introns as an ancestral feature of eukaryotic genomes and refutes radical versions of introns-late. Perhaps, most importantly, I argue that the intron invasion triggered other pivotal events of eukaryogenesis, including the emergence of the spliceosome, the nucleus, the linear chromosomes, the telomerase, and the ubiquitin signaling system. This concept of eukaryogenesis, in a sense, revives some tenets of the exon hypothesis, by assigning to introns crucial roles in eukaryotic evolutionary innovation.

Conclusion

The scenario of the origin and evolution of introns that is best compatible with the results of comparative genomics and theoretical considerations goes as follows: self-splicing introns since the earliest stages of life's evolution – numerous spliceosomal introns invading genes of the emerging eukaryote during eukaryogenesis – subsequent lineage-specific loss and gain of introns. The intron invasion, probably, spawned by the mitochondrial endosymbiont, might have critically contributed to the emergence of the principal features of the eukaryotic cell. This scenario combines aspects of the introns-early and introns-late views.

Reviewers

this article was reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole.

Background

Only one spliceosomal-type intron has previously been identified in the unicellular eukaryotic parasite, Giardia lamblia (a diplomonad). This intron is only 35 nucleotides in length and is unusual in possessing a non-canonical 5' intron boundary sequence, CT, instead of GT.

Results

We have identified a second spliceosomal-type intron in G. lamblia, in the ribosomal protein L7a gene (Rpl7a), that possesses a canonical GT 5' intron boundary sequence. A comparison of the two known Giardia intron sequences revealed extensive nucleotide identity at both the 5' and 3' intron boundaries, similar to the conserved sequence motifs recently identified at the boundaries of spliceosomal-type introns in Trichomonas vaginalis (a parabasalid). Based on these observations, we searched the partial G. lamblia genome sequence for these conserved features and identified a third spliceosomal intron, in an unassigned open reading frame. Our comprehensive analysis of the Rpl7a intron in other eukaryotic taxa demonstrates that it is evolutionarily conserved and is an ancient eukaryotic intron.

Conclusion

An analysis of the phylogenetic distribution and properties of the Rpl7a intron suggests its utility as a phylogenetic marker to evaluate particular eukaryotic groupings. Additionally, analysis of the G. lamblia introns has provided further insight into some of the conserved and unique features possessed by the recently identified spliceosomal introns in related organisms such as T. vaginalis and Carpediemonas membranifera.

Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.

Author Summary

The surprising observation 30 years ago that genes are interrupted by non-coding introns changed our view of gene architecture. Intron number varies dramatically among species; ranging from nine introns/gene in humans to less than one in some simple eukyarotes. Here we ask where new introns come from and how they are maintained in a population. We find that novel introns do not arise from pre-existing introns, although the mechanisms that generate novel introns remain unclear. We also show that novel introns carry only weak signals for their identification and removal, and therefore depend on nonsense-mediated decay (NMD). NMD maintains RNA quality control by degrading transcripts that have not been spliced properly. We propose that NMD shelters novel introns from natural selection. This increases the likelihood that a novel intron will rise in frequency and be maintained within a population, thus increasing the rate of intron gain.

Over 30 years since their discovery, the origin of spliceosomal introns remains uncertain. One nearly universally accepted hypothesis maintains that spliceosomal introns originated from self-splicing group-II introns that invaded the uninterrupted genes of the last eukaryotic common ancestor (LECA) and proliferated by “insertion” events. Although this is a possible explanation for the original presence of introns and splicing machinery, the emphasis on a high number of insertion events in the genome of the LECA neglects a considerable body of empirical evidence showing that spliceosomal introns can simply arise from coding or, more generally, nonintronic sequences within genes. After presenting a concise overview of some of the most common hypotheses and mechanisms for intron origin, we propose two further hypotheses that are broadly based on central cellular processes: 1) internal gene duplication and 2) the response to aberrant and fortuitously spliced transcripts. These two nonmutually exclusive hypotheses provide a powerful way to explain the establishment of spliceosomal introns in eukaryotes without invoking an exogenous source.

The removal of non-coding sequences, introns, from the mRNA precursors is an essential step in eukaryotic gene expression. U12-type introns are a minor subgroup of introns, distinct from the major or U2-type introns. U12-type introns are present in most eukaryotes but only account for less than 0.5% of all introns in any given genome. They are processed by a specific U12-dependent spliceosome, which is similar to, but distinct from, the major spliceosome. U12-type introns are spliced somewhat less efficiently than the major introns, and it is believed that this limits the expression of the genes containing such introns. Recent findings on the role of U12-dependent splicing in development and human disease have shown that it can also affect multiple cellular processes not directly related to the functions of the host genes of U12-type introns. At the same time, advances in understanding the regulation and phylogenetic distribution of the minor spliceosome are starting to shed light on how the U12-type introns and the minor spliceosome may have evolved. © 2012 John Wiley & Sons, Ltd.

Group II introns act as both large catalytic RNAs and mobile retroelements. They are found in organelle and bacterial genomes and are spliced via a lariat intermediate, in a mechanism similar to that of spliceosomal introns. However, their distribution and insertion patterns, particularly for bacterial group II introns, suggest that they function and behave more like retroelements than organelle introns. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. This group II intron is excised, in vivo and in vitro, as intron lariats. However, the complete splicing reaction in vivo remains to be elucidated. A lacZ reporter gene system, northern blotting and real-time reverse transcription were carried out to investigate RmInt1 splicing activity. Splicing efficiency of 0.07 ± 0.02% was recorded. These findings suggest that bacterial group II introns function more like retroelements than spliceosomal introns. Their location is consistent with a role for these introns in preventing the spread of other potentially harmful mobile elements in bacteria.

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.

Synopsis

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.

Background

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.

Results

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.

Conclusion

Our results strongly support the introns-late hypothesis of the origin of spliceosomal introns.

The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns' initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 3′ consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 3′ splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures.

Author Summary

The spliceosomal introns that interrupt eukaryotic genes show great number and sequence variation across species, from the rare, highly uniform yeast introns to the ubiquitous and highly variable vertebrate intron sequences. The causes of these differences remain mysterious. We studied sequences of intron branch points and 3′ termini in 50 eukaryotic species. All intron-rich species exhibit variable 3′ sequences. However, intron-poor species range from variable sequences, to uniform branch point motifs, to uniform branch point motifs in uniform positions along the intronic sequence. This is a more complex pattern than the clear relationship between intron number and 5′ intron sequence uniformity found previously. The correspondence of sequence uniformity and intron number extends to species of the green algal genus Ostreococcus, in which the single intron-rich genomic region shows far more variable intron sequences than in the otherwise intron-poor genome. We suggest that different concentrations of spliceosomal complexes may explain these differences. In addition, we report the existence of 3′ polyT tails in diverse eukaryotic protists, suggesting that this structure is ancestral. Together, these results underscore the complexity of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron sequences in modern eukaryotes, and the impressive evolutionary malleability of eukaryotic genes.

Analysis of intron gain and loss in fungal genomes provides support for an intron-rich fungus-animal ancestor.

Background

Eukaryotic protein-coding genes are interrupted by spliceosomal introns, which are removed from transcripts before protein translation. Many facets of spliceosomal intron evolution, including age, mechanisms of origins, the role of natural selection, and the causes of the vast differences in intron number between eukaryotic species, remain debated. Genome sequencing and comparative analysis has made possible whole genome analysis of intron evolution to address these questions.

Results

We analyzed intron positions in 1,161 sets of orthologous genes across 25 eukaryotic species. We find strong support for an intron-rich fungus-animal ancestor, with more than four introns per kilobase, comparable to the highest known modern intron densities. Indeed, the fungus-animal ancestor is estimated to have had more introns than any of the extant fungi in this study. Thus, subsequent fungal evolution has been characterized by widespread and recurrent intron loss occurring in all fungal clades. These results reconcile three previously proposed methods for estimation of ancestral intron number, which previously gave very different estimates of ancestral intron number for eight eukaryotic species, as well as a fourth more recent method. We do not find a clear inverse correspondence between rates of intron loss and gain, contrary to the predictions of selection-based proposals for interspecific differences in intron number.

Conclusion

Our results underscore the high intron density of eukaryotic ancestors and the widespread importance of intron loss through eukaryotic evolution.

The spliceosome is a molecular machine that performs the excision of introns from eukaryotic pre-mRNAs. This macromolecular complex comprises in human cells five RNAs and over one hundred proteins. In recent years, many spliceosomal proteins have been found to exhibit intrinsic disorder, that is to lack stable native three-dimensional structure in solution. Building on the previous body of proteomic, structural and functional data, we have carried out a systematic bioinformatics analysis of intrinsic disorder in the proteome of the human spliceosome. We discovered that almost a half of the combined sequence of proteins abundant in the spliceosome is predicted to be intrinsically disordered, at least when the individual proteins are considered in isolation. The distribution of intrinsic order and disorder throughout the spliceosome is uneven, and is related to the various functions performed by the intrinsic disorder of the spliceosomal proteins in the complex. In particular, proteins involved in the secondary functions of the spliceosome, such as mRNA recognition, intron/exon definition and spliceosomal assembly and dynamics, are more disordered than proteins directly involved in assisting splicing catalysis. Conserved disordered regions in spliceosomal proteins are evolutionarily younger and less widespread than ordered domains of essential spliceosomal proteins at the core of the spliceosome, suggesting that disordered regions were added to a preexistent ordered functional core. Finally, the spliceosomal proteome contains a much higher amount of intrinsic disorder predicted to lack secondary structure than the proteome of the ribosome, another large RNP machine. This result agrees with the currently recognized different functions of proteins in these two complexes.

Author Summary

In eukaryotic cells, introns are spliced out of proteincoding mRNAs by a highly dynamic and extraordinarily plastic molecular machine called the spliceosome. In recent years, multiple regions of intrinsic structural disorder were found in spliceosomal proteins. Intrinsically disordered regions lack stable native three-dimensional structure in solutions, which makes them structurally flexible and/or able to switch between different conformations. Hence, intrinsically disordered regions are the ideal candidate responsible for the spliceosome's plasticity. Intrinsically disordered regions are also frequently the sites of post-translational modifications, which were also proven to be important in spliceosome dynamics. In this article, we describe the results of a structural bioinformatics analysis focused on intrinsic disorder in the spliceosomal proteome. We systematically analyzed all known human spliceosomal proteins with regards to the presence and type of intrinsic disorder. Almost a half of the combined sequence of these spliceosomal proteins is predicted to be intrinsically disordered, and the type of intrinsic disorder in a protein varies with its function and its location in the spliceosome. The parts of the spliceosome that act earlier in the process are more disordered, which corresponds to their role in establishing a network of interactions, while the parts that act later are more ordered.

U12-type introns exist, albeit rarely, in a variety of multicellular organisms. Splicing of U12 intron-containing precursor mRNAs takes place in the U12-type spliceosome that is distinct from the major U2-type spliceosome. Due to incompatibility of these two spliceosomes, alternative splicing involving a U12-type intron may give rise to a relatively complicated impact on gene expression. We studied alternative U12-type intron splicing in an attempt to gain more mechanistic insights. First, we characterized mutually exclusive exon selection of the human JNK2 gene, which involves an unusual intron possessing the U12-type 5′ splice site and the U2-type 3′ splice site. We demonstrated that the long and evolutionary conserved polypyrimidine tract of this hybrid intron provides important signals for inclusion of its downstream alternative exon. In addition, we examined the effects of single nucleotide polymorphisms in the human WDFY1 U12-type intron on pre-mRNA splicing. These results provide mechanistic implications on splice-site selection of U12-type intron splicing. We finally discuss the potential effects of splicing of a U12-type intron with genetic defects or within a set of genes encoding RNA processing factors on global gene expression.

Most of eukaryotic genes are interrupted by introns that need to be removed from pre-mRNAs before they can perform their function. This is done by complex machinery called spliceosome. Many eukaryotes possess two separate spliceosomal systems that process separate sets of introns. The major (U2) spliceosome removes majority of introns, while minute fraction of intron repertoire is processed by the minor (U12) spliceosome. These two populations of introns are called U2-type and U12-type, respectively. The latter fall into two subtypes based on the terminal dinucleotides. The minor spliceosomal system has been lost independently in some lineages, while in some others few U12-type introns persist. We investigated twenty insect genomes in order to better understand the evolutionary dynamics of U12-type introns. Our work confirms dramatic drop of U12-type introns in Diptera, leaving these genomes just with a handful cases. This is mostly the result of intron deletion, but in a number of dipteral cases, minor type introns were switched to a major type, as well. Insect genes that harbor U12-type introns belong to several functional categories among which proteins binding ions and nucleic acids are enriched and these few categories are also overrepresented among these genes that preserved minor type introns in Diptera.

Background

The U12-type spliceosome is responsible for the removal of a subset of introns from eukaryotic mRNAs. U12-type introns are spliced less efficiently than normal U2-type introns, which suggests a rate-limiting role in gene expression. The Drosophila genome contains about 20 U12-type introns, many of them in essential genes, and the U12-type spliceosome has previously been shown to be essential in the fly.

Methodology/Principal Findings

We have used a Drosophila line with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, to investigate the impact of U12-type introns on gene expression at the organismal level during fly development. This line exhibits progressive accumulation of unspliced U12-type introns during larval development and the death of larvae at the third instar stage. Surprisingly, microarray and RT-PCR analyses revealed that most genes containing U12-type introns showed only mild perturbations in the splicing of U12-type introns. In contrast, we detected widespread downstream effects on genes that do not contain U12-type introns, with genes related to various metabolic pathways constituting the largest group.

Conclusions/Significance

U12-type intron-containing genes exhibited variable gene-specific responses to the splicing defect, with some genes showing up- or downregulation, while most did not change significantly. The observed residual U12-type splicing activity could be explained with the mutant U6atac allele having a low level of catalytic activity. Detailed analysis of all genes suggested that a defect in the splicing of the U12-type intron of the mitochondrial prohibitin gene may be the primary cause of the various downstream effects detected in the microarray analysis.

U12-dependent introns are spliced by the minor U12-type spliceosome and occur in a variety of eukaryotic organisms, including Arabidopsis. In this study, a set of putative U12-dependent introns was compiled from a large collection of cDNA/EST- confirmed introns in the Arabidopsis thaliana genome by means of high-throughput bioinformatic analysis combined with manual scrutiny. A total of 165 U12-type introns were identified based upon stringent criteria. This number of sequences well exceeds the total number of U12-type introns previously reported for plants and allows a more thorough statistical analysis of U12-type signals. Of particular note is the discovery that the distance between the branch site adenosine and the acceptor site ranges from 10 to 39 nt, significantly longer than the previously postulated limit of 21 bp. Further analysis indicates that, in addition to the spacing constraint, the sequence context of the potential acceptor site may have an important role in 3′ splice site selection. Several alternative splicing events involving U12-type introns were also captured in this study, providing evidence that U12-dependent acceptor sites can also be recognized by the U2-type spliceosome. Furthermore, phylogenetic analysis suggests that both U12-type AT-AC and U12-type GT-AG introns occurred in Na+/H+ antiporters in a progenitor of animals and plants.

In many eukaryotes, spliceosomal introns are able to influence the level and site of gene expression. The mechanism of this Intron Mediated Enhancement (IME) has not yet been elucidated, but regulation of gene expression is likely to occur at several steps during and after transcription. Different introns have different intrinsic enhancing properties, but the determinants of these differences remain unknown. Recently, an algorithm called IMEter, which is able to predict the IME potential of introns without direct testing, has been proposed. A computer program was developed for Arabidopsis thaliana and rice (Oryza sativa L.), but was only tested experimentally in Arabidopsis by measuring the enhancement effect on GUS expression of different introns inserted within otherwise identical plasmids. To test the IMEter potential in rice, a vector bearing the upstream regulatory sequence of a rice β-tubulin gene (OsTub6) fused to the GUS reporter gene was used. The enhancing intron interrupting the OsTub6 5′-UTR was precisely replaced by seven other introns carrying different features. GUS expression level in transiently transformed rice calli does not significantly correlate with the calculated IMEter score. It was also found that enhanced GUS expression was mainly due to a strong increase in the mRNA steady-state level and that mutations at the splice recognition sites almost completely abolished the enhancing effect. Splicing also appeared to be required for IME in Arabidopsis cell cultures, where failure of the OsTub6 5′ region to drive high level gene expression could be rescued by replacing the poorly spliced rice intron with one from Arabidopsis.

Accurate gene prediction in eukaryotes is a difficult and subtle problem. Here we point out a useful feature of expected distributions of spliceosomal intron lengths. Since introns are removed from transcripts prior to translation, intron lengths are not expected to respect coding frame, thus the number of genomic introns that are a multiple of three bases (‘3n introns’) should be similar to the number that are a multiple of three plus one bases (or plus two bases). Skewed predicted intron length distributions thus suggest systematic errors in intron prediction. For instance, a genome-wide excess of 3n introns suggests that many internal exonic sequences have been incorrectly called introns, whereas a deficit of 3n introns suggests that many 3n introns that lack stop codons have been mistaken for exonic sequence. A survey of genomic annotations for 29 diverse eukaryotic species showed that skew in intron length distributions is a common problem. We discuss several examples of skews in genome-wide intron length distributions that indicate systematic problems with gene prediction. We suggest that evaluation of length distributions of predicted introns is a fast and simple method for detecting a variety of possible systematic biases in gene prediction or even problems with genome assemblies, and discuss ways in which these insights could be incorporated into genome annotation protocols.

Background

Spliceosomal introns are an ancient, widespread hallmark of eukaryotic genomes. Despite much research, many questions regarding the origin and evolution of spliceosomal introns remain unsolved, partly due to the difficulty of inferring ancestral gene structures. We circumvent this problem by using genes originated by endosymbiotic gene transfer, in which an intron-less structure at the time of the transfer can be assumed.

Results

By comparing the exon-intron structures of 64 mitochondrial-derived genes that were transferred to the nucleus at different evolutionary periods, we can trace the history of intron gains in different eukaryotic lineages. Our results show that the intron density of genes transferred relatively recently to the nuclear genome is similar to that of genes originated by more ancient transfers, indicating that gene structure can be rapidly shaped by intron gain after the integration of the gene into the genome and that this process is mainly determined by forces acting specifically on each lineage. We analyze 12 cases of mitochondrial-derived genes that have been transferred to the nucleus independently in more than one lineage.

Conclusions

Remarkably, the proportion of shared intron positions that were gained independently in homologous genes is similar to that proportion observed in genes that were transferred prior to the speciation event and whose shared intron positions might be due to vertical inheritance. A particular case of parallel intron gain in the nad7 gene is discussed in more detail.

Background

Spliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir) genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns.

Results

Uncommonly large size of spliceosomal intron was observed in V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE) activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars.

Conclusions

In this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and repress TE removal mechanisms such as recombination. The intron size expansion observed in domesticated grapevines provided an example of rapid plant genome evolution in response to artificial selection and propagation, and may shed light on the important genomic changes during domestication. In addition, the transcriptome approach used to gather intron size data significantly improved annotations of the V. vinifera genome.

Background

Inteins and introns are genetic elements that are removed from proteins and RNA after translation or transcription, respectively. Previous studies have suggested that these genetic elements are found in conserved parts of the host protein. To our knowledge this type of analysis has not been done for group II introns residing within a gene. Here we provide quantitative statistical support from an analyses of proteins that host inteins, group I introns, group II introns and spliceosomal introns across all three domains of life.

Results

To determine whether or not inteins, group I, group II, and spliceosomal introns are found preferentially in conserved regions of their respective host protein, conservation profiles were generated and intein and intron positions were mapped to the profiles. Fisher's combined probability test was used to determine the significance of the distribution of insertion sites across the conservation profile for each protein. For a subset of studied proteins, the conservation profile and insertion positions were mapped to protein structures to determine if the insertion sites correlate to regions of functional activity. All inteins and most group I introns were found to be preferentially located within conserved regions; in contrast, a bacterial intein-like protein, group II and spliceosomal introns did not show a preference for conserved sites.

Conclusions

These findings demonstrate that inteins and group I introns are found preferentially in conserved regions of their respective host proteins. Homing endonucleases are often located within inteins and group I introns and these may facilitate mobility to conserved regions. Insertion at these conserved positions decreases the chance of elimination, and slows deletion of the elements, since removal of the elements has to be precise as not to disrupt the function of the protein. Furthermore, functional constrains on the targeted site make it more difficult for hosts to evolve immunity to the homing endonuclease. Therefore, these elements will better survive and propagate as molecular parasites in conserved sites. In contrast, spliceosomal introns and group II introns do not show significant preference for conserved sites and appear to have adopted a different strategy to evade loss.

Background

Eukaryotic pre-mRNA gene transcripts are processed by the spliceosome to remove portions of the transcript, called spliceosomal introns. The spliceosome recognizes intron boundaries by the presence of sequence signals (motifs) contained in the actual transcript, thus sequence changes in the genome that affect existing splicing signals or create new signals may lead to changes in transcript splicing patterns. Such changes may lead to previously excluded (intronic) transcript regions being included (exonic) or vice versa. Such changes can affect the encoded protein sequence and/or post-transcriptional regulation, and are thus a potentially important source of genomic and phenotypic novelty. Two recent papers suggest that such changes may be a major force in remodeling of eukaryotic gene structures, however the rate of occurrence of such changes has not been assessed at the genomic level.

Results

I studied four closely related species of Cryptoccocus fungi. Among 28,256 studied introns, canonical GT/C...AG boundaries are nearly universally conserved across all four species. Among only 40 observed cases of cDNA-confirmed non-conserved intron boundaries, most are likely to involve alternative splicing. I find only five cases of "intronization," intron creation from an internal exonic region by de novo emergence of new splicing boundaries, and no cases of the reverse process, "de-intronization." I find no more than ten clear cases of true movement of an intron boundary of a possibly constitutively spliced intron, and no clear cases of true "intron sliding," in which changes in the positions of both intron boundaries could lead to a movement of the intron position along the coding sequence.

Conclusion

These results suggest that intronization, de-intronization, and intron boundary movement are rare events in evolution.

Background

Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups.

Results

U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost.

Conclusions

The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost.

U12-dependent introns are found in small numbers in most eukaryotic genomes, but their scarcity makes accurate characterisation of their properties challenging. A computational search for U12-dependent introns was performed using the draft version of the human genome sequence. Human expressed sequences confirmed 404 U12-dependent introns within the human genome, a 6-fold increase over the total number of non-redundant U12-dependent introns previously identified in all genomes. Although most of these introns had AT-AC or GT-AG terminal dinucleotides, small numbers of introns with a surprising diversity of termini were found, suggesting that many of the non-canonical introns found in the human genome may be variants of U12-dependent introns and, thus, spliced by the minor spliceosome. Comparisons with U2-dependent introns revealed that the U12-dependent intron set lacks the ‘short intron’ peak characteristic of  U2-dependent introns. Analysis of this U12-dependent intron set confirmed reports of a biased distribution of U12-dependent introns in the genome and allowed the identification of several alternative splicing events as well as a surprising number of apparent splicing errors. This new larger reference set of U12-dependent introns will serve as a resource for future studies of both the properties and evolution of the U12 spliceosome.

U12 intron-specific spliceosomes contain U11 and U12 small nuclear ribonucleoproteins and mediate the removal of U12 introns from precursor-mRNAs. Among the several proteins unique to the U12-type spliceosomes, an Arabidopsis thaliana AtU11/U12-31K protein has been shown to be indispensible for proper U12 intron splicing and for normal growth and development of Arabidopsis plants. Here, we assessed the functional roles of the rice (Oryza sativa) OsU11/U12-31K protein in U12 intron splicing and development of plants. The U11/U12-31K transcripts were abundantly expressed in the shoot apical meristems (SAMs) of Arabidopsis and rice. Ectopic expression of OsU11/U12-31K in AtU11/U12-31K-defecient Arabidopsis mutant complemented the incorrect U12 intron splicing and abnormal development phenotypes of the Arabidopsis mutant plants. Impaired cell division activity in the SAMs and inflorescence stems observed in the AtU11/U12-31K-deficient mutant was completely recovered to normal by the expression of OsU11/U12-31K. Similar to Arabidopsis AtU11/U12-31K, rice OsU11/U12-31K was determined to harbor RNA chaperone activity. Collectively, the present findings provide evidence for the emerging idea that the U11/U12-31K protein is an indispensible RNA chaperone that functions in U12 intron splicing and is necessary for normal development of monocotyledonous plants as well as dicotyledonous plants.