Intron size, abundance, and distribution within untranslated regions of genes

Most research concerning the evolution of introns has largely considered introns within coding sequences (CDSs), without regard for introns located within untranslated regions (UTRs) of genes. Here, we directly determined intron size, abundance, and distribution in UTRs of genes using full-length cDNA libraries and complete genome sequences for four species, Arabidopsis thaliana, Drosophila melanogaster, human, and mouse. Overall intron occupancy (introns/exon kbp) is lower in 5' UTRs than CDSs, but intron density (intron occupancy in regions containing introns) tends to be higher in 5' UTRs than in CDSs. Introns in 5' UTRs are roughly twice as large as introns in CDSs, and there is a sharp drop in intron size at the 5' UTR-CDS boundary. We propose a mechanistic explanation for the existence of selection for larger intron size in 5' UTRs, and outline several implications of this hypothesis. We found introns to be randomly distributed within 5' UTRs, so long as a minimum required exon size was assumed. Introns in 3' UTRs were much less abundant than in 5' UTRs. Though this was expected for human and mouse that have intron-dependent nonsense-mediated decay (NMD) pathways that discourage the presence of introns within the 3' UTR, it was also true for A. thaliana and D. melanogaster, which may lack intron-dependent NMD. Our findings have several implications for theories of intron evolution and genome evolution in general.     

The evolution of spliceosomal introns in alveolates

Many issues concerning the evolution of spliceosomal introns remain poorly understood. In this respect, the reconstruction of the evolution of introns in deep branching species such as alveolates is of special significance. In this study, we inferred the intron evolution in alveolates using 3,368 intron positions in 162 orthologs from 10 species (9 alveolates and 1 outgroup, Homo sapiens). We found that although very few intron gains and losses have occurred in Theileria and Plasmodium recently, many intron gains and losses have occurred in the evolution of alveolates. Thus, the rates of intron gain and loss in alveolates have varied greatly across time and lineage. Our results seem to support the notion that massive intron gains and losses have occurred during short episodes, perhaps coinciding with major evolutionary events.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

The evolution of spliceosomal introns: patterns, puzzles and progress

The origins and importance of spliceosomal introns comprise one of the longest-abiding mysteries of molecular evolution. Considerable debate remains over several aspects of the evolution of spliceosomal introns, including the timing of intron origin and proliferation, the mechanisms by which introns are lost and gained, and the forces that have shaped intron evolution. Recent important progress has been made in each of these areas. Patterns of intron-position correspondence between widely diverged eukaryotic species have provided insights into the origins of the vast differences in intron number between eukaryotic species, and studies of specific cases of intron loss and gain have led to progress in understanding the underlying molecular mechanisms and the forces that control intron evolution.     

New maximum likelihood estimators for eukaryotic intron evolution

The evolution of spliceosomal introns remains poorly understood. Although many approaches have been used to infer intron evolution from the patterns of intron position conservation, the results to date have been contradictory. In this paper, we address the problem using a novel maximum likelihood method, which allows estimation of the frequency of intron insertion target sites, together with the rates of intron gain and loss. We analyzed the pattern of 10,044 introns (7,221 intron positions) in the conserved regions of 684 sets of orthologs from seven eukaryotes. We determined that there is an average of one target site per 11.86 base pairs (bp) (95% confidence interval, 9.27 to 14.39 bp). In addition, our results showed that: (i) overall intron gains are ~25% greater than intron losses, although specific patterns vary with time and lineage; (ii) parallel gains account for ~18.5% of shared intron positions; and (iii) reacquisition following loss accounts for ~0.5% of all intron positions. Our results should assist in resolving the long-standing problem of inferring the evolution of spliceosomal introns.     

A 25-bp ancient spliceosomal intron in the TvRab1a gene of Trichomonas vaginalis

Spliceosomal introns play a key role in eukaryotic genome evolution and protein diversity. A large Rab GTPase family has been identified in a unicellular eukaryote Trichomonas vaginalis. However, the characteristics of introns in Rab genes of T. vaginalis have not been investigated previously. In this study, we identified a 25-bp spliceosomal intron in the T. vaginalis Rab1a (TvRab1a) gene, the smallest intron in T. vaginalis to be characterized to date. This intron contains a canonical splice site at both 5' (GT) and 3' (AG) ends, and a putative branch-point sequence (TCTAAC) that matches the Trichomonad consensus sequence of ACTAAC except for the first nucleotide. The position and phase of the TvRab1a intron are evolutionarily conserved in Rab1 homologous genes across at least five eukaryotic supergroups, including Opisthokonta, Amoebozoa, Excavata, Chromalveolata, and Plantae. These results strongly suggest that the TvRab1a intron is likely to be an ancient spliceosomal intron, and it can therefore be used as a phylogenetic marker to evaluate particular eukaryotic groupings. Identification and characterization of the TvRabla intron may provide an insight into the evolution of the large Rab repertoire in T. vaginalis.     

Patterns of intron loss and gain in plants: intron loss-dominated evolution and genome-wide comparison of O. sativa and A. thaliana

Numerous previous studies have elucidated 2 surprising patterns of spliceosomal intron evolution in diverse eukaryotes over the past roughly 100 Myr. First, rates of recent intron gain in a wide variety of eukaryotic lineages have been surprisingly low, far too low to explain modern intron densities. Second, intron losses have outnumbered intron gains over a variety of lineages. For several reasons, land plants might be expected to have comparatively high rates of intron gain and thus to represent a possible exception to this pattern. However, we report several studies that indicate low rates of intron gain and an excess of intron losses over intron gains in a variety of plant lineages. We estimate that intron losses have outnumbered intron gains in recent evolution in Arabidopsis thaliana (roughly 12.6 times more losses than gains), Oryza sativa (9.8 times), the green alga Chlamydomonas reinhardtii (5.1 times), and the Bigelowiella natans nucleomorph, an enslaved green algal nucleus (2.8 times). We estimate that during recent evolution, A. thaliana and O. sativa have experienced very low rates of intron gain of around one gain per gene per 2.6-8.0 billion years. In addition, we compared 8,258 pairs of putatively orthologous A. thaliana-O. sativa genes. We found that 5.3% of introns in conserved coding regions are species-specific. Observed species-specific A. thaliana and O. sativa introns tend to be exact and to lie adjacent to each other along the gene, in a pattern suggesting mRNA-mediated intron loss. Our results underscore that low intron gain rates and intron number reduction are common features of recent eukaryotic evolution. This pattern implies that rates of intron creation were higher during earlier periods of evolution and further focuses attention on the causes of initial intron proliferation.     

Intron loss and gain in Drosophila

Although introns were first discovered almost 30 years ago, their evolutionary origin remains elusive. In this work, we used multispecies whole-genome alignments to map Drosophila melanogaster introns onto 10 other fully sequenced Drosophila genomes. We were able to find 1,944 sites where an intron was missing in one or more species. We show that for most (>80%) of these cases, there is no leftover intronic sequence or any missing exonic sequence, indicating exact intron loss or gain events. We used parsimony to classify these differences as 1,754 intron loss events and 213 gain events. We show that lost and gained introns are significantly shorter than average and flanked by longer than average exons. They also display quite distinct phase distributions and show greater than average similarity between the 5' splice site and its 3' partner splice site. Introns that have been lost in one or more species evolve faster than other introns, occur in slowly evolving genes, and are found adjacent to each other more often than would be expected for independent single losses. Our results support the cDNA recombination mechanism of intron loss, suggest that selective pressures affect site-specific loss rates, and show conclusively that intron gain has occurred within the Drosophila lineage, solidifying the "introns-middle" hypothesis and providing some hints about the gain mechanism.     

Intron length evolution in Drosophila

I present data on the evolution of intron lengths among 3 closely related Drosophila species, D. melanogaster, Drosophila simulans, and Drosophila yakuba. Using D. yakuba as an outgroup, I mapped insertion and deletion mutations in 148 introns (spanning approximately 30 kb) to the D. melanogaster and D. simulans lineages. Intron length evolution in the 2 sister species has been different: in D. melanogaster, X-linked introns have increased slightly in size, whereas autosomal ones have decreased slightly in size; in D. simulans, both X-linked and autosomal introns have decreased in size. To understand the possible evolutionary causes of these lineage- and chromosome-specific patterns of intron evolution, I studied insertion-deletion (indel) polymorphism and divergence in D. melanogaster. Small insertion mutations segregate at elevated frequencies and enjoy elevated probabilities of fixation, particularly on the X chromosome. In contrast, there is no detectable X chromosome effect on fixations in D. simulans. These findings suggest X chromosome-specific selection or biased gene conversion-gap repair favoring insertions in D. melanogaster but not in D. simulans. These chromosome- and lineage-specific patterns of indel substitution are not easily explained by existing general population genetic models of intron length evolution. Genomic data from D. melanogaster further suggest that the forces described here affect introns and intergenic regions similarly.     

Prevalence of intron gain over intron loss in the evolution of paralogous gene families

The mechanisms and evolutionary dynamics of intron insertion and loss in eukaryotic genes remain poorly understood. Reconstruction of parsimonious scenarios of gene structure evolution in paralogous gene families in animals and plants revealed numerous gains and losses of introns. In all analyzed lineages, the number of acquired new introns was substantially greater than the number of lost ancestral introns. This trend held even for lineages in which vertical evolution of genes involved more intron losses than gains, suggesting that gene duplication boosts intron insertion. However, dating gene duplications and the associated intron gains and losses based on the molecular clock assumption showed that very few, if any, introns were gained during the last ~100 million years of animal and plant evolution, in agreement with previous conclusions reached through analysis of orthologous gene sets. These results are generally compatible with the emerging notion of intensive insertion and loss of introns during transitional epochs in contrast to the relative quiet of the intervening evolutionary spans.     

Analysis of ribosomal protein gene structures: implications for intron evolution

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.     

Splicing-Related Features of Introns Serve to Propel Evolution

The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron''s ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution.     

Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena.

The origin of present day introns is a subject of spirited debate. Any intron evolution theory must account for not only nuclear spliceosomal introns but also their antecedents. The evolution of group II introns is fundamental to this debate, since group II introns are the proposed progenitors of nuclear spliceosomal introns and are found in ancient genes from modern organisms. We have studied the evolution of chloroplast introns and twintrons (introns within introns) in the genus Euglena. Our hypothesis is that Euglena chloroplast introns arose late in the evolution of this lineage and that twintrons were formed by the insertion of one or more introns into existing introns. In the present study we find that 22 out of 26 introns surveyed in six different photosynthesis-related genes from the plastid DNA of Euglena gracilis are not present in one or more basally branching Euglena spp. These results are supportive of a late origin for Euglena chloroplast group II introns. The psbT gene in Euglena viridis, a basally branching Euglena species, contains a single intron in the identical position to a psbT twintron from E.gracilis, a derived species. The E.viridis intron, when compared with 99 other Euglena group II introns, is most similar to the external intron of the E.gracilis psbT twintron. Based on these data, the addition of introns to the ancestral psbT intron in the common ancester of E.viridis and E.gracilis gave rise to the psbT twintron in E.gracilis.     

The recent transfer of a homing endonuclease gene

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.