Intron Transposition Research Articles

Intron lariat spliceosomes convert lariats to true circles: implications for intron transposition.

Rare, full-length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envisioned and tested a hypothesis for their formation using Saccharomyces cerevisiae, documenting full-length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3' processing machinery) attacks the branch, joining the intron 3' end to the 5' splice site in a 3'-5' linked circle. Human U2 and U12 spliceosomes produce analogous full-length and processed circles. Postsplicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

Spliceosomal intronogenesis

The presence of intervening sequences, termed introns, is a defining characteristic of eukaryotic nuclear genomes. Once transcribed into pre-mRNA, these introns must be removed within the spliceosome before export of the processed mRNA to the cytoplasm, where it is translated into protein. Although intron loss has been demonstrated experimentally, several mysteries remain regarding the origin and propagation of introns. Indeed, documented evidence of gain of an intron has only been suggested by phylogenetic analyses. We report the use of a strategy that detects selected intron gain and loss events. We have experimentally verified, to our knowledge, the first demonstrations of intron transposition in any organism. From our screen, we detected two separate intron gain events characterized by the perfect transposition of a reporter intron into the yeast genes RPL8B and ADH2, respectively. We show that the newly acquired introns are able to be removed from their respective pre-mRNAs by the spliceosome. Additionally, the novel allele, RPL8Bint, is functional when overexpressed within the genome in a strain lacking the Rpl8 paralogue RPL8A, demonstrating that the gene targeted for intronogenesis is functional.

The Complex Intron Landscape and Massive Intron Invasion in a Picoeukaryote Provides Insights into Intron Evolution

Genes in pieces and spliceosomal introns are a landmark of eukaryotes, with intron invasion usually assumed to have happened early on in evolution. Here, we analyze the intron landscape of Micromonas, a unicellular green alga in the Mamiellophyceae lineage, demonstrating the coexistence of several classes of introns and the occurrence of recent massive intron invasion. This study focuses on two strains, CCMP1545 and RCC299, and their related individuals from ocean samplings, showing that they not only harbor different classes of introns depending on their location in the genome, as for other Mamiellophyceae, but also uniquely carry several classes of repeat introns. These introns, dubbed introner elements (IEs), are found at novel positions in genes and have conserved sequences, contrary to canonical introns. This IE invasion has a huge impact on the genome, doubling the number of introns in the CCMP1545 strain. We hypothesize that each IE class originated from a single ancestral IE that has been colonizing the genome after strain divergence by inserting copies of itself into genes by intron transposition, likely involving reverse splicing. Along with similar cases recently observed in other organisms, our observations in Micromonas strains shed a new light on the evolution of introns, suggesting that intron gain is more widespread than previously thought.

Evidence for Extensive Recent Intron Transposition in Closely Related Fungi

Though spliceosomal introns are a major structural component of most eukaryotic genes and intron density varies by more than three orders of magnitude among eukaryotes [1-3], the origins of introns are poorly understood, and only a few cases of unambiguous intron gain are known [4-8]. We utilized population genomic comparisons of three closely related fungi to identify crucial transitory phases of intron gain and loss. We found 74 intron positions showing intraspecific presence-absence polymorphisms (PAPs) for the entire intron. Population genetic analyses identified intron PAPs at different stages of fixation and showed that intron gain or loss was very recent. We found direct support for extensive intron transposition among unrelated genes. A substantial proportion of highly similar introns in the genome either were recently gained or showed a transient phase of intron PAP. We also identified an intron transfer among paralogous genes that created a new intron. Intron loss was due mainly to homologous recombination involving reverse-transcribed mRNA. The large number of intron positions in transient phases of either intron gain or loss shows that intron evolution is much faster than previously thought and provides an excellent model to study molecular mechanisms of intron gain.

Large-Scale tRNA Intron Transposition in the Archaeal Order Thermoproteales Represents a Novel Mechanism of Intron Gain

Recently, diverse arrangements of transfer RNA (tRNA) genes have been found in the domain Archaea, in which the tRNA is interrupted by a maximum of three introns or is even fragmented into two or three genes. Whereas most of the eukaryotic tRNA introns are inserted strictly at the canonical nucleotide position (37/38), archaeal intron-containing tRNAs have a wide diversity of small tRNA introns, which differ in their numbers and locations. This feature is especially pronounced in the archaeal order Thermoproteales. In this study, we performed a comprehensive sequence comparison of 286 tRNA introns and their genes in seven Thermoproteales species to clarify how these introns have emerged and diversified during tRNA gene evolution. We identified 46 intron groups containing sets of highly similar sequences (>70%) and showed that 16 of them contain sequences from evolutionarily distinct tRNA genes. The phylogeny of these 16 intron groups indicates that transposition events have occurred at least seven times throughout the evolution of Thermoproteales. These findings suggest that frequent intron transposition occurs among the tRNA genes of Thermoproteales. Further computational analysis revealed limited insertion positions and corresponding amino acid types of tRNA genes. This has arisen because the bulge-helix-bulge splicing motif is required at the newly transposed position if the pre-tRNA is to be correctly processed. These results clearly demonstrate a newly identified mechanism that facilitates the late gain of short introns at various noncanonical positions in archaeal tRNAs.

A group II intron has invaded the genus Azotobacter and is inserted within the termination codon of the essential groEL gene.

A group II intron that was previously identified within Azotobacter vinelandii by polymerase chain reac-tion with consensus primers has been completely sequenced, together with its flanking exons. In contrast to other bacterial members of group II, which are associated with mobile or other presumably non-essential DNA, the A. vinelandii intron is inserted within the termination codon of the groEL coding sequence, which it changes from UAA to UAG. Both the host gene and the intron appear to be functional as (i) the ribozyme component of the intron self-splices in vitro and (ii) both intron-carrying and intronless versions of the single-copy groEL gene from A. vinelandii complement groEL mutations in Escherichia coli. Moreover, analysis of nucleotide substitutions within and around a closely related intron sequence that is present at the same site in Azotobacter chroococcum provides indirect evidence of intron transposition posterior to the divergence of the two Azotobacter taxa. Somewhat surprisingly, however, analyses of RNA extracted from cells that had or had not undergone a heat shock show that the bulk of groEL transcripts end within the first 140 nucleotides of the intron. These findings are discussed in the light of our current knowledge of the biochemistry of group II introns.

Erratum: correction: Retrotransposition of a bacterial group II intron

Nature, 404, 1018–1021 (2000). In this Letter, we demonstrated active group II intron transposition to multiple ectopic sites and showed that movement to these new sites occurs via an RNA intermediate in a process termed retrotransposition. Of interest was whether retrotransposition occurs by reverse splicing of the intron RNA into RNA, single-stranded DNA and/or double-stranded DNA targets.

Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites.

Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.

Barriers to intron promiscuity in bacteria.

“The field of bacterial viruses is a fine playground for serious children who ask ambitious questions.” Max Delbruck The first bacterial intron, a self-splicing group I intron, was found to interrupt the thymidylate synthase (td) gene of the Escherichia coli phage T4 (11). The second and third bacterial group I introns were found to interrupt the aerobic (nrdB) and anaerobic (nrdD [initially named sunY]) ribonucleotide reductases of phage T4 (29, 90), and another group I intron was soon discovered in the DNA polymerase gene of SPO1, a Bacillus phage (25). From this (admittedly) small sampling of phage genomes, one might have naively expected that group I introns would be abundant in phage or bacterial genomes, especially since subsequent laboratory experiments demonstrated that group I introns could propagate themselves (by a process called homing) throughout populations of intron-minus alleles with near 100% efficiency (5, 68). That a similar homing phenomenon had also been previously demonstrated for a group I intron in the large rRNA gene of yeast mitochondria (34) gave additional support to the notion that group I introns should be able to spread efficiently throughout populations. However, this expected outcome has never been realized in natural phage populations; some phage populations harbor many introns, while other related phage populations are strangely lacking in any introns whatsoever (Table ​(Table1).1). Why do group I introns have an unusual distribution in phage and bacterial genomes, and what potential barriers might exist to prevent their spread? TABLE 1 Distribution of group I introns in bacteria and bacteriophages A similar question might be asked of group II introns in bacteria. Much was made of the initial finding of group II introns in bacteria, as their discovery added fuel to the debate concerning the evolutionary origins of eukaryotic spliceosomal introns (22, 70) which have both structural and functional similarities to group II introns (53, 79, 84). Yet, the number of group II introns in bacteria is small, many of which are inferred only from database matches to reverse transcriptases or maturases encoded within known introns (13, 41, 73, 89), and only two have been shown to splice or be mobile in vivo (48, 49, 55, 80) (Table ​(Table2).2). While group II intron homing is mechanistically distinct from group I intron homing, the principle is similar; group II introns home from intron-containing to intronless alleles. Many elegant biochemical and genetic experiments have unraveled the complexities of bacterial group II intron homing (14, 48, 49), and based on these results, there seems no a priori reason why group II introns should not be able to spread efficiently through populations of intron-minus alleles. The paucity of bacterial group II introns becomes even more perplexing given the recent demonstration of group II intron transposition to novel chromosomal sites (15). That group II introns are abundant in mitochondrial and chloroplast genomes (52) and present in bacterial genomes but at lower levels (and absent in phages) only adds to the mystery surrounding the lack of group II introns in bacteria, as mitochondria and chloroplasts are typically prokaryotic in genome organization and ultimately are derived from two distinct bacterial lineages. Do similar barriers that prevent group I introns from spreading throughout bacterial populations also apply to preventing the spread of group II introns in bacteria? TABLE 2 Distribution of group II introns in bacteria

Transposition and exon shuffling by group II intron RNA molecules in pieces

In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules. Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNA-containing viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking. Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria. We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuffling. The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions.

Generation of Highly Site-Specific DNA Double-Strand Breaks in Human Cells by the Homing Endonucleases I-PpoI and I-CreI

We have determined the ability of two well-characterized eukaryotic homing endonucleases, I-PpoI from the myxomycetePhysarum polycephalumand I-CreI from the green algaChlamydomonas reinhardtii,to generate site-specific DNA double-strand breaks in human cells. These 18-kDa proteins cleave highly conserved 15- or 24-bp rDNA homing sites in their respective hosts to generate homogeneous 4-base, 3′ ends that initiate target intron transposition or “homing.” We show that both endonucleases can be expressed in human cells and can generate site-specific DNA double-strand breaks in 28S rDNA and homing site plasmids. These endonuclease-induced breaks can be repairedin vivo,although break repair is mutagenic with the frequent generation of short deletions or insertions. I-PpoI and I-CreI should be useful for analyzing DNA double-strand break repair in human cells and rDNA.

Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-td Intron Model System

Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage lambda model system was developed to analyze exon homology effects on intron homing and determine the role of the lambda 5'-3' exonuclease complex (Red alpha beta) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type lambda, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.

Mechanisms of Intron Mobility

Mechanisms of Intron Mobility

Homing of a group II intron in yeast mitochondrial DNA is accompanied by unidirectional co-conversion of upstream-located markers.

Group II introns ai1 and ai2 of the Saccharomyces cerevisiae mitochondrial COXI gene encode proteins having a dual function (maturase and reverse transcriptase) and are mobile genetic elements. By construction of adequate donor genomes, we demonstrate that each of them is self-sufficient and practises homing in the absence of homing-type endonucleases encoded by either group I introns or the ENS2 gene. Each of the S. cerevisiae group II self-mobile introns was tested for its ability to invade mitochondrial DNA (mtDNA) from two related Saccharomyces species. Surprisingly, only ai2 was observed to integrate into both genomes. The non-mobility of ai1 was clearly correlated with some polymorphic changes occurring in sequences flanking its insertion sites in the recipient mtDNAs. Importantly, studies of the behaviour of these introns in interspecific crosses demonstrate that flanking marker co-conversion accompanying group II intron homing is unidirectional and efficient only in the 3' to 5' direction towards the upstream exon. Thus, the polar co-conversion and dependence of the splicing proficiency of the intron reported previously by us are hallmarks of group II intron homing, which significantly distinguish it from the strictly DNA-based group I intron homing and strictly RNA-based group II intron transposition.

Reverse transcriptase activity of an intron encoded polypeptide.

A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to retroviral reverse transcriptases (RTs). We have used the yeast transposon (Ty) system to express ORFs for RTs from eukaryotic organelles. This includes the mitochondrial coxI intron i1 from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii. The ORFs were fused with the TYA ORF from the yeast retrotransposon Ty to produce virus-like particles in the recipient strains with detectable amounts of the RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of viral or retrotransposable origin. In vitro assays showed that the intron encoded RT is sensitive to RT inhibitors such as N-ethylmaleimide and dideoxythymidine triphosphate but is insensitive against the DNA polymerase inhibitor aphidicolin. The direct biochemical evidence provided here supports the idea that intron encoded RTs are involved in intron transposition events.

A Chloroplast Group I Intron Undergoes the First Step of Reverse Splicing into Host Cytoplasmic 5·8 S rRNA: Implications for Intron-mediated RNA Recombination, Intron Transposition and 5·8 S rRNA Structure

The chloroplast 23 S rRNA gene of Chlamydomonas reinhardtii contains a self-splicing group I intron, Cr.LSU. Incubation of total RNA from C. reinhardtii [α- 32P]GTP produces a GTP-labeled RNA that is derived from Cr.LSU, but is ∼95 nucleotides longer (and referred to as I A). We show that I A is derived from an intermolecular reaction of Cr.LSU with cytoplasmic 5·8 S rRNA; the 3′-terminal G of the intron becomes ligated to A64. This reaction, which was reproduced using synthetic RNAs, is identical to the first step of reverse splicing. The second step of reverse splicing did not occur with any efficiency, even though both P1 and P10-like helices can form between the reactive region of 5·8 S rRNA and the IGS of Cr.LSU. Cloning of I A provided a chimeric Cr.LSU precursor with the normal 3′ exon substituted by 95 nucleotides of 5·8 S; this precursor spliced poorly despite having a potential P10 helix (between the 3′-exon and the IGS). The inefficient reverse splicing of Cr.LSU into 5·8 S RNA and poor forward splicing of the chimera may be explained by competition between helices P1 (between the 5′-exon and IGS) and P10; competition between P1 and P10 is apparently not a factor in forward splicing of the wild-type LSU precursor. The sequence of the 5·8 S gene of C. reinhardtii was determined, and the published RNA sequence revised. A secondary structure model of C. reinhardtii 5·8 S rRNA was proposed incorporating the requirement for base-pairing to the IGS of Cr.LSU. The resulting structure resembles that recently proposed for yeast 5·8 S rRNA, and has considerable advantages over previous models of C. reinhardtii 5·8 S rRNA. These data describe a novel example of naturally occurring ribozyme reacting with a naturally occurring RNA of the same cell; implications for ribozyme-mediated RNA recombination and intron transposition via reverse splicing are discussed.

Transposition of a group II intron.

Among mobile genetic elements, self-splicing introns are of particular interest. They belong to either group I or group II depending on their three-dimensional structure. Homing, the systematic intron invasion of an intronless gene when it encounters its homologous intron-bearing allele, is the only means for intron mobility so far demonstrated. It depends on the activity of the intron-encoded protein and is very specific for the acceptor site. Intron transposition, the transfer of an intron to a novel site, predicted on the basis of phylogenetic studies and in vitro reverse-splicing experiments, has been proposed to be responsible for evolutionary intron spreading. Here we present results from polymerase chain reaction experiments consistent with transposition of a group II intron. This event is proposed to account for the site-specific deletion in the mitochondrial chromosome of the fungus Podospora anserina that is associated with the premature death syndrome and might also be involved in the senescence process affecting this species.

Transposition of group II intron aI1 in yeast and invasion of mitochondrial genes at new locations.

Intron mobility at the RNA level by splicing reversal at allelic (homing) and non-allelic locations (transposition) has been reported in vitro. In the living cell, however, only intron homing by unidirectional gene conversion has been described. Supposing that intron insertions at non-allelic sites might occur in vivo, we speculated that group II splice-site-associated macro-deletions in fungal mitochondrial DNA might result from group II intron transposition to new locations followed by recombination. We used polymerase chain reaction techniques to detect this critical, infrequent intermediate in mtDNA populations. Here we report on group II intron aI1 transposition to non-allelic, splicing-compatible locations within the cox1 gene of yeast mtDNA. The identified integration sites are preceded by motifs similar to the upstream exon A1. Sequences flanking intron aI1 are not co-converted to the insertion sites and cis- and trans-acting mutations within aI1 reduce intron mobility below detection levels. These findings suggest the involvement of an RNA intermediate in group II intron transposition.

Group I introns interrupt the chloroplast psaB and psbC and the mitochondrial rrnL gene in Chlamydomonas.

The polymerase chain reaction was used to identify novel IAI subgroup introns in cpDNA-enriched preparations from the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii. These experiments along with sequence analysis disclosed the presence, in both green algae, of a single IA1 intron in the psaB gene and of two group I introns (IA2 and IA1) in the psbC gene. In addition, two group I introns (IA1 and IB4) were found in the peptidyltransferase region of the mitochondrial large subunit rRNA gene at the same positions as previously reported Chlamydomonas chloroplast introns. The 188 bp segment preceding the first mitochondrial intron revealed extensive sequence similarity to the distantly spaced rRNA-coding modules L7 and L8 in the Chlamydomonas reinhardtii mitochondrial DNA, indicating that these two modules have undergone rearrangements in Chlamydomonas. The IA1 introns in psaB and psbC were found to be related in sequence to the first intron in the C. moewusii chloroplast psbA gene. The similarity between the former introns extends to the immediate 5' flanking exon sequence, suggesting that group I intron transposition occurred from one of the two genes to the other through reverse splicing.

Integration of a group I intron into a ribosomal RNA sequence promoted by a tyrosyl-tRNA synthetase.

Group I and II introns are mobile elements that propagate by insertion into different genes. Some introns of both types self-splice in vitro by transesterification reactions catalysed by the intron RNA. These transesterifications are reversible, and it has been suggested that reverse splicing followed by reverse transcription and recombination with genomic DNA may be a mechanism for intron transposition. In vivo the splicing of many, if not all, group I and II introns requires protein factors, which may facilitate correct folding of the intron RNAs. Here we show that the Neurospora mitochondrial large rRNA intron, a group I intron that is not self-splicing in vitro, undergoes reverse splicing in a reaction promoted by the CYT-18 protein, the Neurospora mitochondrial tyrosyl-tRNA synthetase, which is required for splicing the intron in vivo. In contrast to known RNA-catalysed reverse splicing reactions, this protein-assisted reverse splicing is sufficiently rapid to compete with forward splicing at low RNA concentrations under physiologically relevant conditions, including high GTP and low Mg2+ concentrations. Our results indicate that proteins that promote splicing could contribute to intron mobility by promoting reverse splicing in vivo.